Actin organization and dynamics: novel regulatory mechanisms from the biophysical to the tissue level

Properly regulated actin organization and dynamics are crucial for cellular processes such as cell migration, cell division, and maintenance of cell–cell junctions. Speakers in the “Actin Organization and Dynamics” Minisymposium at the ASCB 2012 meeting highlighted new and interesting mechanisms that underlie the regulation of actin dynamics. The session was broad in scope, ranging from in vitro biochemical and biophysical studies with purified components to cellular- and tissue-level studies. There was an emphasis on severing mechanisms and mechanics, as well as the complexities of regulatory pathways in cells. Hyeran Kang (Yale University), a postdoc with Enrique M. De La Cruz, presented work on how cations affect filament mechanics and severing via the regulatory protein, cofilin. Kang identified, using structural bioinformatics, two potential filament-specific cation-binding sites and classified them as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent assembly and bending mechanics, respectively. Cofilin enhances actin filament bending and twisting compliance, and it is hypothesized that local mechanical discontinuities in partially decorated filaments promote severing at boundaries of bare and cofilin-decorated segments. Using native and engineered yeast actin mutants, Kang showed that dissociation of stiffness site cations by human cofilin drives changes in filament bending mechanics and that this is required for severing. Pinar Gurel (Geisel School of Medicine at Dartmouth), a graduate student with Harry Higgs, described research investigating the severing activity of INF2, a formin linked to two human genetic diseases: Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis. Using a combination of classic biochemical experiments and total internal reflection fluorescence (TIRF) microscopy, Gurel showed that, in addition to the normal formin binding site at the barbed end, INF2 binds the sides of actin filaments stoichiometrically (one INF2 per subunit) and with submicromolar affinity. INF2-catalyzed filament severing occurs within INF2-decorated regions of partially and fully decorated filaments. This property, along with INF2’s ability to bind ADP-Pi filaments, distinguishes INF2-mediated filament severing from that of cofilin. Dennis Breitsprecher (Brandeis University), a postdoc with Bruce Goode, presented recent work demonstrating a new role for Srv2/cyclase-associated protein. Breitsprecher observed, using dual-color TIRF microscopy, that the N-terminal half of Srv2 (N-Srv2) increases the severing activity of cofilin without appreciably affecting cofilin binding to filaments. Electron microscopy and single-particle analysis revealed that N-Srv2 self-associates into hexameric ring structures. N-terminal truncations weaken N-Srv2 oligomerization, compromise enhanced severing activity, and yield actin organization and cell polarity defects in cells. Peter Bieling (University of California, San Francisco), a postdoc with Dyche Mullins and Dan Fletcher, evaluated how reconstituted dendritic actin network growth responds to external loads. Dendritic actin networks assembled in vitro from micron-sized patterns of surface-immobilized nucleation-promoting factor were challenged with force applied via an atomic force microscope (AFM) cantilever, and actin assembly was monitored using multicolor TIRF microscopy and AFM. Bieling demonstrated that the dendritic actin networks dynamically adapt to external forces; the actin network growth velocity decreases with load, while actin density in the force-generating region of the network increases with load. Barbed-end capping is force-dependent and slows with increasing load. Colleen G. Bilancia (University of North Carolina, Chapel Hill), a postdoc with Mark Peifer, presented work on how Enabled (Ena) and Diaphanous (Dia) coordinate to regulate actin filament barbed-end polymerization. Overexpressing active Dia in Drosophila D16 cells promotes long, stable filopodial protrusions, while overexpressing Ena leads to dynamic, fan-like protrusions. Coexpression of Dia and Ena promotes protrusions distinct from those produced with expression of either regulator independently. Bilancia noted that filopodia retract when active Dia and Ena colocalize, suggesting that Ena negatively regulates Dia, possibly through direct binding interactions. Indeed, Bilancia showed that the Ena EVH1 domain directly binds the Dia FH1 domain, and the EVH1 domain inhibits Dia-mediated in vitro pyrene actin assembly and filopodia formation in cells. Ann Miller (University of Michigan) described her lab's finding that the cytokinesis regulator anillin plays a novel role in regulating epithelial cell–cell junctions. Using Xenopus embryos as a model system, Miller showed that a population of anillin localizes at cell–cell junctions in both dividing and nondividing cells. Further, anillin functionally regulates junctional integrity. Both tight junctions and adherens junctions are disrupted when anillin is knocked down. Anillin binds actin filaments and the small GTPase RhoA, both of which regulate cell–cell junction structure and function. Miller's group tested the effects of anillin expression on RhoA activation and found that anillin knockdown results in increased spontaneous flares of active RhoA that are prominent at cell–cell junctions in both dividing cells and nondividing regions of the epithelium.