Bembenek Lab

The eggshell as a model for membrane trafficking during cell division

Genome-wide screens categorized many genes according to similar phenotypes, suggesting they regulate similar underlying cell biological processes. A strange observation was that numerous cell cycle regulators caused eggshell defects when knocked out in C. elegans. The “osmotic integrity defective” eggshell phenotype was considered non-specific because various genes from ribosome subunits to cell cycle genes cause the phenotype when inactivated. While investigating cell cycle genes in the embryo, we discovered that the eggshell is actually formed in part by cargo delivered to the extracellular matrix by vesicle exocytosis. Oocytes of many species produce secretory vesicles called cortical granules that release cargo required to modify the covering of oocytes to prevent polyspermy. Therefore, the OID phenotype is due to the fact that cortical granule exocytosis is required for eggshell formation in C. elegans.

Several core cell cycle regulators disrupt eggshell formation by causing defects in cortical granule biogenesis, trafficking and exocytosis. The exocytosis of these vesicles occurs during mid-anaphase, after chromosomes initiate their polward journey. Several key regulators of the metaphase to anaphase transition such as the Anaphase Promoting Complex/ Cyclosome, Securin and Separase all disrupt cortical granule trafficking. This pathway is key to chromosome segregation by ensuring that Separase, a large protease, becomes active only at the onset of anaphase when it cleaves the cohesin glue holding sister chromatids together. The original unexpected observation was that separase localizes to cortical granules and is required for exocytosis during anaphase. This same pathway operates during mitosis to control RAB-11 vesicle trafficking required for cytokinesis.

In exciting new work, we have discovered that outer kinetochore proteins assemble into linear elements in the cortical cytoplasm to regulate vesicle transport during prometaphase I. This significantly extends the number of cell cycle components that contribute to vesicle trafficking in the oocyte and suggests that the spindle checkpoint is a bifurcated pathway that controls both chromosomes and membranes during cell division.

These observations form the basis for our NIH R01 funded research project seeking to understand how the core cell cycle machinery coordinates chromosome segregation with membrane trafficking during division. We have three main areas of investigation on this problem:

Investigating the Role of the Protease Activity of Separase

We have sought to understand how separase promotes exocytosis by first asking whether the protease activity is required. On the surface, this is an obvious question. However, in addition to cleaving substrates, separase also regulates signaling pathways that control cytokinesis independently of the protease activity in other systems. To test this, we expressed protease dead separase fused to GFP and unexpectedly found that it is dominant negative. Our results indicate that protease dead separase is a substrate trapping enzyme that interferes with cleavage of endogenous substrates, including a substrate independent of cohesin that is involved in exocytosis. This sets the stage for the next phase of the project to identify the relevant substrates of separase using immunoprecipitation of the substrate trapping enzyme. We are collaborating with John Yates at Scripps to identify candidate interacting proteins. Despite the fundamental importance of separase, few substrates are known, therefore, identification of novel substrates would have a significant impact.

Genetic Screening for Separase Suppressors

            In order to identify new players that are involved in separase regulation and function, we conducted a suppressor screen with our hypomorphic mutants, hoping to identify vesicle trafficking machinery related to separase function. We found 11 intragenic suppressors within the N-terminal domain of separase that may provide new insight into the function of this regulatory region. We also identified numerous alleles of PPH-5, a previously identified suppressor, and an allele of HSP-90 that has a mutation near the C-terminal domain that is known to bind and activate PPH-5. These results suggest that the PPH-5/HSP-90 signaling complex might be important for regulating separase function. Finally, we found at least four unknown suppressors and are using genome wide sequencing to identify them. In the future we will continue our genetic analysis of separase and expect to generate important insights into the regulation and function of this key cell cycle regulator.

Cell Cycle Regulation of Separase

We recently characterized how the spindle checkpoint pathway regulates the function of separase during exocytosis. We found that separase moves from kinetochore structures on chromosomes and linear elements to sites of action at the midbivalent and vesicles at anaphase onset. Separase relocalization is regulated by APC/C and securin. How separase is recruited to different sites at different times is currently not well understood and will be investigated in the future.