Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.

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In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.

Plk1, but not Cdk1, phosphorylates PRC1 on Thr-602 to prevent premature midzone assembly in metaphase. Microtubules enhance this phosphorylation. Plk1, PRC1, and microtubules together regulate homeostatic MT bundling throughout cell division.

Using multiplexed quantitative proteomics, we analyzed cell cycledependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/ Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C CDH1 -dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.

The cytoskeleton globally reorganizes between mitosis (M phase) and cytokinesis (C phase), which presumably requires extensive regulatory changes. To reveal these changes, we undertook a comparative proteomics analysis of cells tightly drug-synchronized in each phase. We identified 25 proteins that bind selectively to microtubules in C phase and identified several novel binding partners including nucleolar and spindle-associated protein. C phase-selective microtubule binding of many of these proteins depended on activity of Aurora kinases as assayed by treatment with the drug VX680. Aurora-B binding partners switched dramatically between M phase to C phase, and we identified several novel C phase-selective Aurora-B binding partners including PRC1, KIF4, and anaphase-promoting complex/cyclosome. Our approach can be extended to other cellular compartments and cell states, and our data provide the first broad biochemical framework for understanding C phase. Concretely, we report a central role for Aurora-B in regulating the C phase cytoskeleton. Molecular & Cellular Proteomics 9: 336 -350, 2010.Cytokinesis requires coordinated reorganization of microtubules, actin, and membranes, which implies global regulation of cellular biochemistry. Animal cells are competent to undergo cytokinesis during a brief window in the cell cycle, called C phase, which starts shortly after cells exit mitosis (M phase) and lasts ϳ30 -60 min (1, 2). The cytoplasm is globally regulated in M phase by the activity of one master kinase, Cyclin B-CDC2 (CDK1), that is strongly activated as cells enter M phase and controls the organization and function of 100s to 1000s of substrates (3, 4). C phase regulation, in contrast, is poorly understood. Given the global cytoplasmic changes that characterize C phase, global regulation by kinases seems likely. CDK1 activity decreases abruptly at anaphase onset, and the phosphatases that oppose it increase in activity (5). Thus, one global change in the transition from M to C phase is likely to be the loss of CDK1 phosphorylation, although the kinetics by which different substrates are dephosphorylated is unclear. Two other kinases, Aurora-B and PLK1, are broadly implicated in the regulation of C phase biochemistry and might function to some extent as global regulators (6). However, both these kinases are also active in M phase, and it is not clear how their activity and substrate specificities change between M and C phase.Many cytoplasmic systems and compartments are likely to be controlled by C phase-regulating kinases. Here, we focus on the microtubule cytoskeleton as one specific compartment, although our methods could easily be adapted to any other compartment that could be isolated. Microtubules change dramatically in organization from the highly dynamic mitotic spindle in M phase to the less dynamic midzone and astral arrays in C phase. Several conserved proteins, including PRC1 and MKLP1, have been identified by cytology and genetics as C phase-specific microtubule-binding proteins that are required...