Sullivan Lab Research

The Sullivan Laboratory
Molecular, Cell, and Developmental Biology
University of California at Santa Cruz

Cytokinesis

The role of membrane addition in
cleavage furrow formation

Furrows (green) encompass each spindle (bright red), preventing inappropriate interaction between neighboring spindles.

The final event in the mitotic cycle is a dramatic constriction of the plasma membrane, which produces two distinct daughter cells. This process, known as cytokinesis, involves formation of an acto-myosin based contractile ring that forms perpendicular and midway to the anaphase spindle. During the past five years, a major advance in our understanding of animal cytokinesis has been the realization that vesicle-mediated membrane addition, as well as acto-myosin based contraction, drives furrow invagination. While it had long been appreciated that Golgi-mediated vesicle fusion drives plant cytokinesis, studies of animal cytokinesis focused on acto-myosin based contraction. However, observational and functional studies from a number of systems revealed that vesicle-based membrane delivery is also an important element of animal cytokinesis. This insight has created new avenues of investigation in the cytokinesis field: what are the sources of membrane, when and where is membrane added during furrow invagination, and how is this process regulated and
coordinated with the cell cycle. These questions are currently a major focus in our lab.

Highlights of progress during the past five years:

Golgi and Recycling endosome-derived vesicles are an important source of membrane for the cytokinesis furrows.

Over the past five years, we discovered that both the Golgi and the recycling endosome, an organelle that traffics vesicles to the plasma membrane, play a key role in Drosophila cellularization by delivering membrane and actin to the invaginating cytokinetic furrow. John Sisson, while a post-doctoral fellow in my lab, identified through affinity chromatography proteins in Drosophila embryo extracts that bind both microfilaments and microtubules. John pursued a novel protein he called Lava-lamp (Lva). He found that it is in a complex with Spectrin and CLIP-170 and is required for vesicle-mediated delivery to the leading edge of the invaginating cellularization furrows. Blocking Lva function prevented elongation of the cellularization furrows. This work is published in the Journal of Cell Biology (Sisson et al. 2000).

Through our screens for mutants that disrupt cytokinesis in the early Drosophila embryo, we discovered a gene, Nuclear-fallout (Nuf), that regulates recycling-endosome (RE) mediated vesicle delivery to the cytokinetic furrows. Nuf is a homolog of the mammalian Arfophilin-2, a Rab11 effector, that binds and co-localizes with Rab11 at the recycling endosome (Hickson et al. 2003). Rab11, a member of the Rab family of small GTPases involved in vesicle targeting, is a well established marker of the RE and is required for recycling endosome organization and function. Several lines of evidence from our lab clearly demonstrate that Nuf and Rab11 act cooperatively at the RE during furrow formation. These findings support the notion that Nuf and Rab11 work together in membrane trafficking and actin remodeling during the initial stages of furrow formation. This work is published in Molecular Biology of the Cell (Hickson et al. 2003) and the Journal of Cell Biology (Riggs et al. 2003).

We are currently in the final stages of a study demonstrating that the cell-cycle regulated location of Nuf at the recycling endosome relies on a physical association with Dynein. Our future work will address the finding from our lab and others that disruption of vesicle-mediated membrane addition during cytokinesis often produces severe disruptions in F-actin organization at the cleavage furrow. The proposed experiments are designed to gain insight into the molecular basis of this linkage between membrane addition and F-actin organization. Our analysis of Nuf also provides an opportunity to determine how vesicle-mediated membrane addition to the cleavage furrow is cell-cycle regulated. We recently found that the phosphorylation state of Nuf and its association with Rab11 at the MTOC is Cdk1 regulated. We are currently performing genetic screens for the kinases that specifically interact with Nuf.

Relevant publications:

Albertson, R., B. Riggs and W. Sullivan 2005 Membrane traffic: a driving force in cytokinesis. Trends in Cell Biology 15: 92-101.

Field, C. M., Coughlin, M., Doberstein, S.. Marty, T., Sullivan, W. 2005 Characterization of anillin mutants reveals essential roles in septin localization and plasma membrane integrity. Development 132: 2849-60.

Royou, A., C. Field, J. Sisson, W. Sullivan and R. Karess 2004 Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos. Mol. Biol. Cell 15:838-50 .

Riggs, B, W. Rothwell, S. Mische, G. Hickson, J. Matheson, T. Hays, G. Gould, and W. Sullivan 2003 Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11. J. Cell Biol. 163:143-54.

Hickson GR, J. Matheson, B. Riggs, VH Maier, AB Fielding, R. Prekeris, W. Sullivan, FA Barr, GW Gould. 2003    Arfophilins are dual arf/rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout. Mol. Biol. Cell 14:2908-20.

Royou, A, W. Sullivan, and R. Karess 2002 Cortical recruitment of nonmuscle myosin II in early syncytial Drosophila embryos: its role in nuclear axial expansion and its regulation by Cdc2 activity.   J. Cell Biol.158:127-37.

Sisson, J. C., Field, C., Ventura, R., and Sullivan W. 2000. Lava lamp, a novel peripheral golgi protein is required for Drosophila melanogaster cellularization. J. Cell Biol. 151: 905-918.

Zhang, C. X., Rothwell, W.F., Sullivan, W., and Hsieh, T. 2000. Discontinuous actin hexagon, a protein essential for cortical furrow formation in Drosophila, is membrane associated and hyper-phosphorylated. Mol. Biol. Cell. 11: 1011-1022.