Centrosome duplication 

Centrosomes are the major microtubule-organizing centers (MTOCs) of animal cells and are composed of two centrioles surrounded by pericentriolar material. Cells are born with a single centrosome, which duplicates during S phase, resulting in bipolar spindle assembly during mitosis. Failure of centrosome duplication can lead to a monopolar spindle, excess centrosome duplication to a multipolar spindle. Despite being crucial for genome integrity, the mechanisms governing centrosome duplication remain poorly understood. C. elegans is a model of choice for investigating these mechanisms through a combination of powerful forward genetic, functional genomic and cell biological approaches. Five proteins are essential for centriole formation in C. elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SPD-2, SAS-5 and SAS-6. We established that centriole formation in C. elegans is an orderly assembly process in which SPD-2, ZYG-1, SAS-5, SAS-6 and SAS-4 are recruited in a step-wise fashion. Most of these proteins have homologues in other metazoans, and we suggest that this assembly pathway is evolutionarily conserved. We also study centrosome duplication in human cells. Thus, we found that siRNA-mediated inactivation of HsSAS-6 abrogates centrosome duplication (see Figure 1), indicating broad functional conservation among SAS-6 related proteins. Moreover, we found that HsSAS-6 localizes to the base of the newly formed centriole and that its overexpression results in the formation of excess centriole-like structures, indicating a key role in driving the centrosome duplication cycle.

Figure 1: Human cells in mitosis treated either with a control siRNA (left) or with an siRNA directed against HsSAS-6 (right), and stained with antibodies against the centriolar protein centrin (red, visible in yellow at spindle poles), a‑tubulin (green), as well as counterstained with a DNA dye (blue). Note that a monopolar spindle assembles after HsSAS-6 depletion.

Asymmetric cell division

Asymmetric divisions are central for generating cell diversity during development. In animal cells, the mitotic spindle must be eccentrically positioned for unequal cell division to occur. How cell polarity modulates the microtubule cytoskeleton to achieve proper spindle positioning is incompletely understood. In the one-cell stage C. elegans embryo, the spindle elongates asymmetrically towards the posterior in response to anterior-posterior (A-P) polarity cues. Laser microsurgery experiments conducted in living embryos showed that asymmetric spindle elongation is achieved by an imbalance of forces pulling on astral microtubules emanating from the spindle pole. As a larger net force pulls on the posterior spindle pole, the spindle elongates asymmetrically and the first division is unequal. We established that microtubule depolymerization and cytoplasmic dynein are both required for generating extensive pulling forces. Moreover, we established that pulling forces rely on GOA-1 and GPA-16 (see Figure 2), two alpha subunits of the heterotrimeric G proteins, as well as the interacting proteins GPR-1/2, LIN-5 and RIC-8. Homologues of these components are also required for asymmetric cell division in Drosophila, as well as for spindle positioning in human cells, underscoring the fact that findings in C. elegans provide general insights into the mechanisms of asymmetric cell division.

Figure 2: C. elegans embryos (left: end of one-cell stage; right: four-cell stage) stained with antibodies against the Ga protein GPA-16 (red), a‑tubulin (green) and counterstained with a DNA dye (blue). Note the presence of GPA-16 at the cell membrane.  

Timing of cell division 

Mitosis allows faithful partition of the genetic material to daughter cells. Several mitotic regulators, including Cyclin B1/Cdk1, are present at centrosomes prior to mitosis onset in human cells, but it is unclear whether centrosomes promote mitotic entry in vivo. We developed a sensitive assay in C. elegans embryos for the spatio-temporal analysis of mitotic entry, in which the male and female pronuclei undergo asynchronous entry into mitosis when separated from one another. Using this assay, we found that centrosome integrity is necessary for timely mitotic entry. Furthermore, we established that centrosomes do not function in this instance through their ability to nucleate microtubules. Instead, they serve to focus the Aurora-A kinase AIR-1, which is essential for timely mitotic entry. Importantly, by analyzing mutant embryos in which centrosomes and pronuclei are detached from one another, we demonstrated that centrosomes are also sufficient to promote timely mitosis onset. Together with earlier work, our findings support the view that centrosomes help orchestrate the proper execution of mitosis in time and space.

Keywords

Cell biology, developmental biology, mitosis, centrosome, asymmetric cell division, C. elegans