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10.1126/science.278.5338.675
| 39,474,152
|
A systematic search of the nonrecombining region of the human Y chromosome (NRY) identified 12 novel genes or families, 10 with full-length complementary DNA sequences. All 12 genes, and six of eight NRY genes or families previously isolated by less systematic means, fell into two classes. Genes in the first group were expressed in many organs; these housekeeping genes have X homologs that escape X inactivation. The second group, consisting of Y-chromosomal gene families expressed specifically in testes, may account for infertility among men with Y deletions. The coherence of the NRY's gene content contrasts with the apparently haphazard content of most eukaryotic chromosomes.
|
10.1038/35056058
|
Genes in the non-recombining region of the human Y chromosome conform to a small number of functional themes.
|
10.1073/pnas.88.24.11256
| 3,742,594
|
To investigate the evolution of the mammalian sex chromosomes, we have compared the gene content of the X chromosomes in the mammalian groups most distantly related to man (marsupials and monotremes). Previous work established that genes on the long arm of the human X chromosome are conserved on the X chromosomes in all mammals, revealing that this region was part of an ancient mammalian X chromosome. However, we now report that several genes located on the short arm of the human X chromosome are absent from the platypus X chromosome, as well as from the marsupial X chromosome. Because monotremes and marsupials diverged independently from eutherian mammals, this finding implies that the whole human X short arm region is a relatively recent addition to the X chromosome in eutherian mammals.
|
10.1038/35056058
|
This paper shows that there was a large translocation from autosome to sex chromosome in an ancestor of placental mammals, resulting in great enlargement of the placental sex chromosomes.
|
10.1083/jcb.153.1.159
| 58,551,558
|
The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother–bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions.
|
10.1038/35099020
|
This paper indicates that the presence of cytoplasmic microtubules in the bud neck might provide an inhibitory signal for the MEN by regulating the activity of Bub2–Bfa1. The authors correlate the loss of cytoplasmic microtubules from the bud neck with exit from mitosis. In mutants defective in cytoplasmic microtubule nucleation, exit from mitosis occurs inappropriately. The authors also propose that LTE1 has no role in controlling the activity of the MEN, at least at high temperatures.
|
10.1242/jcs.114.14.2649
| 123,075,918
|
In Saccharomyces cerevisiae, the phosphoprotein phosphatase Cdc14p plays a central role in exit from mitosis, by promoting B-type cyclin degradation and allowing accumulation of the cyclin-dependent kinase inhibitor Sic1p. Cdc14p is sequestered in the nucleolus during interphase, from where it is released at the end of mitosis, dependent upon mitotic exit network function. The CDC14 gene is essential and loss-of-function mutants arrest at the end of mitosis. We have identified a fission yeast orthologue of CDC14 through database searches. A Schizosaccharomyces pombe flp1 (cdc fourteen-like-phosphatase) null mutant is viable, divides at a reduced size and shows defects in septation. flp1p is not the essential effector of the S. pombe septation initiation network, but may potentiate signalling of the onset of septation. In contrast to S. cerevisiae Cdc14p, flp1p is not required for the accumulation or destruction of the B-type cyclin cdc13p, the cyclin-dependent kinase inhibitor rum1p, or for dephosphorylation of the APC/C specificity factor ste9p in G1. Like its budding yeast counterpart, flp1p is restricted to the nucleolus until mitosis, when it is dispersed through the nucleus. In contrast to S. cerevisiae Cdc14p, flp1p is also present on the mitotic spindle and contractile ring. The potential roles of flp1p in cell cycle control are discussed.
|
10.1038/35099020
|
S. pombe orthologue of S. cerevisiae CDC14 , called flp1 + , was identified and proposed to have a reduced role in cell-cycle regulation. Localization of flp1 is similar to that of Cdc14 in S. cerevisiae : it is in the nucleolus and on the SPB during interphase, and becomes re-localized to the mitotic spindles, the nucleus and contractile ring during mitosis. flp1 inhibits mitotic CDKs by regulating the tyrosine 15 phosphorylation of cdc2. The role of the SIN in flp1 release is also discussed, as well as a role for both in the cytokinesis checkpoint. The SIN and flp1 seem to be involved in a feedback loop, and the authors propose that, although flp1 is not the essential effector of the SIN, flp1 might help potentiate SIN signalling.
|
10.1242/jcs.113.10.1695
| 83,419,482
|
We have isolated the Schizosaccharomyces pombe orthologue of the Saccharomyces cerevisiae MOB1 gene in a screen designed to enrich for septation mutants. The gene is essential, and cells lacking it display a phenotype typical of septation signalling network mutants. mob1p is located on both spindle pole bodies throughout mitosis. In addition it is also co-localised with the medial ring later in mitosis, and flanks the septum as the medial ring contracts. We also demonstrate that mob1p can be precipitated from cells in a complex with the septation regulating kinase sid2p.
|
10.1038/35099020
|
A homologue of S. cerevisiae MOB1 was cloned from S. pombe and found to be a member of the SIN pathway. mob1 localizes to the SPB as well as to the medial ring late in the cell cycle. mob1 can form a complex with sid2. The authors propose that the mob1–sid2 complex is involved in carrying the septation signal from the SPB to the septum late in the cell cycle.
|
10.1242/jcs.113.7.1223
| 62,067,200
|
Cell division in Schizosaccharomyces pombe is achieved through the use of a medially positioned actomyosin ring. A division septum is formed centripetally, concomitant with actomyosin ring constriction. Genetic screens have identified mutations in a number of genes that affect actomyosin ring or septum assembly. These cytokinesis-defective mutants, however, undergo multiple S and M phases and die as elongated cells with multiple nuclei. Recently, we have shown that a mutant allele of the S. pombe drc1(+)/cps1(+) gene, which encodes a 1,3-(beta)-glucan synthase subunit, is defective in cytokinesis but displays a novel phenotype. drc1-191/cps1-191 cells are capable of assembling actomyosin rings and completing mitosis, but are incapable of assembling the division septum, causing them to arrest as binucleate cells with a stable actomyosin ring. Each nucleus in arrested cps1-191 cells is able to undergo S phase but these G(2) nuclei are significantly delayed for entry into the M phase. In this study we have investigated the mechanism that causes cps1-191 to block with two G(2) nuclei. We show that the inability of cps1-191 mutants to proceed through multiple mitotic cycles is not related to a defect in cell growth. Rather, the failure to complete some aspect of cytokinesis may prevent the G(2)/M transition of the two interphase-G(2) nuclei. The G(2)/M transition defect of cps1-191 mutants is suppressed by a mutation in the wee1 gene and also by the dominant cdc2 allele cdc2-1w, but not the cdc2-3w allele. Transient depolymerization of all F-actin structures also allowed a significant proportion of the cps1-191 cells to undergo a second round of mitosis. We conclude that an F-actin and Wee1p dependent checkpoint blocks G(2)/M transition until previous cytokinesis is completed.
|
10.1038/35099020
|
The authors characterize the cytokinesis checkpoint, which halts cell cycle progression when a septum fails to form. A mutation in the drc1 + cps1 + gene prevents the cells from forming a septum. Cells lacking drc1 + cps1 + arrest with two nuclei with a 2n DNA content. The authors show that this arrest is mediated by wee1 and cdc2. The authors also show that a transient depolymerization of the actin cytoskeleton can allow the arrested cells to escape the checkpoint arrest, indicating that the checkpoint is somehow sensing the state of the actin cytoskeleton.
|
10.1126/science.1057330
| 109,156,801
|
As an organelle coupling nuclear and cytoplasmic divisions, the centrosome is essential to mitotic fidelity, and its inheritance could be critical to understanding cell transformation. Investigating the behavior of the centrosome in living mitotic cells, we documented a transient and remarkable postanaphase repositioning of this organelle, which apparently controls the release of central microtubules from the midbody and the completion of cell division. We also observed that the absence of the centrosome leads to defects in cytokinesis. Together with recent results in yeasts, our data point to a conserved centrosome-dependent pathway that integrates spatial controls into the decision of completing cell division, which requires the repositioning of the centrosome organelle.
|
10.1038/35099020
|
This paper shows that the mammalian centrosome has an important role in cytokinesis. The behaviour of GFP-tagged centrioles shows a post-anaphase repositioning of the old centriole to the midbody that is essential for cytokinesis, as ablation of the centriole causes defects in cytokinesis.
|
10.1083/jcb.153.1.237
| 101,173,535
|
When centrosomes are destroyed during prophase by laser microsurgery, vertebrate somatic cells form bipolar acentrosomal mitotic spindles (Khodjakov, A., R.W. Cole, B.R. Oakley, and C.L. Rieder. 2000. Curr. Biol. 10:59–67), but the fate of these cells is unknown. Here, we show that, although these cells lack the radial arrays of astral microtubules normally associated with each spindle pole, they undergo a normal anaphase and usually produce two acentrosomal daughter cells. Relative to controls, however, these cells exhibit a significantly higher (30–50%) failure rate in cytokinesis. This failure correlates with the inability of the spindle to properly reposition itself as the cell changes shape. Also, we destroyed just one centrosome during metaphase and followed the fate of the resultant acentrosomal and centrosomal daughter cells. Within 72 h, 100% of the centrosome-containing cells had either entered DNA synthesis or divided. By contrast, during this period, none of the acentrosomal cells had entered S phase. These data reveal that the primary role of the centrosome in somatic cells is not to form the spindle but instead to ensure cytokinesis and subsequent cell cycle progression.
|
10.1038/35099020
|
This study investigates the effects of destroying centrosomes after prophase. The authors find that although anaphase occurs normally and can produce two acentrosomal daugher cells, cytokinesis is markedly affected with 30–50% of cells failing to execute cytokinesis properly.
