Dissecting Recruitment of Actin into the Contractile Ring

Oregon State University, Corvallis OR

Investigators

Abstract

Scientific Research: Cytokinesis in animal cells is brought about by assembly of an actomyosin contractile ring that constricts the cell around the spindle equator. The molecular basis underlying the organization and constriction of the contractile ring is well documented, yet the mechanics of how actin filaments redistribute and assemble into the ring remain poorly understood. This project aims to elucidate the mechanics of contractile ring assembly by learning how microtubules interact with actin filaments and how the filaments incorporate into the ring. Knowledge gained from this project can be applied to analogous cellular processes, such as polarized growth and wound healing of a single cell. The working model is as follows. During early anaphase, dynamic astral microtubules exclude actin filaments from the poles, ultimately resulting in actin accumulation at the equatorial cortex. Meanwhile, at the plus ends of the relatively stable central-spindle microtubules, actin filaments are assembled de novo, and delivered to the equatorial cortex by laterally splaying microtubule bundles. There they coalesce with the actin excluded from the polar cortex to assemble the contractile ring. Based on this Microtubule Induction model, the project aims to dissect the mechanics of microtubule-mediated transport of preexisting actin filaments. Exclusion of these filaments from the poles by microtubules may occur via two distinct mechanisms. If actin filaments are driven towards the equatorial cortex by elongating microtubules, mechanical interactions between microtubules and actin filaments will be demonstrated and perturbed in living cytokinetic cells. In cells deprived of the spindle apparatus, a microneedle will be used as a surrogate for microtubules to recruit actin filaments, in order to rescue furrow positioning. Alternatively, if actin filaments are transported by motor proteins along spindle microtubules that are contiguous to the cortex, direct observations of their movement on microtubules will be made in living cytokinetic cells. The project will use a newly-developed approach that employs multimode micro-techniques to surgically remodel spindles and cells so as to mechanically dissect microtubule-mediated assembly of the contractile ring. This unique approach combines classic micromanipulation and microinjection with modern laser microbeam surgery - on a microscope equipped with both digital-enhanced polarization and spinning disc confocal microscopy. Thus, the approach permits direct micromanipulation of both unlabeled and fluorescently labeled cytoskeletal components in living cells and in vitro. Broader Impacts: The general impact of this research project will be increased in multiple ways. Concomitant with achieving the specific aim, one to two graduate students and one postdoctoral fellow will be trained in designing and conducting experiments, presenting data at scientific conferences, and publishing papers in respected journals. The research will also be used as an effective teaching tool for science education in a variety of contexts - in undergraduate and graduate Cell Biology courses, the HHMI-supported undergraduate summer research program, the NSF-supported OSU outreach programs to GK-12 students and teachers, the HHMI-supported Science & Math Investigative and Learning Experiences (SMILE) program, and in communicating with the general public. This research generates striking, aesthetically compelling data via cutting edge technology, making it ideally suited for educational outreach. Furthermore, because this basic research uses grasshopper spermatocytes as a model for understanding cell division, it may steer applied research toward novel ways of suppressing reproduction in agriculturally destructive insects.