Revising the actin disassembly machinery : The role of GMF and twinfilin in turnover of dendritic actin arrays

Polymerization of actin filaments against cellular membranes and contraction of actomyosin fibers generate pushing and pulling forces for cell migration, endocytosis, cell division, and maintenance of cell morphology, as well as for intracellular motility and morphogenesis of organelles. Thus, the actin cytoskeleton is a fundamental cellular component in development, immune responses, and in several other aspects of physiology. Moreover, the actin cytoskeleton is hijacked by viruses and pathogens during the infection process. Owing to their central role in above-mentioned cellular processes, actin and actin-binding proteins have been in the limelight of cancer research. Actin is a globular protein, which can polymerize into filaments and depolymerize back to monomers. Dozens of actin binding proteins regulate actin dynamics in cells. Whereas regulation of actin filament nucleation and filament elongation are relatively well understood, the disassembly is far more enigmatic topic. ADF/cofilin is the key actin disassembly factor. It belongs to a family of six actin depolymerizing homology (ADF-H) domain proteins, which all interact with actin or actin-related proteins. However, apart from ADF/cofilin, biochemical and cellular functions of members of this protein family have remained elusive. In this work, I studied the cellular and biochemical roles of two ADF-H domain proteins, glia maturation factor (GMF) and twinfilin. I show that they both promote the disassembly of dendritic actin networks in cells, but by distinct mechanisms. GMF, which binds actin-related proteins (Arp) in the Arp2/3 complex, debranches dendritic actin networks in vitro. The data presented here show that GMF regulates the dynamics of lamellipodial, dendritic actin network in Drosophila cells and promotes collective border cell migration in vivo. Moreover, Drosophila GMF display a strong genetic interaction in cells and in vivo with another actinregulatory protein, actin-interacting protein 1 (Aip1), indicating that they facilitate actin disassembly in a synergistic manner. Twinfilin interacts with actin monomers and actin filament barbed ends to inhibit actin polymerization. Moreover, it binds heterodimeric Capping Protein (CP) and membrane phosphatidylinositol phosphates (PIPs), which inhibit the actin-binding function of twinfilin. However, the molecular mechanism of this interaction has remained unknown. Thus, in the second part of the thesis I utilized a combination of mutagenesis and biochemistry, supplemented with molecular dynamics simulations, to reveal how PIPs inhibit twinfilin. Interestingly, twinfilin interacts with PIPs with a two-step mechanism. First, the CP-interaction motif in the carboxy-terminal tail of twinfilin anchors the protein to plasma membrane. Subsequently, the actin-binding interface interacts with lipids, leading to inhibition of both the CPand actin-binding activities of twinfilin. Cellular functions of twinfilin have remained elusive despite extensive studies in past decades. In the third part of the thesis, I generated mouse twinfilin knockout cell lines and showed that twinfilin regulates both actin and CP turnover in lamellipodia. Surprisingly, twinfilin promotes CP dynamics in cells and in vitro by uncapping filament barbed ends, thus providing an explanation why the localization of CP in cells is restricted to the very distal edge of lamellipodia. Moreover, twinfilin itself does not accelerate filament depolymerization after uncapping, but instead allows filaments to disassemble after removal of CP from actin filament barbed ends, explaining the diminished filament disassembly rates in twinfilin-deficient cells. Together, the work presented here highlights the important roles of twinfilin and GMF in regulation of lamellipodial actin networks. Their distinct roles in actin disassembly show that actin turnover in dendritic arrays is maintained by several functionally different proteins which, in concert, facilitate the turnover of branched actin filament networks in cells.