How do different F-actin binding proteins (F-ABPs) interact with F-actin?

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Two billion years ago, a simple protein forming helical polymers in archaeal cells produced a progenitor cytoskeleton; the same early cytoskeleton that engulfed ancient bacteria to give rise to the very first eukaryotic cells. That protein evolved into actin. ******Actin is the most abundant protein in eukaryotic cells. Evidence of its primordial legacy is found in its universal presence in all eukaryotes, extreme conservation of sequence, and essential participation in almost every major physiological process in eukaryotic life. Throughout the evolution of life, cells have gathered and developed hundreds of different actin binding proteins (ABPs) to control when and where actin functions. As a result, understanding how ABPs interact with actin tells us how eukaryotic life works. ******My research has been squarely focused on building that understanding for the past 2 decades. My approach has been to 1) produce short F-actin complexes (SFACs) that overcome F-actin not forming crystals for atomic structure determination, 2) identify F-actin binding proteins (F-ABPs), and then 3) characterize those F-ABPs biochemically and structurally. ******My previous NSERC-funded research showed that uniformly modified SFAC does not bind myosin, while others suggested an SFAC with a combination of modifications interacts with myosin. To further our work with myosin, we will confirm that this combination approach produces an active SFAC and then perform biochemical and structural characterizations. ******Several near-atomic pictures of F-actin with myosin bound were determined in the last 5 years using cryo-electron microscopy; however, specific interactions at each stage of the actomyosin cycle still need to be described. Beyond myosin, how do different ABPs interact with F-actin? ******Building on our deep experience with actin and working with local and national collaborations, we will tackle these questions using modern single molecule approaches. Ultimately, our goal is to determine how F-actin interacts with binding partners, starting with unanswered questions around myosin binding and expanding the research program to other ABP classes. ***The specific Objectives of this research are: ****** 1. To produce an SFAC that supports myosin force generation and determine the role of the C-terminus of actin in the actomyosin cycle. ****** 2. To identify and characterize ABPs that interact with SFACs with mass spectroscopy. ****** 3. To determine the high-resolution structure of ABPs bound to actin with cryo-EM. ******Interactions between F-actin and ABPs is fundamental for life. Our research will expand our understanding of the molecular dance between actin and myosin and the modes of binding to F-actin by ABPs using state-of-the art proteomic and structural biology techniques. This knowledge is not only critical for understanding how cells function but can also be applied to the development of nanotechnologies.