Speakers at the Minisymposium on “Actin Dynamics and Function” reported recent discoveries ranging from the structure and mechanics of actin filaments, mechanisms regulating filament polymerization, depolymerization, and interaction with actin-binding proteins (ABPs) to the regulation and function of actin in the contexts of diverse cellular physiologies such as nuclear movement, phagocytosis, autophagy, secretion, and axon turning.
Pinar Gurel (Greg Alushin lab, Rockefeller University, New York) used cryo–electron microscopy to investigate the conformation of single actin filaments under compressive and tensile forces. A reconstitution system in which actin filaments were placed under mechanical load using immobilized myosin V or myosin VI revealed that both compressive and tensile forces produced a novel, persistent actin structural state characterized by oscillating areas of high filament curvature called “squiggles.” Cryo–electron tomography revealed that squiggles were three-dimensional corkscrew structures. Efforts are underway to resolve a high-resolution structure of this state.
Denise Hilton (Bruce Goode lab, Brandeis University, Waltham, MA) sought to understand how capping protein functions when there is an abundance of known capping antagonists, such as V1 and CARMIL, present in cells. TIRF microscopy and anisotropy binding assays were used to demonstrate that the protein Twinfilin binds to capping protein with high affinity and antagonizes the inhibitory effects of CARMIL and V1. This “pro-capping” biochemical activity of Twinfilin was consistent with the phenotypes observed in Twinfilin-depleted cells.
Kaitlin Homa (David Kovar lab, University of Chicago, Chicago, IL) discussed the role of the actin assembly factors formin and Arp2/3 complex in sorting the actin-binding proteins fimbrin and tropomyosin to distinct actin networks in fission yeast. With the use of in vitro reconstitutions with purified proteins, competition between fimbrin and tropomyosin was found to be necessary for the sorting of fimbrin to actin filaments assembled by Arp2/3 complex and for tropomyosin recruitment to actin filaments assembled by formin. Complementary work in fission yeast cells showed that the actin-binding proteins also exhibited preferences for specific F-actin networks mediated by distinct assembly factors.
Nicholas Boyer (Stephanie Gupton lab, University of North Carolina, Chapel Hill) demonstrated that the brain-enriched E3 ubiquitin ligase TRIM67 regulates the function of the actin polymerase VASP and the regulation of growth cone filopodia in embryonic cortical neurons. Genetic deletion of Trim67 resulted in a surprising increase in VASP ubiquitination, aberrant filopodia and growth responses to the axon guidance cue netrin, a lack of netrin-dependent axon branching, and failure of the axon to turn in a gradient of netrin-1, as revealed by live cell microscopy approaches, ubiquitination assays, and microfluidic axon turning assays.
Yosuke Senju (Pekka Lappalainen lab, University of Helsinki, Helsinki, Finland) reported the molecular mechanisms by which ABPs such as profilin, cofilin, Dia2, N-WASP, ezrin, and moesin interact with PI(4,5)P2-rich membranes. A combination of biochemical, biophysical, and molecular dynamics simulation approaches showed that although these ABPs interact with PI(4,5)P2-rich membranes through multivalent electrostatic interactions, they exhibit differences in the affinities and dynamics of membrane interactions. These distinct membrane-interaction kinetics correlate with specific functions of these ABPs in cytoskeletal dynamics.
Kenneth Campellone (University of Connecticut, Storrs, CT) described his lab's recent work investigating the molecular and cellular basis of an inherited disease called Amish Galloway-Mowat Syndrome (GMS). Patients with this disorder encode truncated forms of the actin nucleation factor WHAMM that render it incapable of promoting actin assembly, and cells from these individuals exhibited cytoskeletal irregularities and defects in autophagy. Inactivation of WHAMM in healthy cell lines inhibited lipidation of the autophagosomal protein LC3 and the clearance of protein aggregates. Normal WHAMM function required binding to the phospholipid PI(3)P at sites of autophagosome biogenesis.
Valentin Jaumouillé (Clare Waterman lab, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD) reported on the mechanical coupling of β2 integrins to the actin cytoskeleton during macrophage phagocytosis of complement-opsonized particles. Live cell imaging demonstrated that Arp2/3-dependent protrusion drove the formation of the phagocytic cup and that retrograde F-actin flow slowed in the newly formed cup. Slower flow coincided with recruitment of β2 integrins and several adhesion components to the phagocytic site. Traction force microscopy and the sensitivity of phagocytosis to inhibition of specific tyrosine kinases supported the conclusion that the focal adhesion–lamellipodium machinery is coopted during phagocytosis of large particles.
Seham Ebrahim (Richard Weigert lab, National Cancer Institute, National Institutes of Health, Bethesda, MD) examined how large secretory granules in mouse exocrine glands fused with narrow tube-like canaliculi at the plasma membrane. Intravital subcellular microscopy revealed that granules were encased in a geodesic lattice with triskelia-like tripolar vertices. STED microscopy further showed that the lattice contained distinct cages of F-actin, myosin II, and septins. Depolymerization of actin with cytochalasin enlarged the granules, but the septin and myosin II lattices remained. Myosin II recruitment appeared to be dependent on septins, suggesting novel roles for these factors in remodeling vesicles in vivo.
Susumu Antoku (Gregg Gundersen lab, Columbia University, New York) sought to understand how the formin FHOD1 interacts with nesprin-2G and regulates the formation of transmembrane actin-associated nuclear (TAN) lines and nuclear movement in fibroblasts. Using purified recombinant proteins, the spectrin repeats within nesprin-2G that interact with FHOD1 were shown to promote an actin bundling activity and enhance an anti-polymerization activity of FHOD1. These studies also uncovered a novel actin-binding site within FHOD1 required for actin bundling. The phosphorylation of FHOD1 was further hypothesized to regulate actin bundling.