|
10.1126/science.285.5424.113
| 125,294,056
|
Lymphocyte development is critically influenced by self-antigens. T cells are subject to both positive and negative selection, depending on their degree of self-reactivity. Although B cells are subject to negative selection, it has been difficult to test whether self-antigen plays any positive role in B cell development. A murine model system of naturally generated autoreactive B cells with a germ line gene–encoded specificity for the Thy-1 (CD90) glycoprotein was developed, in which the presence of self-antigen promotes B cell accumulation and serum autoantibody secretion. Thus, B cells can be subject to positive selection, generated, and maintained on the basis of their autoreactivity.
|
10.1038/35105052
|
The first demonstration that self-antigen drives the selection of innate lymphocytes.
|
10.1084/jem.153.3.694
| 41,151,638
|
The antiphosphocholine (PC) antibody in normal mouse sera (NMS) provides protection against intravenous infection with encapsulated strain WU2 of type 3 Streptococcus pneumoniae. Mice unable to make anti-PC antibody, as a result of suppression with anti-T-15 idiotype or inheritance of the xid gene of CAB/N mice, are highly susceptible to infection with strain WU2. Mice inheriting the xid gene can be protected with NMS from immunologically normal mice or with IgM hybridoma anti-PC antibody. The protective effect of NMS can be removed with PC-containing immunoabsorbents.
|
10.1038/35105052
|
The first demonstration that innate lymphocytes confer natural protection against infection.
|
10.1084/jem.193.8.893
| 18,633,189
|
To define the phenotype and T cell receptor (TCR) repertoire of CD1d-dependent T cells, we compared the populations of T cells that persisted in major histocompatibility complex (MHC)-deficient mice, which lack mainstream T cells, with those from MHC/CD1d doubly deficient mice, which lack both mainstream and CD1d-dependent T cells. Surprisingly, up to 80% of the CD1d-dependent T cells were stained by tetramers of CD1d/α-galactosylceramide, which specifically identify the previously described CD1d autoreactive Vα14-Jα18/Vβ8 natural killer (NK) T cells. Furthermore, zooming in on the CD1d-dependent non-Vα14 T cells, we found that, like Vα14 NK T cells, they mainly expressed recurrent, CD1d autoreactive TCR families and had a natural memory phenotype. Thus, CD1d-restricted T cells differ profoundly from MHC-peptide–specific T cells by their predominant use of autoreactive and semiinvariant, rather than naive and diverse, TCRs. They more closely resemble other lineages of innate lymphocytes such as B-1 B cells, γδ T cells, and NK cells, which express invariant or semiinvariant autoreactive receptors. Finally, we demonstrate that the MHC-restricted TCR repertoire is essentially non–cross-reactive to CD1d. Altogether, these findings imply that lipid recognition by CD1d-restricted T cells may have largely evolved as an innate rather than an adaptive arm of the mouse immune system.
|
10.1038/35105052
|
The intriguing demonstration that most of the mouse CD1d-restricted T-cell receptor repertoire is innate rather than adaptive.
|
10.1126/science.7973709
| 104,301,637
|
The role played in immune surveillance by γδ T cells residing in various epithelia has not been clear. It is shown here that activated γδ T cells obtained from skin and intestine express the epithelial cell mitogen keratinocyte growth factor (KGF). In contrast, intraepithelial αβ T cells, as well as all lymphoid αβ and γδ T cell populations tested, did not produce KGF or promote the growth of cultured epithelial cells. These results suggest that intraepithelial γδ T cells function in surveillance and in repair of damaged epithelial tissues.
|
10.1038/35105052
|
A demonstration of the crosstalk between tissues and their resident lymphocytes.
|
10.1152/jn.1995.73.1.1
| 42,503,747
|
1. On the basis of its anatomic connections and single-unit properties, the superior temporal polysensory area (STP) would seem to be primarily involved in visuospatial functions. We have examined the effects of lesions of STP on saccadic eye movements, visual fixation, and smooth pursuit eye movements to directly test the hypothesis that STP is involved in visuospatial and visuomotor behavior. 2. Seven monkeys were trained to make saccades to targets 8, 15, and 22 degrees from a central fixation point along the horizontal meridian and 8 degrees from the central fixation point along the vertical meridian. One monkey was also trained to make saccades to auditory targets. The same monkeys were trained to foveate a stationary central fixation point and to follow it with a smooth pursuit eye movement when it began moving 5, 13, or 20 degrees/s. Four monkeys received unilateral STP lesions, one received a bilateral STP lesion, and as a control, two received unilateral inferior temporal cortex (IT) lesions. After testing, three of the animals with unilateral STP lesions received an additional STP lesion in the hemisphere contralateral to the first lesion. Similarly, one animal with a unilateral IT lesion received an additional IT lesion in the hemisphere contralateral to the first lesion. 3. All monkeys with complete removal of STP showed a significant increase in saccade latency to the most peripheral contralateral target, and most also had increased saccade latencies to the other contralateral targets. Saccades directed to targets along the vertical meridian or toward targets in the hemifield ipsilateral to the lesion were not impaired by removal of STP. By contrast, IT lesions did not impair the monkeys' ability to make saccadic eye movements to visual stimuli at any location, showing that saccades to visually guided targets are not impaired nonspecifically by damage to visual cortex. 4. The deficit in making eye movements after STP lesions was specific to saccade latency, with little effect on the accuracy of saccades to visual targets. 5. In the one monkey trained to make saccades to auditory targets, removal of STP did not impair saccades to auditory targets contralateral to its lesion, despite this monkey showing the largest increase in saccades latencies to visual targets. 6. There was complete recovery of saccade latency to the baseline level of performance on the saccade task after all STP lesions.(ABSTRACT TRUNCATED AT 400 WORDS)
|
10.1038/35086057
|
The first study to investigate the behavioural effects of lesions confined to the STP in monkeys. Saccade latency was found to be increased for orienting to contralesional targets, whereas responses towards ipsilesional targets were unaffected.
|
10.1152/jn.1996.76.1.109
| 41,441,699
|
1. Processing of visual information in primates is believed to occur in at least two separate cortical pathways, commonly labeled the "form" and "motion" pathways. This division lies in marked contrast to our everyday visual experience, in which we have a unified percept of both the form and motion of objects, implying integration of both types of information. We report here on a neuronal population in the anterior part of the superior temporal polysensory area (STPa) both sensitive to form (heads and bodies) and selective for motion direction. 2. A total of 161 cells were found to be sensitive to body form and motion. The majority of cells (125 of 161, 78%) responded to only one combination of view and direction (termed unimodal cells, e.g., left profile view moving left, not right profile moving left, or left profile moving right). We show that the response of some of these cells is selective for both the motion and the form of a single object, not simply the juxtaposition of appropriate form and motion signals. 3. A smaller number of cells (9 of 161, 6%) responded selectively to two opposite combinations of view and direction (e.g., left profile moving left and right profile moving right, but no other view and direction combinations). A few cells (4 of 161, 2%) showed "object-centered" selectivity to view and direction combinations, responding to all directions of motion where the body moves in a direction compatible with the direction it faces, for example, responding to left profile going left, right profile going right, face view moving toward the observer, back view moving away from the observer, but not other view and direction combinations. 4. The majority of the neurons (106 of 138, 77%) selective for specific body view and direction combinations responded best to compatible motion (e.g., left profile moving left), and one fourth (23%) showed selectivity for incompatible motion (e.g., right profile moving left). 5. The relative strengths of motion and form inputs to cells in STPa conjointly sensitive to information about form and motion were assessed. The majority of the responses (95%) were characterized as showing nonlinear summation of form and motion inputs. 6. The capacity to discriminate different directions and different forms was compared across three populations of STPa cells, namely those sensitive to 1) form only, 2) motion only, and 3) both form and motion. The selectivity of the latter class could be predicted from combinations of the other two classes. 7. The response latencies of cells selective for form and motion are on average coincident with cells selective for direction of motion (but not stimulus form). Both these cell populations have response latencies on average 20 ms earlier than cells selective for static form. 8. Calculation of the average of early response latency cells (cell whose response latency was under the sample mean) suggests that direction information is present in cell responses some 35 ms before form information becomes evident. Direction information and form information become evident within 5 ms of each other in the average late response latency cells (those cells whose response latency was greater than the sample mean). Inputs relating to movement show an initial response period that does not discriminate direction. The quality of initial direction discrimination appeared to be independent of response latency. The initial discrimination of form was related to response latency in that cells with longer response latencies showed greater initial discrimination of form in their responses. We argue that these findings are consistent with form inputs arriving to area STPa approximately 20 ms after motion inputs into area STPa.
|
10.1038/35086057
|
Functional evidence is reported that information from the dorsal and ventral systems converge in the superior temporal polysensory area at the single-unit level, providing a conjoint representation of object identity and direction of motion.
|
10.1093/oso/9780198524533.001.0001
| 149,178,222
|
Abstract As we move around in our environment, and interact with it, many of the most important problems we face involve the processing of spatial information. We have to be able to navigate by perceiving and remembering the locations and orientations of the objects around us relative to ourself; we have to sense and act upon these objects; and we need to move through space to position ourselves in favourable locations or to avoid dangerous ones. While this appears so simple that we don't even think about it, the difficulty of solving these problems has been shown in the repeated failure of artificial systems to perform these kinds of tasks efficiently. In contrast, humans and other animals routinely overcome these problems every single day. This book examines some of the neural substrates and mechanisms that support these remarkable abilities. The hippocampus and the parietal cortex have been implicated in various core spatial behaviours, such as the ability to localise an object and navigate to it. Damage to these areas in humans and animals leads to impairment of these spatial functions. This collection of papers, written by internationally recognized experts in the field, reviews the evidence that each area is involved in spatial cognition, examines the mechanisms underlying the generation of spatial behaviours, and considers the relative roles of the parietal and hippocampal areas, including how each interacts with the other. The papers integrate a wide range of theoretical and experimental approaches, and touch on broader issues relating to memory and imagery. As such, this book represents the state of the art of current research into the neural basis of spatial cognition. It should be of interest to anyone - researchers or graduate students - working in the areas of cognitive neuroscience, neuroanatomy, neuropsychology, and cognition generally.
