Polar Exploration in Establishing This Polarity Are Poorly for the Asymmetric Localization of Understood
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RESEARCH HIGHLIGHTS CELL POLARITY Many bacteria show polar character- C. crescentus cells. By analogy to istics, such as the positioning of a eukaryotic actin, MreB molecules flagellum at one end of the cell. might have an intrinsic polarity, so However, the mechanisms involved the spirals they form could be used Polar exploration in establishing this polarity are poorly for the asymmetric localization of understood. A recent study from molecules required for the develop- Lucy Shapiro and colleagues has ment of polar structures. To test this, made important progress in under- the authors analysed the effect of standing these processes by identify- MreB depletion on the distribution ing a master regulator of polarity in of four signalling proteins — PleC, Caulobacter crescentus. DivJ, CckA and DivK — that are The C. crescentus life cycle involves required for polar development in two different cell types, both with C. crescentus.Depletion of MreB specialized structures located at one abolished the polar foci that are usu- pole of the cell. Several proteins that ally formed by all four proteins at cer- are involved in the development of tain points in the cell cycle, consistent these structures and that have corre- with a role for MreB as a global regu- sponding polar distributions have lator of polarity. been identified, providing useful Importantly, MreB seems to be markers of polarity. Shapiro and col- actively required for specifying polar- leagues made use of these features to ity, rather than having a passive role investigate whether the actin-like in protein localization. Unlike CckA protein MreB is required for polar- and DivK, which form foci at both ization in C. crescentus. poles of C. crescentus cells, PleC and MreB has a distinctive localiza- DivJ are asymmetrically distributed tion pattern, forming a spiral struc- at certain points in the cell cycle, ture that extends along the length of localizing to only one pole. When CYTOKINESIS homologues. The functional relevance of the include proteins that are involved in lipid- candidate midbody proteins became raft formation and vesicle trafficking. They apparent when the authors analysed gene suggest that raft-associated factors could function by RNA interference using double- target and activate specific membrane events A good place stranded (ds)RNA that corresponded to each in cytokinesis. of the C. elegans genes. Most of these Among the midbody proteins, 24% were to start dsRNAs produced a disrupted cytokinesis Golgi-associated proteins, which led the phenotype. authors to speculate that there might be some A new report in Science describes the rapid By analysing the various mutant parallels between cytokinesis in animals and identification and characterization of phenotypes, the authors found that a plants, as Golgi-derived vesicles are involved proteins that function in cytokinesis, using significant percentage of midbody proteins in cell-wall formation after cell division in an approach that combines functional- that function in cytokinesis also produce plants. proteomic and comparative-genomic defects in germline development when they Finally, the functional analysis of analysis. are disrupted. They went on to show that midbody proteins revealed some From synchronized Chinese hamster ovary gonad development and sterility mutant unexpected phenotypes — 20% of the (CHO) cells, Ahna Skop and colleagues phenotypes are, in fact, frequently caused by identified proteins caused defects in isolated midbodies — microtubule-rich, defects in cytokinesis in the germline or in coordination, which implicates these transient structures that are derived from the early embryos. In addition, 16 proteins that proteins in muscle or neuronal spindle midzone and that exist after cell are essential for embryo and germline development. In addition, 14% of the division, just before the daughter cells detach. cytokinesis were also required for polar-body mammalian proteins that were identified The proteins in the midbody preparation extrusion (the polarized, asymmetric cell are known to have a role in human diseases, were identified by a protein identification division that occurs during meiosis). So, mostly those that are associated with technology — MudPIT — that is used for the cytokinesis, gonad organization and polar- membrane and cytoskeletal pathologies. So analysis of proteins in complex protein body extrusion seem to use a common set of this study provides plenty of starting points mixtures. proteins. for further investigations — including into After eliminating proteins that have Although membrane and cytoskeletal cytokinesis, development or disease general housekeeping functions, 160 