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Abstract

The actin cytoskeleton is a dynamic system involved in a variety of cellular processes, including endocytosis, motility, polarity, and cytokinesis. In a single crowded cytoplasm, the cell assembles multiple different filamentous actin (F-actin) networks at the correct time and place and with the proper architecture and dynamics. How the cell is capable of assembling multiple distinct F-actin networks simultaneously remains an unanswered question. In a cell, actin monomers are assembled into actin filaments, and these actin filaments are organized into defined and distinct networks by the coordinated action of the associated actin binding proteins (ABPs). ABPs are involved in nucleating, bundling, severing, pulling, and capping actin filaments, and therefore, the ABPs associated with an actin network define its specific properties. Therefore, it is crucial that ABPs properly localize to the correct actin network. However, the mechanisms behind how different sets of ABPs sort to different actin networks are unclear. My hypothesis is that competition between ABPs for association with F-actin is a driving factor behind proper ABP sorting. Fission yeast is an ideal simplified system in which to study the underlying molecular mechanisms behind F-actin network self-organization. Fission yeast has three primary actin cytoskeleton networks, in which all of the actin filaments are assembled by a distinct actin assembly factor: endocytic actin patches (Arp2/3 complex), polarizing actin cables (formin For3), and the cytokinetic contractile ring (formin Cdc12). Moreover, each of these F-actin networks contains a distinct set of ABPs. We hypothesize that ABP competition for association with actin filaments is critical for their proper sorting to distinct F-actin networks. Previous work identified three ABPs (two that localize to endocytic actin patches, fimbrin Fim1 and cofilin Adf1, and one, tropomyosin Cdc8, that localizes to the contractile ring) that have competitive interactions (Skau and Kovar, 2010). I investigated the molecular mechanisms behind their competitions and determined that fimbrin Fim1 actively displaces tropomyosin Cdc8 specifically from bundled regions. I additionally found that tropomyosin Cdc8 inhibits cofilin Adf1-mediated severing by blocking the initial association of cofilin Adf1 with F-actin. Finally, I found that Fim1 and Adf1 compete for the same binding site on F-actin, and that this competition results in the generation of Adf1 boundaries, facilitating rapid severing by Adf1 and bundling of the F-actin network by Fim1. This dense actin network generated by Fim1 and Adf1 rapidly displaces Cdc8 from the network. I speculate that this mechanism causes Cdc8 to be removed from actin patches and associate instead with the contractile ring. Additionally, I performed a survey in fission yeast to determine other potential competitive interactions between ABPs. I identified a competitive interaction between fimbrin Fim1 and contractile ring ABP α-actinin Ain1. I found that fimbrin Fim1 outcompetes α-actinin Ain1 for association with F-actin, likely via competition for the same binding site on the actin filament. This competition can occur at both the contractile ring and actin patches. Additionally, I found that contractile ring ABP tropomyosin Cdc8 enhances Ain1-mediated bundling. Finally, I found that Cdc8 synergizes with Ain1 to prevent Fim1 from associating with F-actin. Though my primary work has been on investigating ABP competition, I also completed a project characterizing the formin (CrFor1) and profilin (CrPRF) from Chlamydomonas reinhardtii. I found that CrPRF is an unusual profilin that prevents both the nucleation of CrPRF-bound actin monomers as well as F-actin elongation. CrFor1 is capable of overcoming this inhibition of actin polymerization and rapidly assembles CrPRF-bound actin monomers into F-actin. These findings suggest that CrPRF and CrFor1 are tailor-made to rapidly assemble F-actin at the correct time and place. Additionally, CrPRF inhibits actin assembly by the fission yeast Arp2/3 complex, suggesting that CrPRF favors CrFor1-mediated actin assembly both by directly enhancing CrFor1’s ability to assemble F-actin and by inhibiting the activity of competing actin assembly factors.

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