|
10.1038/35086057
|
A review and analysis of 'object-centred' and egocentric effects in patients with spatial neglect. It is suggested that the pathological egocentric bias produced by the lesion that leads to neglect can be superimposed on relatively preserved visual object-segmentation processes.
|
10.1126/science.7701330
| 104,003,151
|
Neurons in the superior temporal gyrus of anesthetized rhesus monkeys were exposed to complex acoustic stimuli. Bandpassed noise bursts with defined center frequencies evoked responses that were greatly enhanced over those evoked by pure tones. This finding led to the discovery of at least one new cochleotopic area in the lateral belt of the nonprimary auditory cortex. The best center frequencies of neurons varied along a rostrocaudal axis, and the best bandwidths of the noise bursts varied along a mediolateral axis. When digitized monkey calls were used as stimuli, many neurons showed a preference for some calls over others. Manipulation of the calls' frequency structure and playback of separate components revealed different types of spectral integration. The lateral areas of the monkey auditory cortex appear to be part of a hierarchical sequence in which neurons prefer increasingly complex stimuli and may form an important stage in the preprocessing of communication sounds.
|
10.1038/35086057
|
Single-unit recordings in the superior temporal gyrus of the rhesus monkey revealed neurons showing preferences for vocalizations from the monkeys' own repertoire (species-specific communication calls).
|
10.1034/j.1399-0004.2000.580112.x
| 35,883,641
|
Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by a CAG repeat expansion in the CACNA1A gene. The neurodegeneration that occurs in CAG repeat diseases is considered to share a common mechanism that may result in the gain of a toxic function related to the expanded polyglutamine tracts. However, the phenotypic expression in homozygotes for CAG repeat diseases has been controversial, and is not clearly related to a gain of functional mechanism. We identified a Japanese family with two sisters who were homozygous for the SCA6 with identical CAG repeat expansion (25/25). They showed an earlier age of onset (27 years in both) than their father (44 years), a heterozygote with an expanded allele showing the same CAG repeat length as the homozygotes (25/14). Interestingly, the two sisters showed differences in disease progression and severity, although the age of onset and CAG repeat length were identical. These findings strongly suggest that the gene dosage influences the age of onset, but other unknown factors are also important in the phenotypic expression of homozygous SCA6 .
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10.1038/35039051
|
Whereas onset of neurological symptoms in two sisters homozygous for SCA6 mutant alleles was identical, disease progression and symptoms varied significantly, supporting a role for genetic, environmental or stochastic modifiers.
|
10.1073/pnas.94.8.3872
| 20,022,738
|
Huntington disease (HD) is associated with abnormal expansions of a CAG repeat close to the 5′ end of the IT15 gene. We have assembled a set of 293 HD subjects whose ages of onset were known and sized their HD CAG repeats. These repeats accounted for 69% of the variance of age of onset when we used the most parsimonious model, which relates the logarithm of age of onset to a function of CAG repeat number. Since other familial factors have been proposed to influence the age of onset of HD, we have examined a number of candidate loci. The CAG repeat number on normal chromosomes, the Δ2642 polymorphism in the HD gene, and apolipoprotein E genotypes did not affect the age of onset of HD. Although mitochondrial energy production defects in HD have led to suggestions that variants in the mitochondrial genome may be associated with clinical variability in HD, this suggestion was not supported by our preliminary experiments that examined the Dde I mitochondrial restriction fragment length polymorphism at position 10,394. Excitotoxicity has been a favored mechanism to explain the cell death in HD, particularly since intrastriatal injection of excitatory amino acids in animals creates HD-like pathology. Accordingly, we investigated the GluR6 kainate receptor. Of the variance in the age of onset of HD that was not accounted for by the CAG repeats, 13% could be attributed to GluR6 genotype variation. These data implicate GluR6-mediated excitotoxicity in the pathogenesis of HD and highlight the potential importance of this process in other polyglutamine repeat expansion diseases.
|
10.1038/35039051
|
This paper identifies the first genetic factor other than the disease gene that modifies the onset of a polyglutamine disorder, confirmed in a different population by MacDonald and colleagues 78
|
10.1083/jcb.152.4.809
| 104,410,971
|
Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA–gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.
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10.1038/35096009
|
Describes the model for melanosome biogenesis discussed in this review and is the first paper to demonstrate a distinction between melanosomes and lysosomes.
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10.1083/jcb.141.5.1121
| 58,551,668
|
The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus–transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1–2 μm), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules.
|
10.1038/35096009
|
This paper describes the late endosomal/ lysosomal protein sorting defects observed in Chediak Higashi syndrome, using B lymphocytes as a model.
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10.1083/jcb.130.4.807
| 16,725,327
|
The structural and functional integrity of cytoplasmic organelles is maintained by intracellular mechanisms that sort and target newly synthesized proteins to their appropriate cellular locations. In melanocytic cells, melanin pigment is synthesized in specialized organelles, melanosomes. A family of melanocyte-specific proteins, known as tyrosinase-related proteins that regulate melanin pigment synthesis, is localized to the melanosomal membrane. The human brown locus protein, tyrosinase-related protein-1 or gp75, is the most abundant glycoprotein in melanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sorting of glycoprotein (gp)75, we constructed protein chimeras containing the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fibroblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelles. Signals in the gp75 cytoplasmic tail alone, were sufficient for intracellular retention and targeting of the chimeric proteins to the endosomal/lysosomal compartment. Analysis of subcellular localization of carboxy-terminal deletion mutants of gp75 and the CD8/gp75 chimeras showed that deletion of up amino acids from the gp75 carboxyl terminus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acids were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conserved among other melanosomal proteins. Further evidence showed that this hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanosomal proteins along the endosomal/lysosomal pathway lies within this sequence. These data provide evidence for common signals for intracellular sorting of melanosomal and lysosomal proteins, and support the notion that lysosomes and melanosomes share a common endosomal pathway of biogenesis.
|
10.1038/35096009
|
The first paper to show that targeting signals for integral membrane transport to melanosomes and to lysosomes are similar.
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10.1083/jcb.143.7.1899
| 59,302,826
|
Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va−) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.
|
10.1038/35096009
|
This paper was the first tp propose the current model for melanosome transport in melanocytes.
|
10.1083/jcb.152.4.795
| 40,524,185
|
Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myosinVa colocalize on the cytoplasmic face of peripheral melanosomes in wild-type melanocytes. However, the amount of myosinVa in melanosomes from Rab27a-deficient ashen melanocytes is greatly reduced. These results, together with recent data implicating myosinVa in the peripheral capture of melanosomes, suggest that Rab27a is necessary for the recruitment of myosinVa, so allowing the peripheral retention of melanosomes in melanocytes.
|
10.1038/35096009
|
Together with references 89 and 101 , this paper provides mechanistic insights into the role of Rab27a and myosin Va in melanosome transport.
|
10.1083/jcb.152.4.825
| 100,813,025
|
Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the α subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.
|
10.1038/35096009
|
Together with reference 103 , this paper shows the requirement for Rab27a in lysosome-like organelle secretion, using cytotoxic T cells as a model system.
|
10.1126/science.8009217
| 83,408,569
|
Malaria is a disease caused by repeated cycles of growth of the parasite Plasmodium in the erythrocyte. Various cellular and molecular strategies allow the parasite to evade the human immune response for many cycles of parasite multiplication. Under certain circumstances Plasmodium infection causes severe anemia or cerebral malaria; the expression of disease is influenced by both parasite and host factors, as exemplified by the exacerbation of disease during pregnancy. This article provides an overview of malaria pathogenesis, synthesizing the recent field, laboratory, and epidemiological data that will lead to the development of strategies to reduce mortality and morbidity.
|
10.1038/35100540
|
This paper provides a concise overview of the pathogenesis of malaria.
|
10.1126/science.2417315
| 41,624,245
|
Red blood cells that are infected with the malaria parasite Plasmodium falciparum express new antigens on their surface. In a study of these antigens in the erythrocytes of naturally infected children in the Gambia, an antibody-mediated agglutination assay revealed an extreme degree of antigenic diversity. Serum samples from each of ten children in the convalescent stage of malaria infection reacted with infected cells from the same child but generally not with infected cells from the other children. The Gambian children's erythrocytes also expressed shared determinants: sera from Gambian adults often reacted with the surface of infected cells from all of the children and were shown by adsorption and elution experiments to contain antibodies that recognized several isolates. Conserved determinants exposed on infected erythrocytes may be important for development of antimalarial immunity either naturally or through vaccination.
|
10.1038/35100540
|
This paper provides some of the best early insights into the nature of natural immunity to malaria and provides an explanation for why prolonged exposure to the parasite is required for this to develop.
|
10.1126/science.8100366
| 123,801,339
|
CD4 + T cells play a major role in protective immunity against the blood stage of malaria, but the mechanism of protection is unclear. By adoptive transfer of cloned T cell lines, direct evidence is provided that both T H 1 and T H 2 subsets of CD4 + T cells can protect mice against Plasmodium chabaudi chabaudi infection. T H 1 cells protect by a nitric oxide-dependent mechanism, whereas T H 2 cells protect by the enhancement and accelerated production of specific immunoglobulin G1 antibody.
|
10.1038/35100540
|
Indicates that T H 1- and T H 2-type T cells can control malaria infections through different mechanisms.
|
10.1073/pnas.95.4.1715
| 38,573,535
|
The immune response to malaria parasites includes T cell responses that reduce parasites by effector T cell responses and by providing help for antibody responses. Some parasites are more sensitive to antibody and others are more sensitive to cell-mediated immunity. We demonstrate that cultured CD4 + T cells that produce interferon γ and interleukin 2, but not interleukin 4, in response to stimulation with the rodent parasite Plasmodium berghei can reduce but not eliminate parasites in vivo after adoptive transfer. Although cells can persist in vivo for up to 9 months in uninfected mice, infection results in elimination of up to 99% of specific T cells in different tissues, as judged by tracking T cells labeled with the fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester. T cells specific for ovalbumin are unaffected. In vivo activation and division of transferred T cells per se are not responsible for deletion because T cells positive for 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester divide up to six times within 7 days in uninfected mice and are not deleted. Understanding the factors responsible for parasite-mediated specific deletion of T cells would enhance our knowledge of parasite immunity.
