Cytoskeletal Dynamics and Cell Division

AMY SHAUB MADDOX , Ph.D.

• Research Assistant Professor, Department of Pathology and Cell Biology, Faculty of Medicine, Université de Montréal

We study the molecular mechanisms of cell shape change in cytokinesis: the assembly, organization and function of the actomyosin contractile ring.  With live-cell imaging of fluorescent probes, we monitor the dynamics of the ring in early C. elegans embryos.  Because of the highly stereotypical nature of cell division events in C. elegans, we can devise novel quantitative assays for cytokinesis.

The conserved actin-, myosin and septin-binding protein Anillin (see Fig. 1) is a prime candidate for optimizing organization in the contractile ring.  We demonstrated that Anillin is required to break circumferential symmetry in the contractile ring (see Fig. 2).  While abnormal ring symmetry can be compensated for by other behaviors of the ring, asymmetry appears to help make cytokinesis robust to random errors.  Thus, by taking different views of the contractile ring and quantifying speed, geometry, and protein localization, we uncovered a novel facet of the redundant mechanisms that ensure successful cytokinesis.

Here are some of the projects currently underway:

We performed biochemical purifications to identify proteins with novel contributions to organization and function of the contractile ring.  We are now characterizing the defects in cytokinesis following depletion of our candidate proteins, making GFP fusion strains and antibodies for them, and aim to confirm the interactions by “reverse” co-IP.

The C. elegans early embryo is always the same size and shape, and is essentially a cylinder.  Therefore, it is highly amenable to descriptive and predictive mathematical modeling.  For example, preliminary studies correlating contractile ring dynamics with cell shape change indicate that actomyosin organization, not just quantity, affects the kinetics of cell shape transitions.  We are now devising image analysis software to track cell shape and protein localization, and aim to create a predictive mathematical model for contractile ring function.

We are pioneering the use of C. elegans tissue/cell types other than the early embryo for cell division studies.  We are beginning by optimizing protein depletion and live-cell imaging in these tissues. We aim to compare cell division kinetics and dynamics with those in the embryo and cultured somatic cells.

All our projects entail molecular biology, micromanipulation and dissection, live-cell imaging, and computer-based analysis.  We also use Matlab to create original programs.

FIGURE 1. A C.elegans embryo undergoing its first mitotic cytokinesis was fixed and stained for Anillin (red), a conserved structural component of the contractile ring, and DNA (blue).

FIGURE 2. An end-on view of a C. elegans embryo in cytokinesis shows the contractile ring closing asymmetrically within the division plane. This occurred despite a centrally placed mitotic spindle, and requires Anillin and the septins. Fluorescence: GFP-tagged PH domain that marks the plasma membrane, time elapsed: ~4 minutes.

T + 514 343.6111, ext. 1-0317
F + 514 343.6843
amy.maddox@umontreal.ca