Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the paper will be accessible via the Research Library.
The series of saltatory steps that occur as neurons migrate along glial fibers to form the neuronal laminae, involve the release of adhesion followed by a forward movement of the soma. Solecki et al. have now uncovered a possible mechanism underlying this unusual type of neuronal movement.
The authors introduced fluorescently-tagged -tubulin into cerebellar granule cells in order to visualize the microtubule dynamics of neurons migrating along astroglial fibres. Live cell imaging revealed a cage-like arrangement of microtubules around the nucleus, with microtubules also extending into the leading process. As the neurons migrated along the glia, the microtubules underwent dynamic shape changes, and the well-timed peri-nuclear tubulin cage and nuclear movements resembled that of previously observed saltatory motions.
The authors examined the roles of mPar6 — a known regulator of cell polarity and motility — and p50 dynactin — a component of the dynein-dynactin complex involved in centrosomal positioning — in glial-guided neuronal migration. Fluorescently-tagged mPar6 and p50 dynactin both localized to the centrosome, which was located in front of the nucleus in the direction of the motility. Centrosome relocalization was observed to precede the movement of the nucleus.
Solecki et al. used a combination of gain- and loss-of-function studies to demonstrate an important role for mPar6 in regulating centrosomal organization and positioning during neuronal migration. In addition, when the authors disrupted mPar6 function in cerebellar slice cultures, granule cells had shorter neurites and failed to migrate along Bergmann glial fibres. Overexpression of p50-dynactin also caused a similar but less severe phenotype.
These elegant imaging studies by Solecki et al. provide an insight into the mechanisms underlying glial-guided neuronal migration. The unique two-stroke motion, whereby the centrosome moves before the nucleus and perinuclear microtubule cage, may explain the saltatory migration exhibited by glial-guided neurons both in culture and in situ. Central to this process is mPar6 signaling, which appears to directly regulate nucleokinesis.
This pathway appears to be specific to neurons and has not yet been observed in fibroblast or astrocyte migration, where centrosome and nuclear relocalization is thought to be caused by mPar6-signaling and dynein-mediated pulling of microtubules at the leading edge. It remains to be seen whether Lis1 (a frequently mutated gene in lissencephaly) and its binding partner Nudel, which have been recently implicated in centrosome positioning and in binding dynein in vitro, also have roles in the regulation of glial-guided neuronal migration.
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article David J Solecki, Lynn Model, Jedidiah Gaetz, Tarun M Kapoor & Mary E Hatten Par6 signaling controls glial-guided neuronal migration Nature Neuroscience, 7, 1195 – 1203
(2004); doi:10.1038/nn1332 Full Text (HTML) | PDF | Subscribe to Nature Neuroscience
The transcription factor p53, which is frequently mutated in human cancers, regulates apoptosis, cell cycle progression and DNA repair in response to cellular stress. Its activity is tightly controlled, mainly through different post-translational modifications. Chuikov et al. now report a novel mechanism of p53 regulation through methylation.
Chuikov et al. found that the methyltransferase Set9, which targets histone H3 in vitro, also methylates p53 at a specific lysine residue, K372 (p53-K372). By using specific anti-methylated p53-K372 antibodies the authors demonstrated that this methylation also occurs in vivo, and that levels of methylated p53-K372 protein increase in a Set9-dependent manner in response to DNA damage.
To investigate the function of p53-K372 methylation, the authors examined the transcriptional activity of the p53 target gene p21/WF/CIP. Set9 overexpression resulted in increased expression of p21, correlating with increased levels of methylated p53-K372. This increase required the catalytic activity of Set9 and was more pronounced in response to DNA damage, which caused higher amounts of methylated p53-K372 to bind to the p21 promoter. Stimulation of p53-target gene expression by Set9 was not restricted to p21, but also extended to BAX and MDM2, suggesting that this is a general phenomenon.
Further investigation into the mechanism of Set9-dependent regulation of p53 levels revealed that methylation of p53 results in the stabilization of a chromatin-bound p53 fraction. Hence, the authors hypothesized that ‘hyper-stabilization’ of p53 through the action of Set9, would induce increased activation of p53, cell cycle arrest and apoptosis. Indeed, cells overexpressing Set9 were more apoptotic compared to wild type cells, even in the absence of DNA damage. The observed increase in apoptosis was further exacerbated by treating the Set9-expressing cells with DNA damaging agents.
Thus, Set9-induced methylation stabilizes p53 to enhance expression of target genes important for G1 arrest and apoptosis, such as p21 and BAX, respectively. Methylation appears to have found its place within the plethora of p53 modifications, which include phosphorylation and ubiquitination, and may even play a role in human cancers. Future research will also unveil whether methylation of transcription factors or other cellular targets, in addition to histones, is a universal function of lysine methyltransferases.
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article Sergei Chuikov, Julia K. Kurash, Jonathan R. Wilson, Bing Xiao, Neil Justin, Gleb S. Ivanov, Kristine McKinney, Paul Tempst, Carol Prives, Steven J. Gamblin, Nickolai A. Barlev & Danny Reinberg Regulation of p53 activity through lysine methylation Nature, 432, 353 – 360
(18 November 2004); doi:10.1038/nature03117 Full Text (HTML) | PDF | Subscribe to Nature
Shaping blood vessels: A Netrin – UNC5B affair
During development, blood vessels are guided to their targets in a way similar to that of axonal growth cones — by responding to attractive and repulsive cues. Lu et al. now report that the repulsive netrin receptor UNC5B is expressed on endothelial tip cells to control morphogenesis of the vascular system.