|
10.1038/35100540
|
Indicates that malaria infection can cause deletion of parasite-specific CD4 + T cells. This might prove to be a useful parasite-defence mechanism.
|
10.1126/science.286.5441.950
| 84,427,523
|
Posttranscriptional gene silencing (PTGS) is a nucleotide sequence–specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.
|
10.1038/35052556
|
A potential mediator of post-transcriptional gene silencing (PTGS) was identified for the first time. This small ∼ 25-nucleotide RNA has become a hallmark of silencing processes that are mediated by double-stranded (ds) RNA molecules
|
10.1073/pnas.96.24.14147
| 82,988,841
|
In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.
|
10.1038/35052556
|
This is one of several papers that establishes a second biological function for RNAi by linking dsRNA-induced silencing to virus resistance in plants
|
10.1126/science.285.5429.901
| 104,265,497
|
The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
|
10.1038/35066084
|
A report on the progress of an international consortium that disrupted each annotated open reading frame in the yeast genome using a targeted PCR-based gene-deletion strategy.
|
10.1126/science.290.5500.2306
| 59,323,339
|
Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo. We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively. The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12. Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance.
|
10.1038/35066084
|
References 42 and 43 describe a new genomic approach that combines chromatin immunoprecipitation and microarray hybridization to map the binding sites of chromosomal proteins in vivo.
|
10.1126/science.272.5262.735
| 125,208,521
|
The P 2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P 2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X 7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P 2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X 7 (or P 2Z ) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.
|
10.1038/35058521
|
Cloning and expression of the P2X 7 subunit, and the demonstration of its ability to change its permeability.
|
10.1085/jgp.113.5.695
| 121,938,134
|
The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.
|
10.1038/35058521
|
The first detailed study of the single-channel behaviour of P2X 2 receptors.
|
10.1523/jneurosci.20-20-07517.2000
| 10,339,048
|
Within the Caenorhabditis elegans genome there exist at least 42 genes encoding TWK (two-P domain K + ) channels, potassium channel subunits that contain two pore regions and four transmembrane domains. We now report the first functional characterization of a TWK channel from C. elegans . Although potassium channels have been reported to be activated by a variety of factors, TWK-18 currents increase dramatically with increases in temperature. Two mutant alleles of the twk-18 gene confer uncoordinated movement and paralysis in C. elegans . Expression of wild-type and mutant TWK-18 channels in Xenopus oocytes showed that mutant channels express much larger potassium currents than wild-type channels. Promoter–green fluorescent protein fusion experiments indicate that TWK-18 is expressed in body wall muscle. Our genetic and physiological data suggest that the movement defects observed in mutant twk-18 animals may be explained by an increased activity of the mutant TWK-18 channels.
|
10.1038/35058574
|
Describes the identification of TWK-18, the first functional two-P/four-transmembrane domain clone of at least 42 potential variants in C. elegans
|
10.1126/science.283.5398.94
| 81,877,078
|
CmPP16 from Cucurbita maxima was cloned and the protein was shown to possess properties similar to those of viral movement proteins. CmPP16 messenger RNA (mRNA) is present in phloem tissue, whereas protein appears confined to sieve elements (SE). Microinjection and grafting studies revealed that CmPP16 moves from cell to cell, mediates the transport of sense and antisense RNA, and moves together with its mRNA into the SE of scion tissue. CmPP16 possesses the characteristics that are likely required to mediate RNA delivery into the long-distance translocation stream. Thus, RNA may move within the phloem as a component of a plant information superhighway.
|
10.1038/35067016
|
First evidence for an endogenous protein in pumpkin that mediates long-distance translocation of mRNAs through the plant's vascular system.
|
10.1126/science.276.5315.1128
| 124,089,832
|
In many organisms, pattern formation in the embryo develops from the polarized distributions of messenger RNAs (mRNAs) in the egg. In Xenopus , the mRNA encoding Vg1, a growth factor involved in mesoderm induction, is localized to the vegetal cortex of oocytes. A protein named Vera was shown to be involved in Vg1 mRNA localization. Vera cofractionates with endoplasmic reticulum (ER) membranes, and endogenous Vg1 mRNA is associated with a subcompartment of the ER. Vera may promote mRNA localization in Xenopus oocytes by mediating an interaction between the Vg1 3′ untranslated region and the ER subcompartment.
|
10.1038/35067016
|
First evidence for an association of a zip-code-binding protein (Vera) with the endoplasmic reticulum (ER) of Xenopus oocytes. This work suggests co-transport of the localized Vg1 mRNA with the ER.
|
10.1101/gad.12.11.1593
| 58,400,618
|
Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3′ UTR cis -acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of β actin mRNA in fibroblasts. These data support Vg1 RBP’s direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting.
|
10.1038/35067016
|
Reference 59 60 and 22 led to the surprising finding that the zip-code-binding proteins (ZBPs) involved in chicken β-actin mRNA or Xenopus Vg1 mRNA localization are homologous, although the localization mechanisms seemed to be different. This led to the idea that ZBPs might be mRNA-binding modules that can associate with different transport machineries.
|
10.1083/jcb.148.3.427
| 79,454,612
|
Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu–Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.
|
10.1038/35067016
|
The first example of a biochemical approach to purify localized RNP complexes from Drosophila
|
10.1126/science.289.5487.2120
| 59,365,103
|
The asymmetric localization of messenger RNA (mRNA) and protein determinants plays an important role in the establishment of complex body plans. In Drosophila oocytes, the anterior localization of bicoid mRNA and the posterior localization of oskar mRNA are key events in establishing the anterior-posterior axis. Although the mechanisms that drive bicoid and oskar localization have been elusive, oocyte microtubules are known to be essential. Here we report that the plus end–directed microtubule motor kinesin I is required for the posterior localization of oskar mRNA and an associated protein, Staufen, but not for the anterior-posterior localization of other asymmetric factors. Thus, a complex containing oskar mRNA and Staufen may be transported along microtubules to the posterior pole by kinesin I.
|
10.1038/35067016
|
The identification of the long-sought microtubule motor required for localization of oskar mRNA and Staufen protein to the posterior end of Drosophila oocytes.
|
10.1073/pnas.96.21.11820
| 41,281,361
|
Direct-force measurements of the interactions between recombinant C-cadherin from Xenopus demonstrated that the ectodomain of cadherin exhibits multiple adhesive contacts that involve successive domains along the extracellular region of the protein. Contacts between the fully interdigitated antiparallel proteins form the strongest adhesive interaction. A second weaker minimum was measured when the interdigitated proteins were separated by a distance equal to the length of one domain of the extracellular (EC) fragment and corresponding to the antiparallel alignment of domains one through four (EC1 through EC4). The successive rupture of these interactions generates an unbinding force profile that may be optimized to impede the abrupt failure of cadherin-mediated junctions under force.
|
10.1038/35040042
|
A recent paper that suggests a new model of cadherin trans -dimer formation.
|
10.1083/jcb.145.3.539
| 41,711,001
|
We have isolated a novel actin filament–binding protein, named afadin, localized at cadherin-based cell–cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament–binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor–related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell–cell AJs in various tissues and cell lines. In E-cadherin–expressing EL cells, PRR was recruited to cadherin-based cell–cell AJs through interaction with afadin. PRR showed Ca2+-independent cell–cell adhesion activity. These results indicate that PRR is a cell–cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell–cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word “necto” meaning “to connect”).
|
10.1038/35040042
|
One of a series of papers (refs 40 44 ) that describe a new protein complex that localizes to adherens junctions, and that might interact with the cadherin?catenin complex functionally.
|
10.1242/dev.126.23.5421
| 104,398,700
|
The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.
|
10.1038/35040042
|
References 55 and 56 show a role for the Flamingo/Starry Night cadherin in planar epithelial polarity. Reference 55 also shows that the subcellular distribution of Flamingo/Starry Night depends on the direction of Wnt/Frizzled signalling.
|
10.1242/dev.125.23.4681
| 41,623,964
|
Paraxial Protocadherin (PAPC) encodes a transmembrane protein expressed initially in Spemann's organizer and then in paraxial mesoderm. Together with another member of the protocadherin family, Axial Protocadherin (AXPC), it subdivides gastrulating mesoderm into paraxial and axial domains. PAPC has potent homotypic cell adhesion activity in cell dissociation and reaggregation assays. Gain- and loss-of-function microinjection studies indicate that PAPC plays an important role in the convergence and extension movements that drive Xenopus gastrulation. Thus, PAPC is not only an adhesion molecule but also a component of the machinery that drives gastrulation movements in Xenopus. PAPC may provide a link between regulatory genes in Spemann's organizer and the execution of cell behaviors during morphogenesis.
|
10.1038/35040042
|
Experiments in this paper indicate an important role for a protocadherin in the convergent extension movements during frog gastrulation.
|
10.1083/jcb.135.3.767
| 45,365,849
|
Molecular mechanisms linking pre- and postsynaptic membranes at the interneuronal synapses are little known. We tested the cadherin adhesion system for its localization in synapses of mouse and chick brains. We found that two classes of cadherin-associated proteins, alpha N- and beta-catenin, are broadly distributed in adult brains, colocalizing with a synaptic marker, synaptophysin. At the ultrastructural level, these proteins were localized in synaptic junctions of various types, forming a symmetrical adhesion structure. These structures sharply bordered the transmitter release sites associated with synaptic vesicles, although their segregation was less clear in certain types of synapses. N-cadherin was also localized at a similar site of synaptic junctions but in restricted brain nuclei. In developing synapses, the catenin-bearing contacts dominated their junctional structures. These findings demonstrate that interneuronal synaptic junctions comprise two subdomains, transmitter release zone and catenin-based adherens junction. The catenins localized in these junctions are likely associated with certain cadherin molecules including N-cadherin, and the cadherin/ catenin complex may play a critical role in the formation or maintenance of synaptic junctions.
