Condensin and the spindle midzone prevent cytokinesis failure induced by chromatin bridges in C. elegans embryos.

BACKGROUND: During cell division, chromosomes must clear the path of the cleavage furrow before the onset of cytokinesis. The abscission checkpoint in mammalian cells stabilizes the cleavage furrow in the presence of a chromatin obstruction. This provides time to resolve the obstruction before the cleavage furrow regresses or breaks the chromosomes, preventing aneuploidy or DNA damage. Two unanswered questions in the proposed mechanistic pathway of the abscission checkpoint concern factors involved in (1) resolving the obstructions and (2) coordinating obstruction resolution with the delay in cytokinesis. RESULTS: We found that the one-cell and two-cell C. elegans embryos suppress furrow regression following depletion of essential chromosome-segregation factors: topoisomerase II(TOP-2), CENP-A(HCP-3), cohesin, and to a lesser degree, condensin. Chromatin obstructions activated Aurora B(AIR-2) at the spindle midzone, which is needed for the abscission checkpoint in other systems. Condensin I, but not condensin II, localizes to the spindle midzone in anaphase and to the midbody during normal cytokinesis. Interestingly, condensin I is enriched on chromatin bridges and near the midzone/midbody in an AIR-2-dependent manner. Disruption of AIR-2, the spindle midzone, or condensin leads to cytokinesis failure in a chromatin-obstruction-dependent manner. Examination of the condensin-deficient embryos uncovered defects in both the resolution of the chromatin obstructions and the maintenance of the stable cleavage furrow. CONCLUSIONS: We postulate that condensin I is recruited by Aurora B(AIR-2) to aid in the resolution of chromatin obstructions and also helps generate a signal to maintain the delay in cytokinesis.

Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis.

In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. In this paper, we show that, contrary to previous models, the Drosophila melanogaster CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, we found that the Sti Citron-Nik1 homology domain interacts with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion caused an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. We propose that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics.

Imaging C. elegans embryos using an epifluorescent microscope and open source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples(1,2). Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage(3), thus providing an ideal experiment model for studying questions in cell biology(4,5)and development(6-9). C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis(10,11)) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis(12-15)). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters(16,17). These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo(18-21). In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Action at a distance during cytokinesis.

Animal cells decide where to build the cytokinetic apparatus by sensing the position of the mitotic spindle. Reflecting a long-standing presumption that a furrow-inducing stimulus travels from spindle to cortex via microtubules, debate continues about which microtubules, and in what geometry, are essential for accurate cytokinesis. We used live imaging in urchin and frog embryos to evaluate the relationship between microtubule organization and cytokinetic furrow position. In normal cells, the cytokinetic apparatus forms in a region of lower cortical microtubule density. Remarkably, cells depleted of astral microtubules conduct accurate, complete cytokinesis. Conversely, in anucleate cells, asters alone can support furrow induction without a spindle, but only when sufficiently separated. Ablation of a single centrosome displaces furrows away from the remaining centrosome; ablation of both centrosomes causes broad, inefficient furrowing. We conclude that the asters confer accuracy and precision to a primary furrow-inducing signal that can reach the cell surface from the spindle without transport on microtubules.

An Afg2/Spaf-related Cdc48-like AAA ATPase regulates the stability and activity of the C. elegans Aurora B kinase AIR-2.

The Aurora B kinase is the enzymatic core of the chromosomal passenger complex, which is a critical regulator of mitosis. To identify novel regulators of Aurora B, we performed a genome-wide screen for suppressors of a temperature-sensitive lethal allele of the C. elegans Aurora B kinase AIR-2. This screen uncovered a member of the Afg2/Spaf subfamily of Cdc48-like AAA ATPases as an essential inhibitor of AIR-2 stability and activity. Depletion of CDC-48.3 restores viability to air-2 mutant embryos and leads to abnormally high AIR-2 levels at the late telophase/G1 transition. Furthermore, CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity from metaphase through telophase. While canonical p97/Cdc48 proteins have been assigned contradictory roles in the regulation of Aurora B, our results identify a member of the Afg2/Spaf AAA ATPases as a critical in vivo inhibitor of this kinase during embryonic development.

Cortical centralspindlin and G alpha have parallel roles in furrow initiation in early C. elegans embryos.

Evidence from various systems suggests that either asters or the midzone of the mitotic spindle are the predominant determinants of cleavage plane position. Disrupting spindle midzone formation in the one-cell Caenorhabditis elegans embryo, such as by using mutants of the centralspindlin component ZEN-4, prevents completion of cytokinesis but does not inhibit furrowing. However, furrowing is inhibited by the simultaneous depletion of ZEN-4 with either PAR-2 or G alpha, which are required for asymmetric divisions. Through studies of other genes required for the presence of an intact spindle midzone containing microtubule bundles, we found that furrowing failed in the absence of PAR-2 or G alpha only when centralspindlin was absent from the furrow. We also found spindle length or microtubule distribution did not correlate with furrow initiation. We propose that centralspindlin acts redundantly with G alpha to regulate furrow initiation.

Midbodies and phragmoplasts: analogous structures involved in cytokinesis.

Cytokinesis is an event common to all organisms that involves the precise coordination of independent pathways involved in cell-cycle regulation and microtubule, membrane, actin and organelle dynamics. In animal cells, the spindle midzone/midbody with associated endo-membrane system are required for late cytokinesis events, including furrow ingression and scission. In plants, cytokinesis is mediated by the phragmoplast, an array of microtubules, actin filaments and associated molecules that act as a framework for the future cell wall. In this article (which is part of the Cytokinesis series), we discuss recent studies that highlight the increasing number of similarities in the components and function of the spindle midzone/midbody in animals and the phragmoplast in plants, suggesting that they might be analogous structures.

SPD-1 is required for the formation of the spindle midzone but is not essential for the completion of cytokinesis in C. elegans embryos.

The process of cytokinesis can be divided into two stages: the assembly and constriction of an actomyosin ring giving rise to a narrow intracellular canal and the final breaking and resealing of this canal. Mutations in several genes of Caenorhabditis elegans disrupt the spindle midzone (anti-parallel microtubules and associated proteins that form between the spindle poles) and give rise to failures in the completion of cytokinesis. We show that loss of function of spd-1 causes midzone disruptions, although cytokinesis generally completes. SPD-1 is a conserved microtubule-bundling protein that localizes to the midzone and also to microtubule bundles in the cytoplasm. The midzone localization of SPD-1 is perturbed in embryos depleted of other midzone components, yet the cytoplasmic bundles are not affected. We found that two other midzone components also localize to the ingressing furrow in wild-type embryos; when SPD-1 is depleted, there is no visible midzone, and only this furrow localization remains. SPD-1 differs from other midzone components in that it is essential for the integrity of the midzone, yet not for cytokinesis. Also, it can localize to the midzone when other midzone components are depleted, suggesting that SPD-1 may play an early role in the pathway of midzone assembly.