Netrins act through the DCC (deleted in colorectal cancer) and UNC-5 receptor families to either attract or repel axons from the midline. The UNC5B homolog can convert netrin attraction to repulsion in Xenopus, but its in vivo function in mammals is poorly understood. Lu et al. observed that, throughout mouse development, Unc5b expression, unlike Unc5a and Dcc, is largely restricted to blood vessels and endothelial tip cells. In addition, the loss of both Unc5b alleles results in an increase in vessel branching and the number of filopodia on endothelial tip cells.
To test whether UNC5B has a role in blood vessel guidance, the authors disrupted Unc5b function during intersegmental blood vessel (ISV) development in zebrafish. Knock-down of either Unc5b or netrin1a resulted in defective ISV path-finding, together with supernumerary and ectopic filopodial extensions in ISV endothelial cells. Aberrant proliferation, apoptosis and cell specification were ruled out as possible causes, suggesting that the observed vessel phenotype was most likely due to defective endothelial branching and navigation.
Through a series of elegant in vitro and in vivo experiments, the authors showed that netrin-1 acts through the UNC5B receptor to induce filopodial retraction of endothelial tip cells, ultimately resulting in the inhibition of cell migration. Injection of recombinant netrin-1 protein into the eyes or hindbrains of mouse embryos resulted in a marked decrease in the number of filopodial extensions of tip cells compared to that of control-injected embryos. This effect was rescued by pre-incubation of netrin-1 protein with recombinant UNC5B. Importantly, injection of netrin-1 into Unc5B mutant hindbrains did not affect the number of filopodial extensions, filopodial-extending tip cells or altered capillary morphogenesis. Thus, netrin-1's effect on endothelial filopodia requires UNC5B signaling.
The results by Lu et al. provide an intriguing mechanism for the control of branching during vessel patterning, which is reminiscent of axonal targeting in the nervous system. UNC5B is clearly important in guiding endothelial growth cones during development. However, whether netrin-1 acts as its endogenous ligand, or whether UNC5B has an equally important role in the adult remains to be seen.
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article
Xiaowei Lu, Ferdinand Le Noble, Li Yuan, Quingjan Jiang, Benjamin De Lafarge, Daisuke Sugiyama, Christiane Bréant, Filip Claes, Frederik De Smet, Jean-Léon Thomas, Monica Autiero, Peter Carmeliet, Marc Tessier-Lavigne and Anne Eichmann The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system Nature, 432, 179 – 186 (11 November 2004); doi:10.1038/nature03080
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Calcium signaling: NO stress for ER
The signaling molecule nitric oxide (NO) competes with oxygen to reversibly bind to cytochrome c oxidase in the electron transport chain. It has a physiological role in the control of cell respiration, but can also interfere with normal mitochondrial function. Xu et al. now demonstrate that NO can induce the endoplasmic reticulum (ER) stress response by changing the mitochondrial calcium flux.
The authors studied two human cell lines that express inducible NO synthase (iNOS). Differential hybridization showed that increases in NO result in enhanced transcription from the chaperone glucose-regulated protein 78 (Grp78) gene promoter. Induced Grp78 expression is mediated by stress response elements in the promoter, which are known to be responsive to the membrane-associated ER stress-responsive transcription factor p50 ATF6. Indeed, the increase in Grp78 protein went hand in hand with increases in the cleaved, active form of ATF6, which is regulated by intracellular calcium levels.
To further investigate the functional significance of these observations, Xu et al. tested the ability of NO to protect cells against cytotoxicity. They showed that the NO-mediated increase in Grp78 conferred marked cytoprotection against a selective ER calcium-ATPase inhibitor, and that this cytoprotection was abolished when cells were treated with a calcium chelator or cyclosporin A, which disrupts mitochondrial calcium flux.
ATF6 cleavage is regulated by intramembrane proteolysis (RIP), which involves the site-1 (S1P) and site-2 (S2P) proteases. Xu et al. used small inhibitory RNA interference to silence the S1P or S2P promoters, demonstrating that absence of S1P and S2P abolishes both NO-mediated cytoprotection and increased Grp78. They concluded that both S1P and S2P are intermediates in the NO-dependent cleavage of ATF6 and in the increase of Grp78 expression.
Finally, the authors demonstrated that NO-induced Grp78 expression is diminished in cells devoid of mitochondrial DNA (rho0), consistent with their findings that NO-mediated mitochondrial disruption is coupled to the activation of the ER stress response.
Thus, NO-mediated disruption of the mitochondrial respiratory chain appears to lead to changes in calcium flux, which activates the calcium sensitive S1P and S2P proteases in the Golgi that cleave and activate ATF6. The cleaved ATF6 component then translocates to the nucleus where it induces expression of ER stress-responsive genes such as Grp78. It will be interesting to see whether future research will define additional interactions of NO with components of the stress response pathway.
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article
Weiming Xu, Lizhi Liu, Ian G. Charles and Salvador Moncada Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response Nature Cell Biology, 6, 1129 – 1134 (2004); doi:10.1038/ncb1188
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-tubulin into cerebellar granule cells in order to visualize the microtubule dynamics of neurons migrating along astroglial fibres. Live cell imaging revealed a cage-like arrangement of microtubules around the nucleus, with microtubules also extending into the leading process. As the neurons migrated along the glia, the microtubules underwent dynamic shape changes, and the well-timed peri-nuclear tubulin cage and nuclear movements resembled that of previously observed saltatory motions.