|
10.1038/35040042
|
References 82 and 83 show that classic cadherins are components of synaptic adherens junctions.
|
10.1111/j.1442-2026.1997.tb00387.x
| 18,894,772
|
Abstract Objective To analyse the significance of commonly used symptoms, signs and radiography in predicting skull fractures, intracranial injuries and the need for neurosurgical intervention in paediatric head injuries. Setting A paediatric tertiary referral hospital in central Sydney. Method A 10 year retrospective chart review of all patients admitted with head injuries. Results Skull fractures were associated with decreasing age, findings of a scalp haematoma, clinical signs of a basal skull fracture and suspected skull depression/penetration. Intracranial injury (ICI) was associated with decreasing GCS scores, eye signs, suspected skull depression/penetration and skull fractures. Neurosurgical operative treatment was associated with decreasing GCS scores, eye signs, and skull fractures. Skull fractures were associated with neurosurgical operative treatment in all age groups. However the association of skull fractures and ICI was not significant in three of the five age groups examined. Conclusions In stratifying the risk of a complicated paediatric head injury, a greater focus should be made on specific physical findings. Less emphasis should be placed on historical features. Abnormal skull radiography may assist in stratifying the risk of intracranial injuries or the need for neurosurgical treatment. However the limitations of this imaging modality need to be acknowledged.
|
10.1038/35040009
|
Provides a historical overview of the morphological and biochemical features of apoptosis.
|
10.1073/pnas.96.7.4125
| 44,855,400
|
Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.
|
10.1038/35040009
|
Uses knock-in mice to identify the gain-of-function action of mutant presenilin-1 as a pro-apoptotic activity that results from perturbed cellular calcium homeostasis and can be suppressed by neurotrophic factors.
|
10.1126/science.285.5435.1870
| 62,065,416
|
Long-term potentiation of synaptic transmission in the hippocampus is the leading experimental model for the synaptic changes that may underlie learning and memory. This review presents a current understanding of the molecular mechanisms of this long-lasting increase in synaptic strength and describes a simple model that unifies much of the data that previously were viewed as contradictory.
|
10.1038/35036191
|
References 6 and 7 are two classic summaries of the state of research on E-LTP.
|
10.1126/science.167.3926.1740
| 123,454,860
|
A behavioral reflex mediated by identified motor neurons in the abdominal ganglion of Aplysia undergoes two simple forms of short-term modification. When the gill-withdrawal reflex was repeatedly evoked by a tactile stimulus to the siphon or mantle shelf, the amplitude of the response showed marked decrement (habituation). After a period of rest the response showed spontaneous recovery. The amplitude of a habituated response was facilitated by the presentation of a strong tactile stimulus to another part of the animal (dishabituation). Many characteristics of habituation and dishabituation in Aplysia are similar to those in vertebrates.
|
10.1038/35036191
|
References 11 and 12 were the first systematic attempts to address the questions of behavioural relevance and time course of synaptic plasticity related to memory storage at the level of single identified nerve cells.
|
10.1126/science.287.5451.248
| 38,447,845
|
The memory consolidation hypothesis proposed 100 years ago by Müller and Pilzecker continues to guide memory research. The hypothesis that new memories consolidate slowly over time has stimulated studies revealing the hormonal and neural influences regulating memory consolidation, as well as molecular and cellular mechanisms. This review examines the progress made over the century in understanding the time-dependent processes that create our lasting memories.
|
10.1038/35036191
|
Reviews the importance of modulatory transmitters for attentional and motivational significance of long-term memory storage.
|
10.1084/jem.187.1.129
| 29,131,174
|
T helper cells type 1 (Th1s) that produce interferon-γ predominantly mediate cellular immune responses and are involved in the development of chronic inflammatory conditions, whereas Th2s which produce large amounts of IL-4 and IL-5 upregulate IgE production and are prominent in the pathogenesis of allergic diseases. The precise factors determining whether Th1- or Th2-mediated immune responses preferentially occur at a peripheral site of antigen exposure are largely unknown. Chemokines, a superfamily of polypeptide mediators, are a key component of the leukocyte recruitment process. Here we report that among four CXC (CXCR1-4) and five CC (CCR1-5) chemokine receptors analyzed, CXCR3 and CCR5 are preferentially expressed in human Th1s. In contrast, Th2s preferentially express CCR4 and, to a lesser extent, CCR3. In agreement with the differential chemokine receptor expression, Th1s and Th2s selectively migrate in response to the corresponding chemokines. The differential expression of chemokine receptors may dictate, to a large extent, the migration and tissue homing of Th1s and Th2s. It may also determine different susceptibility of Th1s and Th2s to human immunodeficiency virus strains using different fusion coreceptors.
|
10.1038/35100503
|
References 21 and 22 are the initial studies that identified the preferential expression of specific chemokine receptors on polarized T lymphocytes. They outline the basic principle of preferential expression on the basis of a specific cytokine environment.
|
10.1084/jem.179.3.881
| 101,582,940
|
Eosinophil accumulation is a prominent feature of allergic inflammatory reactions, such as those occurring in the lung of the allergic asthmatic, but the endogenous chemoattractants involved have not been identified. We have investigated this in an established model of allergic inflammation, using in vivo systems both to generate and assay relevant activity. Bronchoalveolar lavage (BAL) fluid was taken from sensitized guinea pigs at intervals after aerosol challenge with ovalbumin. BAL fluid was injected intradermally in unsensitized assay guinea pigs and the accumulation of intravenously injected 111In-eosinophils was measured. Activity was detected at 30 min after allergen challenge, peaking from 3 to 6 h and declining to low levels by 24 h. 3-h BAL fluid was purified using high performance liquid chromatography techniques in conjunction with the skin assay. Microsequencing revealed a novel protein from the C-C branch of the platelet factor 4 superfamily of chemotactic cytokines. The protein, "eotaxin," exhibits homology of 53% with human MCP-1, 44% with guinea pig MCP-1, 31% with human MIP-1 alpha, and 26% with human RANTES. Laser desorption time of flight mass analysis gave four different signals (8.15, 8.38, 8.81, and 9.03 kD), probably reflecting differential O-glycosylation. Eotaxin was highly potent, inducing substantial 111In-eosinophil accumulation at a 1-2 pmol dose in the skin, but did not induce significant 111In-neutrophil accumulation. Eotaxin was a potent stimulator of both guinea pig and human eosinophils in vitro. Human recombinant RANTES, MIP-1 alpha, and MCP-1 were all inactive in inducing 111In-eosinophil accumulation in guinea pig skin; however, evidence was obtained that eotaxin shares a binding site with RANTES on guinea pig eosinophils. This is the first description of a potent eosinophil chemoattractant cytokine generated in vivo and suggests the possibility that similar molecules may be important in the human asthmatic lung.
|
10.1038/35100503
|
A protein mediator that preferentially induced eosinophil accumulation in the airway was first discovered in guinea-pig airways, but the importance for human disease was quickly realized. Much of the early therapy in the chemokine field has been geared towards inhibiting the action of eotaxin with its specific receptor, CCR3.
|
10.1084/jem.185.4.785
| 28,073,833
|
The chemokines are a large group of chemotactic cytokines that regulate leukocyte trafficking and have recently been shown to inhibit human immunodeficiency virus entry into cells. Eotaxin is a C–C chemokine implicated in the recruitment of eosinophils in a variety of inflammatory disorders and, unlike all other eosinophil chemoattractants, is eosinophil specific. However, given the large number of chemoattractants that have activities on eosinophils, it is unclear whether eotaxin has an important role in vivo. Furthermore, it remains unclear why there is constitutive expression of eotaxin in healthy states in the absence of eosinophilic inflammation. To begin to determine the significance of eotaxin at baseline and during eosinophil-mediated disease processes, we have used targeted gene disruption to generate mice that are deficient in eotaxin. Such mice demonstrate that eotaxin enhances the magnitude of the early (but not late) eosinophil recruitment after antigen challenge in models of asthma and stromal keratitis. Surprisingly, a role for eotaxin in regulating the constitutive number of eosinophils in the peripheral circulation is also demonstrated. These results indicate a contributory role for eotaxin in the generation of peripheral blood and antigen-induced tissue eosinophilia.
|
10.1038/35100503
|
The finding that eotaxin-deficient mice showed virtually normal allergic airway responses allowed investigators to realize that targeting the receptor, CCR3, might be more efficacious than an individual chemokine ligand.
|
10.1084/jem.188.1.157
| 18,503,791
|
The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness (BHR) that characterize asthma is achieved by the regulated accumulation and activation of different leukocyte subsets in the lung. The development and maintenance of these processes correlate with the coordinated production of chemokines. Here, we have assessed the role that different chemokines play in lung allergic inflammation and BHR by blocking their activities in vivo. Our results show that blockage of each one of these chemokines reduces both lung leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES (regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1α, reduces only slightly lung eosinophilia and BHR. Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators. These results suggest that different chemokines activate different cellular and molecular pathways that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their individual blockage results in intervention at different levels of these processes.
|
10.1038/35100503
|
One of the earliest studies showing that chemokines were not just a family of cytokines with overlapping functions, but rather induced factors that participated in a response in a coordinated manner. Multiple chemokines have a significant role in different aspects of the allergen-induced responses in the lung.
|
10.4049/jimmunol.165.1.10
| 5,828,335
|
Abstract The in vivo function of Th cell subsets is largely dependent on the ability of differentiated CD4+ T cells to be recruited to specific sites and secrete restricted sets of cytokines. In this paper we demonstrate that Th1 and Th2 cells secrete discrete patterns of chemokines, small m.w. cytokines that function as chemoattractants in inflammatory reactions. Th2 cells secrete macrophage-derived chemokine and T cell activation gene 3, and acquisition of this pattern of expression is dependent on Stat6. In contrast, Th1 cells secrete lymphotactin and RANTES, though unlike IFN-γ, expression of these chemokines is independent of Stat4. We further show that supernatants from activated Th2 cells preferentially induce the chemotaxis of Th2 over Th1 cells, corresponding with Stat6-dependent expression of CCR4 and CCR8 in Th2 cells. These data provide the basis for restricted and direct T cell-mediated cellular recruitment to sites of inflammation.
