MOLECULAR MECHANISMS OF ACTIN FILAMENT ASSEMBLY AND TURNOVER

Actin filaments are a universal cytoskeletal factor across the eukaryotic kingdom powering cellular locomotion, endocytosis, mechano-transduction, and many other essential cellular functions. This versatility stems primarily from the myriads of actin binding proteins (ABPs) that bind to and regulate actin. A substantial proportion of these proteins modify actin turnover at filament ends, defined as the flux between actin’s monomeric G-actin state and its filamentous F-actin state. Our understanding of the mechanisms of many of these proteins so far has been limited to static crystallographic or low-resolution cryo-electron microscopy (cryo-EM) structures. Using novel cryo-EM techniques, the dynamic function of several classes of ABPs are now resolved. Structures of formin at the fast-growing barbed end, as well as formin with incoming profilin actin reveal a step-by-step mechanism of actin polymerization. In addition, we show how inverted formin-2 (INF2) is uniquely adapted for filament severing and why other formins are not. At the pointed end, it was typically thought that only capping or depolymerization can occur in the cell. However, three structures of muscle leiomodin-2 (Lmod2) inform a mechanism of pointed-end elongation. Lmod2 is observed priming the penultimate actin subunit for monomer addition, recruiting an actin monomer, and being expelled by the allosteric interactions of a new Lmod2 subunit. Finally, the synergistic depolymerization by cofilin, cyclase-associated protein (CAP), and capping protein are revealed by twelve structures. Cofilin binds cooperatively along an actin filament, shifting F-actin protomers to their monomeric form while keeping filaments intact. At the pointed-end, cofilin weakens the strongest intermolecular interactions between F-actin protomers. CAP binds to the pointed-end, causing a steric clash between the terminal actin and its neighbor, pushing away the last interactions holding it to the filament. At the barbed-end, capping protein prevents depolymerization. However, cofilin progressively weakens capping protein’s affinity for the barbed end by reverting both terminal subunits into a monomeric conformation. Ultimately, capping protein is removed, allowing for filament disassembly. Combined, this work establishes the mechanism of actin filament turnover at both the barbed and pointed end.

Author dc:creator

• Palmer, Nicholas, Joseph

Advisor dc:contributor.advisor

• Dominguez, Roberto

• Biochemistry, Biophysics, and Structural Biology

Language dc:language.iso

en

Repository record dc:identifier.uri

https://repository.upenn.edu/handle/20.500.14332/62684

OAI identifier oai:identifier

oai:repository.upenn.edu:20.500.14332/62684