|Ph.D Student||Ofer Noa|
|Subject||Lamellipodial Fragments as a Model System of|
Self-Organization in Actin-Based Motility
|Department||Department of Nanoscience and Nanotechnology||Supervisor||Professor Kinneret Keren|
|Full Thesis text|
Cell migration is involved in important biological and pathological processes such as wound healing, fertilization and cancer. The majority of animal cells, as well as many unicellular organisms, move by actin-based crawling. While the key molecular components involved in actin-based motility are known, we are far from understanding this fascinating self-organization process. Keratocyte cells cultured from fish scales are a well-established model system for studying cell motility due to their high speed and consistent behavior. Remarkably, keratocyte fragments, which lack the cell body and most organelles, retain the ability to crawl with speed and persistence similar to whole cells. These fragments, consisting of little else than the motility machinery enclosed by a membrane, likely present the simplest natural system exhibiting actin-based motility. In this study, we developed efficient ways for generating keratocyte fragments allowing us to harness this excellent model system to study self-organization in actin-based cell motility. We found that shape and speed are highly correlated over time within individual fragments, whereby faster crawling is accompanied by larger front-to-rear lamellipodial length. Furthermore, we found that the actin network density decays exponentially from front-to-rear indicating a constant net disassembly rate. These findings led us to a simple hypothesis of a disassembly clock mechanism in which rear position is determined by where the actin network has disassembled enough for membrane tension to crush it and haul it forward. This model allowed us to directly relate membrane tension with actin network treadmilling and elucidate the role of the cell membrane as a global mechanical regulator which coordinates protrusion and retraction. To advance our understanding of the underlying dynamics of the actin network, we developed a method for differentially labeling the free pointed and barbed ends of actin filaments which are the primary sites for actin assembly and disassembly in cellular networks. This method allowed us to map the distributions of barbed ends and pointed ends in the lamellipodial fragments. In addition, we mapped the distribution of actin monomers and filaments and used photobleaching experiments to investigate the turnover between them. This analysis of actin turnover in lamellipodial fragments revealed that a considerable fraction of the actin resides in the form of oligomers which play an important role as intermediates in the actin disassembly process.