|
10.1038/35100503
|
Identifies a primary role for T H 2 cytokine-induced STAT6 regulation of chemokines and their receptors on T lymphocytes, indicating that their expression might be dependent on gene regulation and not on differentiation state.
|
10.1084/jem.191.2.265
| 106,921,717
|
Isolated peripheral blood CD4 cells from allergic individuals express CC chemokine receptor (CCR)3 and CCR4 after expansion in vitro. In addition, human T helper type 2 (Th2) cells polarized in vitro selectively express CCR3 and CCR4 at certain stages of activation/differentiation and respond preferentially to the ligands eotaxin and monocyte-derived chemokine (MDC). However, controversy arises when the in vivo significance of this distinct expression is discussed. To address the functional role of CCR3/eotaxin and CCR4/MDC during the in vivo recruitment of Th2 cells, we have transferred effector Th cells into naive mice to induce allergic airway disease. Tracking of these cells after repeated antigen challenge has established that both CCR3/eotaxin and CCR4/MDC axes contribute to the recruitment of Th2 cells to the lung, demonstrating the in vivo relevance of the expression of these receptors on Th2 cells. We have shown that involvement of the CCR3/eotaxin pathway is confined to early stages of the response in vivo, whereas repeated antigen stimulation results in the predominant use of the CCR4/MDC pathway. We propose that effector Th2 cells respond to both CCR3/eotaxin and CCR4/MDC pathways initially, but that a progressive increase in CCR4-positive cells results in the predominance of the CCR4/MDC axis in the long-term recruitment of Th2 cells in vivo.
|
10.1038/35100503
|
The first study to show that different chemokine receptors are used at distinct phases of disease progression to promote T-lymphocyte accumulation. Complements the earlier studies by this group that show coordinated chemokine production.
|
10.4049/jimmunol.166.3.1457
| 60,537,418
|
Abstract Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-α stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-γ potentiated the TNF-α-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca2+ flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, 125I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.
|
10.1038/35100503
|
One of the first studies to show that the chemokine receptor CCR3 can be expressed on airway epithelial cells during asthmatic responses. Interestingly, these same cells produce an abundant amount of CCR3-specific ligands during asthmatic responses. The consequences and mechanisms associated with this finding will probably have a significant impact on airway biology and physiology during disease.
|
10.1084/jem.193.5.573
| 28,258,190
|
Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (−/−) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8−/− mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.
|
10.1038/35100503
|
The first study to indicate that chemokine receptors can be targeted for therapy to alter the T H 2-specific asthmatic responses. The preferential expression of CCR8 on T H 2 cells and reduced T H 2 cytokine phenotype within the lung of CCR8-deficient mice is a vital clue for therapeutic targeting of a specific receptor.
|
10.1083/jcb.135.6.1685
| 62,477,012
|
We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.
|
10.1038/35103000
|
The first description of an experimental system to study a chromatin region in living cells.
|
10.1083/jcb.147.5.929
| 79,454,512
|
Inositol-1,4,5-trisphosphate (InsP3)-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP3 itself is not generally available. In particular, it has not been determined how the interplay of InsP3 generation, diffusion, and degradation within complex cellular geometries can control the patterns of InsP3 signaling. Here, we explore the spatial and temporal characteristics of [InsP3]cyt during a bradykinin-induced calcium wave in a neuroblastoma cell. This is achieved by using a unique image-based computer modeling system, Virtual Cell, to integrate experimental data on the rates and spatial distributions of the key molecular components of the process. We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP3]cyt in the neurite. This requisite InsP3 spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane. In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.
|
10.1038/35103000
|
A clear and compelling examination of the importance of partial differential equation models when studying large cells or cells with long processes in which one must account for simultaneous diffusion and spatially distributed chemical reactions.
|
10.1083/jcb.143.6.1485
| 59,310,840
|
Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments. The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG– GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG–GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG–GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.
|
10.1038/35103000
|
This was among the first studies to combine the power of green fluorescent protein chimaeras, photobleaching techniques and kinetic analysis to answer questions about protein transport in living cells.
|
10.1126/science.277.5322.55
| 125,327,431
|
Angiogenesis is thought to depend on a precise balance of positive and negative regulation. Angiopoietin-1 (Ang1) is an angiogenic factor that signals through the endothelial cell–specific Tie2 receptor tyrosine kinase. Like vascular endothelial growth factor, Ang1 is essential for normal vascular development in the mouse. An Ang1 relative, termed angiopoietin-2 (Ang2), was identified by homology screening and shown to be a naturally occurring antagonist for Ang1 and Tie2. Transgenic overexpression of Ang2 disrupts blood vessel formation in the mouse embryo. In adult mice and humans, Ang2 is expressed only at sites of vascular remodeling. Natural antagonists for vertebrate receptor tyrosine kinases are atypical; thus, the discovery of a negative regulator acting on Tie2 emphasizes the need for exquisite regulation of this angiogenic receptor system.
|
10.1038/35067005
|
Shows that Ang2 is an antagonist of Tie2, providing the first evidence that related growth factors might have opposing actions towards receptor tyrosine kinases in vertebrates.
|
10.1101/gad.8.16.1897
| 122,856,231
|
The receptor tyrosine kinases (RTKs) expressed on the surface of endothelial cells are likely to play key roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. Expression of the Tek RTK during mouse development is restricted primarily to endothelial cells and their progenitors, the angioblasts, suggesting that Tek is a key participant in vasculogenesis. To investigate the role that Tek plays within the endothelial cell lineage, we have disrupted the Tek signaling pathway using two different genetic approaches. First, we constructed transgenic mice expressing a dominant-negative form of the Tek receptor. Second, we created a null allele of the tek gene by homologous recombination in embryonic stem (ES) cells. Transgenic mice expressing dominant-negative alleles of Tek or homozygous for a null allele of the tek locus both died in utero with similar defects in the integrity of their endothelium. By crossing transgenic mice that express the lacZ reporter gene under the transcriptional control of the endothelial cell-specific tek promoter, we found that the extraembryonic and embryonic vasculature was patterned correctly. However, homozygous tek embryos had approximately 30% and 75% fewer endothelial cells at day 8.5 and 9.0, respectively. Homozygous null embryos also displayed abnormalities in heart development, consistent with the conclusion that Tek is necessary for endocardial/myocardial interactions during development. On the basis of the analysis of mice carrying either dominant-negative or null mutations of the tek gene, these observations demonstrate that the Tek signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells in the mouse embryo.
|
10.1038/35067005
|
Description of the importance of Tie2 in regulating the survival and integrity of the endothelium during development.
|
10.1242/dev.126.20.4569
| 62,574,744
|
TEK (TIE2) and TIE (TIE1) are structurally related receptor tyrosine kinases expressed in endothelial cells and their precursors. Genetic studies in the mouse have revealed essential functions of both receptors in angiogenic expansion of the vasculature during development. As previously shown, mouse embryos homozygous for a disrupted Tek allele die by day 10.5 of embryogenesis due to endocardial defects, hemorrhaging, and impaired vascular network formation. Furthermore, TIE is required cell autonomously for endothelial cell survival and extension of the vascular network during late embryogenesis. Here we have investigated possible redundancy in the TEK and TIE signalling pathways during vascular development. Vasculogenesis proceeds normally in embryos lacking both TEK and TIE, although such embryos die early in gestation of multiple cardiovascular defects. Mosaic analysis revealed an absolute requirement for TEK in the endocardium at E10.5, whereas TEK and TIE are dispensable for the initial assembly of the rest of the vasculature. In contrast, both receptors are required in the microvasculature during late organogenesis and in essentially all blood vessels of the adult. This analysis demonstrates essential functions for TEK and TIE in maintaining the integrity of the mature vasculature.
|
10.1038/35067005
|
Shows that the functions mediated by the Tie1 and Tie2 receptors are not redundant through analysis of chimeric embryos. An interesting endocardial developmental phenotype is also revealed.
|
10.1126/science.286.5449.2511
| 83,130,753
|
Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) are endothelial cell–specific growth factors. Direct comparison of transgenic mice overexpressing these factors in the skin revealed that the VEGF-induced blood vessels were leaky, whereas those induced by Ang1 were nonleaky. Moreover, vessels in Ang1-overexpressing mice were resistant to leaks caused by inflammatory agents. Coexpression of Ang1 and VEGF had an additive effect on angiogenesis but resulted in leakage-resistant vessels typical of Ang1. Ang1 therefore may be useful for reducing microvascular leakage in diseases in which the leakage results from chronic inflammation or elevated VEGF and, in combination with VEGF, for promoting growth of nonleaky vessels.
|
10.1038/35067005
|
Shows that Ang1 can block vascular permeability induced by VEGF.
|
10.1126/science.284.5422.1994
| 81,976,899
|
In contrast with the prevailing view that most tumors and metastases begin as avascular masses, evidence is presented here that a subset of tumors instead initially grows by coopting existing host vessels. This coopted host vasculature does not immediately undergo angiogenesis to support the tumor but instead regresses, leading to a secondarily avascular tumor and massive tumor cell loss. Ultimately, however, the remaining tumor is rescued by robust angiogenesis at the tumor margin. The expression patterns of the angiogenic antagonist angiopoietin-2 and of pro-angiogenic vascular endothelial growth factor (VEGF) suggest that these proteins may be critical regulators of this balance between vascular regression and growth.
|
10.1038/35067005
|
Shows that Ang2 is associated with tumour angiogenesis in a model where the tumour co-opts existing host vessels.
