The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.

Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo

Pierre Gönczy

We have investigated the role of cytoplasmic dynein in microtubule organizing center (MTOC) positioning using RNA-mediated interference (RNAi) in Caenorhabditis elegans to deplete the product of the dynein heavy chain gene dhc-1. Analysis with time-lapse differential interference contrast microscopy and indirect immunofluorescence revealed that pronuclear migration and centrosome separation failed in one cell stage dhc-1 (RNAi) embryos. These phenotypes were also observed when the dynactin components p50/dynamitin or p150(Glued) were depleted with RNAi. Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus. When dynein heavy chain function was diminished only partially with RNAi, centrosome separation took place, but orientation of the mitotic spindle was defective. Therefore, cytoplasmic dynein is required for multiple aspects of MTOC positioning in the one cell stage C. elegans embryo. In conjunction with our observation of cytoplasmic dynein distribution at the periphery of nuclei, these results lead us to propose a mechanism in which cytoplasmic dynein anchored on the nucleus drives centrosome separation.

1999

Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III

Pierre Gönczy

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.

2000

PLK-1 asymmetry contributes to asynchronous cell division of C. elegans embryos

Yemima Suryani Budirahardja, Pierre Gönczy

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

2008