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Local activity of the small GTPase Cdc42 is critical for cell polarization. Gallo Castro and Martin describe a new Cdc42 GTPase-activating protein (GAP) Rga3, which together with two other GAPs, constrains Cdc42-GTP zones during mitotic cycles but not during sexual reproduction., The formation of a local zone of Cdc42 GTPase activity, which governs cell polarization in many cell types, requires not only local activation but also switch-off mechanisms. In this study, we identify Rga3, a paralog of Rga4, as a novel Cdc42 GTPase-activating protein (GAP) in the fission yeast Schizosaccharomyces pombe. Contrary to Rga4, Rga3 localizes with Cdc42-GTP to sites of polarity. Rga3 is dispensable for cell polarization during mitotic growth, but it limits the lifetime of unstable Cdc42-GTP patches that underlie cell pairing during sexual reproduction, masking a partly compensatory patch-wandering motion. In consequence, cells lacking rga3 hyperpolarize and lose out in mating competition. Rga3 synergizes with the Cdc42 GAPs Rga4 and Rga6 to restrict Cdc42-GTP zone sizes during mitotic growth. Surprisingly, triple-mutant cells, which are almost fully round, retain pheromone-dependent dynamic polarization of Cdc42-GTP, extend a polarized projection, and mate. Thus, the requirement for Cdc42-GTP hydrolysis by GAPs is distinct during polarization by intrinsic or extrinsic cues.

The dynein motor is recruited to the kinetochore to capture spindle microtubules and control the spindle assembly checkpoint. Gama et al. reveal the molecular mechanism of how the Rod–Zw10–Zwilch complex and Spindly mediate dynein recruitment in Caenorhabditis elegans and human cells., The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod–Zw10–Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod’s N-terminal β-propeller and the associated Zwilch subunit bind Spindly’s C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod β-propeller–Zwilch complex in vitro. Spindly’s N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein–dynactin–Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein–dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo.

The Journal of Cell Biology, 198 (5), ISSN:0021-9525, ISSN:1540-8140

As a component of chromatin-modifying complexes with histone acetyltransferase (HAT) activity, TRRAP has been shown to be involved in various cellular processes including gene transcription and oncogenic transformation. Inactivation of Trrap, the murine ortholog of TRRAP, in mice revealed its function in development and cell cycle progression. However, the underlying mechanism is unknown. Here, we show that the loss of Trrap in mammalian cells leads to chromosome missegregation, mitotic exit failure and compromised mitotic checkpoint. These mitotic checkpoint defects are caused by defective Trrap-mediated transcription of the mitotic checkpoint proteins Mad1 and Mad2. The mode of regulation by Trrap involves acetylation of histones H4 and H3 at the gene promoter of these mitotic players. Trrap associated with the HAT Tip60 and PCAF at the Mad1 and Mad2 promoters in a cell cycle-dependent manner and Trrap depletion abolished recruitment of these HATs. Finally, ectopic expression of Mad1 and Mad2 fully restores the mitotic checkpoint in Trrap-deficient cells. These results demonstrate that Trrap controls the mitotic checkpoint integrity by specifically regulating Mad1 and Mad2 genes.

The multiple functions of kinetochores are reflected in their complex composition, with over a hundred different proteins, which self-associate in several functional subcomplexes. Most of these kinetochore proteins were identified over the last 10-12 years using a combination of genetic, cell biological, biochemical, and bioinformatic approaches in various model organisms. The key challenge since then has been to determine the structural architecture of kinetochores, define the functions of its different subcomponents, and understand its regulation, both in response to the rapid changes in microtubule dynamics or to sense erroneous attachments for spindle checkpoint signalling. Here, we present some of the key advances obtained in the last six years on the biology of kinetochores, both through our work and through the work of many other groups studying this exciting structure.

Previous studies have shown that neuronal cells in culture can switch neurotransmitters when grown in the presence of different target cells. To examine whether this plasticity extends to structural proteins, we cocultured hippocampal neurons and pituitary-derived neuroendocrine (AtT20) cells with astrocytes, kidney epithelial cells, or skeletal muscle cells. As a marker of phenotypic change we used the cytoskeletal protein peripherin, a type III intermediate filament (IF) subunit which is not expressed in hippocampal neurons and AtT20 cells. We show here that soluble factor(s) secreted specifically from skeletal muscle cells can induce the expression and de novo assembly of peripherin in a subset of post-mitotic neurons. We further demonstrate that one of these factors is the Leukemia Inhibitory Factor/Cholinergic Neuronal Differentiation Factor. The environmentally regulated expression of peripherin implies a remarkable degree of plasticity in the cytoskeletal organization of postmitotic CNS cells and provides a noninvasive model system to examine the de novo assembly of IF proteins under in vivo conditions.

Summary Background SAK/PLK4 is a distinct member of the polo-like kinase family. SAK −/− mice die during embryogenesis, whereas SAK +/− mice develop liver and lung tumors and SAK +/− MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. Results Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. Conclusions SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.

Background: Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans , differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P 1 . How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated. Results : We establish that early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1 . We demonstrate that preferential activation of this checkpoint in the P 1 blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, we show that preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Gα signaling. Conclusion: Our findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development.

Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis elegans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanisms by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL-1/CHK-1 dependent checkpoint in P1, but how the remaining time difference is controlled is not known. Here, we establish that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by A-P polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1, but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, our findings support a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1-dependent preferential retardation in P1 and PLK-1-dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development

Cytokinesis in Schizosaccharomyces pombe begins at mitotic entry, when the site of division is defined by formation of the contractile acto-myosin ring (CAR) at the cell cortex. Contraction of the CAR and formation of the division septum are triggered at the end of mitosis by septation initiation network (SIN) proteins associated with the spindle pole body (SPB). SIN signalling requires activation of the GTPase Spg1p, which is regulated by the bipartite GTPase-activating protein (GAP) Byr4p-Cdc16p. We show that, for Spg1p to associate with the SPB, it must be bound to its GAP or to its mitotic effector, the protein kinase Cdc7p. Analysis of the GAP proteins reveals that the steady-state level of Byr4p reflects that of Spg1p. Furthermore, if the interaction of Byr4p with Spg1p is compromised, the level of Byr4p decreases dramatically. The adaptation of the level of Byr4p to that of Spg1p requires the presence of Cdc16p and is mediated by proteasome-dependent destruction. It requires neither association with the SPB nor an active SIN. We propose a mechanism that limits the amount of the Byr4p-Cdc16p GAP to the amount required to inhibit Spg1p signalling.