|
10.1073/pnas.220418097
| 79,468,883
|
Epifluorescence microscopy studies of mixtures of phospholipids and cholesterol at the air–water interface often exhibit coexisting liquid phases. The properties of these liquids point to the formation of “condensed complexes” between cholesterol and certain phospholipids, such as sphingomyelin. It is found that monolayers that form complexes can incorporate a low concentration of a ganglioside G M1 . This glycolipid is visualized by using a fluorescently labeled B subunit of cholera toxin. Three coexisting liquid phases are found by using this probe together with a fluorescent phospholipid probe. The three liquid phases are identified as a phospholipid-rich phase, a cholesterol-rich phase, and a condensed complex-rich phase. The cholera toxin B labeled ganglioside G M1 is found exclusively in the condensed complex-rich phase. Condensed complexes are likely present in animal cell membranes, where they should facilitate the formation of specialized domains such as rafts. Condensed complexes also have a major effect in determining the chemical activity of cholesterol. It is suggested that this chemical activity plays an essential role in the regulation of cholesterol biosynthesis. Gradients in the chemical activity of cholesterol should likewise govern the rates and direction of intracellular intermembrane cholesterol transport.
|
10.1038/35080071
|
This paper, and several other papers from the same group in 1999/2000, bridge a gap between physical chemistry and cell biology by showing liquid–liquid phase separations in lipid mixtures of biological relevance. McConnell first demonstrated such phase separations 20 years ago.
|
10.1177/42.2.8288861
| 123,762,744
|
The ultrastructural distribution of the ganglioside GM1 was investigated in A431 cells. After fixation, the cells were frozen in liquid nitrogen, freeze-substituted, and then embedded in Lowicryl resin at -45 degrees C. By use of the cholera toxin-binding subunit adsorbed to gold as a specific probe to label on the sections, GM1 was shown to be present in endocytic organelles, in the trans-Golgi network, and on the plasma membrane, but was not detectable in the endoplasmic reticulum. GM1 was not distributed uniformly over the plasma membrane but was concentrated approximately four-fold in non-coated invaginations. These were identified as caveolae by labeling frozen sections of cholera toxin-gold surface-labeled cells with antibodies to VIP-21/caveolin. The results strengthen the functional analogy between caveolae and sorting domains of the TGN in polarized epithelial cells.
|
10.1038/35080071
|
This paper provides solid morphological evidence for the concentration of a glycolipid in caveolae.
|
10.1126/science.283.5407.1530
| 38,424,185
|
Long interspersed nuclear elements (LINE-1s or L1s) are the most abundant retrotransposons in the human genome, and they serve as major sources of reverse transcriptase activity. Engineered L1s retrotranspose at high frequency in cultured human cells. Here it is shown that L1s insert into transcribed genes and retrotranspose sequences derived from their 3′ flanks to new genomic locations. Thus, retrotransposition-competent L1s provide a vehicle to mobilize non-L1 sequences, such as exons or promoters, into existing genes and may represent a general mechanism for the evolution of new genes.
|
10.1038/35038572
|
Use of a cultured-cell assay to show that L1s insert into transcribed genes and can retrotranspose sequences derived from their 3′ flanks to new genomic locations.
|
10.1126/science.280.5361.301
| 65,233,555
|
Assembly of immunoglobulin and T cell receptor genes from separate gene segments [V(D)J recombination] begins with DNA double-strand breakage by the RAG1 and RAG2 proteins, acting at a pair of recombination signal sequences (RSSs). Here, the RAG proteins are shown to reverse the cleavage reaction by joining an RSS to a broken coding sequence end. These “hybrid joints” have also been found in lymphoid cells, even when the normal pathway of DNA double-strand break repair is inactive, and can now be explained by this activity of the RAG proteins.
|
10.1038/35038572
|
Reference 82 and 83 show that the RAG1 and RAG2 proteins, together, form a DNA transposase which can excise a piece of DNA flanked by recombination signal sequences and move it to another DNA molecule.
|
10.1126/science.1064252
| 4,579,897
|
A classic model proposes that the mammalian neocortex is divided into areas early in neurogenesis, but the molecular mechanisms that generate the area map have been elusive. Here we provide evidence that FGF8 regulates development of the map from a source in the anterior telencephalon. Using electroporation-mediated gene transfer in mouse embryos, we show that augmenting the endogenous anterior FGF8 signal shifts area boundaries posteriorly, reducing the signal shifts them anteriorly, and introducing a posterior source of FGF8 elicits partial area duplications, revealed by ectopic somatosensory barrel fields. These findings support a role for FGF signaling in specifying positional identity in the neocortex.
|
10.1038/35104078
|
This study shows that the anteroposterior positioning of areal boundaries in mammalian neocortex can be regulated by a diffusible signalling molecule, FGF8, secreted from a 'signal centre' in the anterior telencephalon.
|
10.1126/science.287.5454.864
| 62,191,077
|
Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.
|
10.1038/35104078
|
By deleting a molecule required for synaptic vesicle fusion, this work shows that activity is not essential for many general aspects of neural development, including the formation of morphological synapses, but is required for the maintenance of connectivity.
|
10.1126/science.2497519
| 81,369,235
|
A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development.
|
10.1038/35093548
|
The first example of enhancer-trap and gene-trap mutagenesis in mouse ES cells, and of ES-cell-derived mice. It was published the same year as the first targeted mutagenesis of non-selectable genes.
|
10.1101/gad.5.9.1513
| 38,450,621
|
A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.
|
10.1038/35093548
|
The development of the ROSA β-geo vectors and the first application of gene trapping to isolate lethal mutations.
|
10.1101/gad.6.6.919
| 60,544,332
|
Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.
|
10.1038/35093548
|
This paper provided the foundation for the current promoter-trapping efforts.
|
10.1073/pnas.92.14.6592
| 18,799,109
|
A strategy based on the gene trap was developed to prescreen mouse embryonic stem cells for insertional mutations in genes encoding secreted and membrane-spanning proteins. The "secretory trap" relies on capturing the N-terminal signal sequence of an endogenous gene to generate an active beta-galactosidase fusion protein. Insertions were found in a cadherin gene, an unc6-related laminin (netrin) gene, the sek receptor tyrosine kinase gene, and genes encoding two receptor-linked protein-tyrosine phosphatases, LAR and PTP kappa. Analysis of homozygous mice carrying insertions in LAR and PTP kappa showed that both genes were effectively disrupted, but neither was essential for normal embryonic development.
|
10.1038/35093548
|
An elegant method to trap a specific class of gene. This project has evolved into one of the gene-trap resource centres.
|
10.1093/genetics/139.2.889
| 83,486,014
|
Abstract We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.
|
10.1038/35093548
|
This work served as proof of principle for high-throughput gene-trap screens.
|
10.1073/pnas.93.26.15279
| 65,352,696
|
A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/ loxP ) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus–thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.
|
10.1038/35093548
|
This paper shows a use of gene trapping other than for mutagenesis.
|
10.1126/science.2274784
| 38,518,077
|
Metaphase chromosomes are dynamically modified in interphase. This review focuses on how these structures can be modified, and explores the functional mechanisms and significance of these changes. Current analyses of genes often focus on relatively short stretches of DNA and consider chromatin conformations that incorporate only a few kilobases of DNA. In interphase nuclei, however, orderly transcription and replication can involve highly folded chromosomal domains containing hundreds of kilobases of DNA. Specific "junk" DNA sequences within selected chromosome domains may participate in more complex levels of chromosome folding, and may index different genetic compartments for orderly transcription and replication. Three-dimensional chromosome positions within the nucleus may also contribute to phenotypic expression. Entire chromosomes are maintained as discrete, reasonably compact entities in the nucleus, and heterochromatic coiled domains of several thousand kilobases can acquire unique three-dimensional positions in differentiated cell types. Some aspects of neoplasia may relate to alterations in chromosome structure at several higher levels of organization.
|
10.1038/35066075
|
An excellent attempt to integrate a multitude of experimental findings in the framework of a functional higher-order chromatin architecture. This paper is still a 'must read'.
|
10.1177/21.8.715
| 98,795,174
|
In many immunohistochemical studies horseradish peroxidase (HRP) is used as the marker enzyme. An in situ and indirect quantitation of antigen or antibody would be possible by determination of the HRP activity. For this reason quantitative aspects of histochemical reaction procedures to localize HRP activity have been studied in a model system consisting of polyacrylamide films into which the enzyme had been incorporated. A linear relation between the amount of HRP activity and the spectrophotometrically determined amount of reaction product could be established for reactions using 3,3'-amino-9-ethylcarbazole as the hydrogen donor. A similar relationship could not be found after staining with benzidine-containing media. These results indicate that for certain substrates the quantities of dyes formed during the cytochemical HRP reaction can be considered as a measure for the activity of the enzyme. This opens perspectives for the in situ quantitation of antigens or antibodies.
|
10.1038/35066075
|
A thorough historical account of in situ hybridization from its beginnings to present-day technologies.
|
10.1242/jcs.113.9.1565
| 104,368,901
|
The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.
|
10.1038/35066075
|
Activation of specific genes from the major histocompatibility locus correlates with an expansion of the gene-harbouring chromatin loops from the surface of chromosome-6 territories.
|
10.1083/jcb.144.5.813
| 59,313,388
|
Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome.
|
10.1038/35066075
|
This in vivo study relates the gross structure of a chromosome territory to that of a prophase chromosome.
|
10.1083/jcb.146.6.1211
| 20,690,944
|
We investigated the nuclear higher order compartmentalization of chromatin according to its replication timing (Ferreira et al. 1997) and the relations of this compartmentalization to chromosome structure and the spatial organization of transcription. Our aim was to provide a comprehensive and integrated view on the relations between chromosome structure and functional nuclear architecture. Using different mammalian cell types, we show that distinct higher order compartments whose DNA displays a specific replication timing are stably maintained during all interphase stages. The organizational principle is clonally inherited. We directly demonstrate the presence of polar chromosome territories that align to build up higher order compartments, as previously suggested (Ferreira et al. 1997). Polar chromosome territories display a specific orientation of early and late replicating subregions that correspond to R- or G/C-bands of mitotic chromosomes. Higher order compartments containing G/C-bands replicating during the second half of the S phase display no transcriptional activity detectable by BrUTP pulse labeling and show no evidence of transcriptional competence. Transcriptionally competent and active chromatin is confined to a coherent compartment within the nuclear interior that comprises early replicating R-band sequences. As a whole, the data provide an integrated view on chromosome structure, nuclear higher order compartmentalization, and their relation to the spatial organization of functional nuclear processes.