proteins are known to be involved in mechanisms. candidate midbody proteins remained, of cleavage-furrow formation, among other Arianne Heinrichs which 57 were known cytokinesis proteins. cytokinesis processes, how these proteins are References and links Most candidate midbody proteins seemed to recruited to the cleavage plane has been ORIGINAL RESEARCH PAPER Skop, A. R. et al. Dissection of the mammalian midbody proteome reveals be conserved in evolution as 147 out of 160 unclear. Skop and co-workers identified 40 conserved cytokinesis mechanisms. Science 27 May 2004 proteins had clear Caenorhabditis elegans membrane and cytoskeletal proteins, which (doi:10.1126/science.1097931) 512 | JULY 2004 | VOLUME 5 www.nature.com/reviews/molcellbio RESEARCH HIGHLIGHTS MreB was depleted and then re- expressed, although polar foci of PleC and DivJ were restored, these were located at the wrong pole in ~50% of cells. This indicates that when MreB is depleted, cells lose all memory of their initial polarity, so that when MreB is re-expressed polarity becomes randomized. MreB must therefore be required for the initial decision that determines which pole of the cell is which. This study shows intriguing simi- larities between the establishment of polarity in C. crescentus and the corre- sponding processes in eukaryotic cells, in which actin has a central role. It will be interesting to see whether similar CYTOSKELETON mechanisms operate in other bacteria. Louisa Flintoft Assistant Editor, Nature Reviews References and links A new pathway to explore ORIGINAL RESEARCH PAPER Gitai, Z., Dye, N. & Shapiro, L. An actin-like gene can determine cell polarity in bacteria. Proc. Natl Acad. Sci. USA 101, When placed in a new environment, a cell will extend increase filopodia formation in Abl- and Arg-null 8643–8648 (2004) long actin-rich structures called filopodia to help cells but could do when Abl was re-expressed in WEB SITE Lucy Shapiro’s laboratory: explore its surroundings, especially whilst it’s spread- these cells. Expressing Y361F Dok1 did not http://caulo.stanford.edu/shaplab/ ing. The tyrosine kinase Abl promotes these finger- enhance the number of filopodia in either case. like filopodia, and Woodring et al.now suggest that The authors also noticed fewer filopodia in Dok1–/– this is achieved by Abl signalling to actin at the cell fibroblasts, and this number could not be periphery through the phosphorylation of Dok1, increased by Abl expression. Furthermore, treat- which then recruits the adaptor protein Nck. ment of wild-type fibroblasts, but not Dok1–/– Filopodia formation is dependent on Abl being fibroblasts, with an Abl inhibitor decreased the active, so the hunt for an Abl substrate began. Using number of filopodia. So, Dok1 and Abl seem to be the Src-homology-2 (SH2) domain of Abl as an required for each other’s effects in forming and affinity matrix in lysates from spreading fibroblasts, maintaining filopodia during cell spreading. the authors detected a group of phosphotyrosine- Consistent with this, Dok1 and Abl were both containing proteins in the ~60-kDa range. The 62- present along and at the tips of filopodia. kDa Dok1 protein is tyrosine phosphorylated in Finally, because Dok1 phosphorylated on Y361 Abl-transformed cells, so the authors checked can interact with p120RasGAP or Nck, Woodring whether Dok1 might be one of the proteins in this et al. carried out immunoprecipitation assays using group. A series of experiments confirmed that Dok1 lysates from spreading cells and found an Abl-stim- was indeed a substrate for Abl during cell spreading. ulated Dok1–Nck association. Nck1–/– Nck2–/–- How, then, does Abl affect Dok1? The authors spreading cells had fewer filopodia and, whereas the studied this using a series of point mutants in which Abl inhibitor reduced the number of filopodia in tyrosine residues were mutated to non-phosphory- wild-type cells, it didn’t in Nck1–/– Nck2–/– cells, latable phenylalanines. The mutants were expressed implying that Nck is involved in the Abl-mediated in spreading mouse embryonic fibroblasts that were formation of filopodia. There were hints that a null for both Abl and Arg (a related protein kinase), ternary complex of Abl, Dok1 and Nck could form in or in the same cells reconstituted with Abl. All the cells, so the authors propose that Abl-mediated phos- constructs apart from Y361F Dok1 had higher levels phorylation of Dok1 on Y361 results in the recruit- of tyrosine phosphorylation in the latter cell type, ment of Nck to the cell periphery where localized indicating that Abl-mediated phosphorylation of actin polymerization occurs. But how the cell decides Dok1 Y361 occurs during cell spreading, a result that where to extend a filopodium, and what lies down- was confirmed by in vitro biochemical assays. stream of the Dok1–Nck complex to drive actin After