|
10.1038/35066075
|
Describes evidence that chromosome territories contribute to gene-rich and gene-poor higher-order chromatin compartments.
|
10.1126/science.291.5505.843
| 41,155,778
|
Studies of nuclear architecture reveal that the dynamic properties of proteins in the nucleus are critical for their function. The high mobility of proteins ensures their availability throughout the nucleus; their dynamic interplay generates an ever-changing, but overall stable, architectural framework, within which nuclear processes take place. As a consequence, overall nuclear morphology is determined by the functional interactions of nuclear components. The observed dynamic properties of nuclear proteins are consistent with a central role for stochastic mechanisms in gene expression and nuclear architecture.
|
10.1038/35066075
|
A summary of the evidence that movement of proteins in the nucleus to their site of action occurs by simple diffusion.
|
10.1091/mbc.10.1.211
| 103,496,831
|
In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.
|
10.1038/35066075
|
Electron-microscopic evidence that nascent RNA is synthesized at chromatin-domain surfaces.
|
10.1073/pnas.92.7.2710
| 61,890,064
|
Fluorescence in situ hybridization data on distances between defined genomic sequences are used to construct a quantitative model for the overall geometric structure of a human chromosome. We suggest that the large-scale geometry during the G0/G1 part of the cell cycle may consist of flexible chromatin loops, averaging approximately 3 million bp, with a random-walk backbone. A fully explicit, three-parametric polymer model of this random-walk/giant-loop structure can account well for the data. More general models consistent with the data are briefly discussed.
|
10.1038/35066075
|
First quantitative modelling of gene distances in distinct chromosome territories.
|
10.1073/pnas.95.11.6043
| 18,898,851
|
Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10 −7 cm 2 /s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10 −7 cm 2 /s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10 −7 cm 2 /s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.
|
10.1038/35066075
|
Using in vivo fluorescence correlation spectroscopy, the authors show that nuclear poly(A) RNA moves by a diffusion-like process.
|
10.1126/science.292.5522.1728
| 20,371,838
|
Glucose homeostasis depends on insulin responsiveness in target tissues, most importantly, muscle and liver. The critical initial steps in insulin action include phosphorylation of scaffolding proteins and activation of phosphatidylinositol 3-kinase. These early events lead to activation of the serine-threonine protein kinase Akt, also known as protein kinase B. We show that mice deficient in Akt2 are impaired in the ability of insulin to lower blood glucose because of defects in the action of the hormone on liver and skeletal muscle. These data establish Akt2 as an essential gene in the maintenance of normal glucose homeostasis.
|
10.1038/35096067
|
First PKB/Akt knockout paper, demonstrating the unexpected finding of a specific role for PKBβ/Akt2 in glucose homeostasis.
|
10.1101/gad.12.16.2488
| 38,401,288
|
A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. We show here that two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest and that AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation.
|
10.1038/35096067
|
The first genetically identified PKB/AKT target, DAF-16, is a forkhead-like transcription factor ? a prelude to the identification of mammalian homologues of PKB/AKT substrates.
|
10.1126/science.283.5402.689
| 19,282,235
|
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease associated with multiple deletions of skeletal muscle mitochondrial DNA (mtDNA), which have been ascribed to a defect in communication between the nuclear and mitochondrial genomes. Examination of 12 MNGIE probands revealed homozygous or compound-heterozygous mutations in the gene specifying thymidine phosphorylase (TP), located on chromosome 22q13.32-qter. TP activity in leukocytes from MNGIE patients was less than 5 percent of controls, indicating that loss-of-function mutations in TP cause the disease. The pathogenic mechanism may be related to aberrant thymidine metabolism, leading to impaired replication or maintenance of mtDNA, or both.
|
10.1038/35072063
|
The first description of a mutation in a gene involved in nuclear–mitochondrial DNA communication.
|
10.1159/000134066
| 102,703,436
|
Humamrodent somatic cell hybrids carrying a single, intact, selectable human chromosome are valuable both for functional somatic cell genetic analysis and genome mapping procedures. Here, we describe the construction and detailed molecular cytogenetic characterization of a panel of 23 stable hybrids, representing all 22 human autosomes plus the X-chromosome. Individual normal human chromosomes have been tagged with a selectable fusion gene (<i>Hytk</i>) introduced into the chromosome in a small (4.2 kbp) retro viral vector. Use of the <i>Hytk </i>marker permits both positive and negative (“in-out”) selection to be applied to the human chromosome in any mammalian cell background. The panel includes 18 new hybrids isolated by direct microcell transfer from normal human diploid fibroblasts into mouse A9 cells.
|
10.1038/35072063
|
Important new technology in the characterization of the chromosome localization responsible for disease.
|
10.1126/science.280.5369.1564
| 20,505,608
|
The mouse Clock gene encodes a bHLH-PAS protein that regulates circadian rhythms and is related to transcription factors that act as heterodimers. Potential partners of CLOCK were isolated in a two-hybrid screen, and one, BMAL1, was coexpressed with CLOCK and PER1 at known circadian clock sites in brain and retina. CLOCK-BMAL1 heterodimers activated transcription from E-box elements, a type of transcription factor–binding site, found adjacent to the mouse per1 gene and from an identical E-box known to be important for per gene expression in Drosophila. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 formed heterodimers that bound DNA but failed to activate transcription. Thus, CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity.
|
10.1038/35036078
|
A key paper about a basic device, the feedback loop: the positive elements of the loop were finally uncovered.
|
10.1126/science.284.5413.502
| 123,022,837
|
Circadian rhythms of mammals are entrained by light to follow the daily solar cycle (photoentrainment). To determine whether retinal rods and cones are required for this response, the effects of light on the regulation of circadian wheel-running behavior were examined in mice lacking these photoreceptors. Mice without cones ( cl ) or without both rods and cones ( rdta/cl ) showed unattenuated phase-shifting responses to light. Removal of the eyes abolishes this behavior. Thus, neither rods nor cones are required for photoentrainment, and the murine eye contains additional photoreceptors that regulate the circadian clock.
|
10.1038/35036078
|
Along with reference 76, this represented a big leap forward in the search for the mammalian circadian photoreceptor: it is neither the cones nor the rods, and so lies elsewhere in the retina.
|
10.1126/science.278.5343.1632
| 104,123,661
|
Transgenic Drosophila that expressed either luciferase or green fluorescent protein driven from the promoter of the clock gene period were used to monitor the circadian clock in explanted head, thorax, and abdominal tissues. The tissues (including sensory bristles in the leg and wing) showed rhythmic bioluminescence, and the rhythms could be reset by light. The photoreceptive properties of the explanted tissues indicate that unidentified photoreceptors are likely to contribute to photic signal transduction to the clock. These results show that autonomous circadian oscillators are present throughout the body, and they suggest that individual cells in Drosophila are capable of supporting their own independent clocks.
|
10.1038/35036078
|
This paper presents the first description of independent non-neural peripheral clocks in animals.
|
10.1084/jem.187.12.2081
| 100,943,218
|
Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70−/− (K70T) mice, like recombination activating gene (RAG)-1– or RAG-2–deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653–665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921–929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig λ light chain transgene (referred to as K70T/HL mice). Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J. Manis, E. Sonoda, K. Rajewsky, and F. Alt. 1998. Int. Immunol. 10:325–332). K70T/HL mice had significant numbers of peripheral surface IgM+ B cells, which generated serum IgM levels similar to those of R2T/HL mice. However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes. In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells. However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h. We conclude that Ku70 is required for the generation of B cells that have undergone Ig HC class switching. Potential roles for Ku70 in the CSR process are discussed.
|
10.1038/35080033
|
References 22–24 showed that class-switch recombination depends on non-homologous end joining.
|
10.1084/jem.192.10.1509
| 109,159,494
|
Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs)/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PKcs activity.
|
10.1038/35080033
|
Unlike class-switch recombination, somatic hypermutation does not require the catalytic subunit of the DNA-dependent protein kinase (DNA-PK cs ), indicating that different mechanisms of DNA repair might occur between class-switch recombination and somatic hypermutation.
|
10.1126/science.1059852
| 59,706,053
|
Listeria monocytogenes is responsible for severe food-borne infections, but the mechanisms by which bacteria cross the intestinal barrier are unknown. Listeria monocytogenes expresses a surface protein, internalin, that interacts with a host receptor, E-cadherin, to promote entry into human epithelial cells. Murine E-cadherin, in contrast to guinea pig E-cadherin, does not interact with internalin, excluding the mouse as a model for addressing internalin function in vivo. In guinea pigs and transgenic mice expressing human E-cadherin, internalin was found to mediate invasion of enterocytes and crossing of the intestinal barrier. These results illustrate how relevant animal models for human infections can be generated.
|
10.1038/35085062
|
This paper is the culmination of many in vitro studies on Listeria interaction with host cells. It shows that the internalin–E-cadherin interaction is crucial for the establishment of infection in vivo
|
10.1128/mmbr.62.2.379-433.1998
| 83,397,687
|
SUMMARY Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.
|
10.1038/35085062
|
An excellent overview of type III secretion systems and their secreted proteins.
|
10.1083/jcb.119.2.301
| 103,823,462
|
For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.
|
10.1038/35056522
|
This is the first report that yeast induces autophagy that is quite similar to that in mammals under various starvation conditions.
|
10.1083/jcb.151.2.263
| 80,133,073
|
Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule “X” and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.
|
10.1038/35056522
|
This showed that Apg8 associates with the intermediate membranes of the autophagosome by serial modification reactions.
|
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