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Identification of cation binding sites on actin that control polymerization, bending stiffness and severing by vertebrate cofilin.
H. Kang1, M. J. Bradley1, B. R. McCullough1, A. Pierre2, E. E. Grintsevich3, E. Reisler3, E. M. De La Cruz1; 1MB&B, Yale University, New Haven, CT, 2Département de Physique, ENS de
Cachan, Cachan, France, 3Chemistry & Biochemistry, UCLA, Los Angeles, CA
The regulation of actin assembly and modulation of filament mechanical properties are critical for actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve non- specific ionic strength effects or specific ion binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. Cofilin binding is coupled to dissociation of filament-associated cations, stiffness site reorganization and enhanced actin filament bending and twisting dynamics, consistent with displacement of stiffness site cations underlying the effects of cofilin on actin filament mechanics. The work presented reveals the molecular mechanism of salt-dependent actin assembly and cofilin-mediated changes in actin filament bending mechanics and severing. 148
The formin INF2 severs actin filaments through a fundamentally different mechanism from cofilin: relating biochemical function to cellular activity.
P. S. Gurel1, H. N. Higgs1; 1Biochemistry, Geisel School of Medicine at Dartmouth College, Hanover, NH
Formins are a class of proteins that accelerate actin nucleation, then influence filament elongation rate by remaining at the barbed end. INF2 is a biochemically unique mammalian formin in that it accelerates both actin polymerization and depolymerization. Importantly, mutations in INF2 lead to the kidney disease focal and segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot-Marie Tooth Disease (CMTD). I am elucidating the molecular mechanism of INF2’s unique depolymerization activity, using TIRF microscopy and other biochemical assays of actin dynamics. Prior work in our lab has shown that depolymerization requires both the FH2 and WH2/DAD sequences, and occurs in two steps: a) a severing step, which requires phosphate release from the actin subunits; and b) a depolymerization step, which requires the WH2/DAD. My work reveals the following mechanistic features of INF2-mediated severing/depolymerization. First, rapid severing occurs throughout the length of the filament and not progressively from the barbed end. This suggests that INF2 binds filament sides prior to severing and accelerates phosphate release of filaments since it is capable of severing filament segments less than 50s after monomer addition. Second, INF2 can bind filament sides that are both phosphate-bound and phosphate-free, but with different stoichiometries. Additionally, INF2 can alter filament flexibility in the phosphate- free state. These results suggest that phosphate release causes a conformational change to
filament-bound INF2, allowing severing. Lastly, INF2 bound at the barbed end causes catastrophic disassembly of short filaments. Based on these results, I postulate that both filament side binding and barbed end binding by the FH2 domain changes filament architecture, which promotes severing and changes monomer dynamics at filament ends. I postulate further that the WH2/DAD enhances severing by insertional binding between actin subunits in the filament. These results contrast in several aspects with the severing mechanism employed by cofilin. This mechanistic investigation of INF2 severing/depolymerization activity gives insight on recent cellular results from our lab, which reveal a role for INF2-mediated actin dynamics in fission of both Golgi and mitochondria.
149
Srv2/CAP forms six-bladed throwing stars that directly catalyze actin filament severing and disassembly.
F. Chaudhry1, D. Breitsprecher1, K. Little1, O. Sokolova2, B. L. Goode1; 1Biology, Brandeis University, Waltham, MA, 2Moscow State University, Moscow, Russia
Actin filament severing is a critical step in the dynamic turnover of cellular actin networks. Cofilin is sufficient to sever filaments, but it has long been thought that additional factors may be required to enhance severing and account for very high rates of filament turnover observed in vivo. Srv2/CAP (cyclase-associated protein) is a conserved and widely expressed regulator of the actin cytoskeleton that until now has been proposed to function exclusively in binding and recycling actin monomers. Further, these functions have been ascribed to domains found in its C-terminal half (C-Srv2). Here, we have unveiled a new and unanticipated role for Srv2/CAP in directly catalyzing cofilin-mediated severing of actin filaments. This function is mediated by N- Srv2, and is physically and genetically separable from C-Srv2 function in actin monomer recycling. Using dual-color TIRF microscopy, we observed that N-Srv2 promotes filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. To address the structural basis of these effects, we used electron microscopy and single particle analysis, which revealed that N-Srv2 forms novel hexameric structures resembling ninja throwing stars. An N-terminal truncation that disrupts Srv2 oligomerization severely impaired enhanced severing activity, and led to striking defects in cellular actin organization and cell polarity. In addition, genetic analysis revealed that the activities of N-Srv2 but not C-Srv2 are essential in vivo in combination with those of another cofilin co-factor, Aip1. Together, our results define a new cellular role for Srv2/CAP in directly stimulating filament severing and disassembly, and demonstrate that the activity of cofilin alone, while essential, is not sufficient for cell viability, and that additional severing co-factors are critical.
150
The mechanobiochemistry of dendritic actin network assembly.
P. Bieling1,2, T-D. Li2, R. D. Mullins1, D. A. Fletcher2; 1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 2Department of Bioengineering, University of California, Berkeley, Berkeley, CA
Branched actin networks generate protrusive forces required for cell motility and movement of sub-cellular structures. Despite advances in our biochemical knowledge of dendritic actin network assembly, very little is known about how dynamic networks of close-to-physiological architecture respond to mechanical stimuli at the molecular level. We combined micropatterning with atomic force microscopy (AFM) and multi-color TIRF imaging to visualize the active, force- generating region of in vitro reconstituted actin networks in a biochemically and mechanically
defined environment. Our measurements demonstrate that Arp2/3-generated actin networks are highly sensitive to load, particularly at low forces. The network growth velocity decreases exponentially with counteracting forces, while the actin density in the force-generating region of the network increases strongly with elevated loads. This shows that net actin polymerization decreases less drastically than the growth velocity. In addition, the levels of network-associated capping protein increase in proportion to the amount of actin polymer in the network, indicating that average filament length is determined solely by the kinetics of capping protein association and is insensitive to applied force. Our AFM-TIRF measurements provide new insight into the role of capping, branching, and filament elongation in force-dependent changes in dendritic actin network assembly.
151
Enabled negatively regulates diaphanous-driven actin dynamics.
C. G. Bilancia1, J. D. Winkelman2, S. H. Nowotarski1, D. Tsygankov3, J. A. Sees2, T. Elston3, D. R. Kovar2, M. Peifer1; 1Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2
Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL, 3Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC
Cell protrusions are an essential component of dynamic cell movement during development and disease. Actin regulatory proteins control the architecture and dynamics of cell protrusions, forming branched actin networks for lamellipodia or unbranched actin bundles for filopodia. While many actin regulators have been identified and characterized, it remains unclear why cells need multiple actin regulators with similar functions and how this network of proteins works together to create the diverse cell protrusions seen in normal development. Enabled (Ena) and Diaphanous (Dia) provide a superb model, as both can act at the barbed end of actin filaments to promote polymerization of unbranched filaments. We previously found that Ena and Dia form a protein complex, and discovered they act in complex, non-additive ways to shape the balance of lamellipodia and filopodia in migrating epidermal cells. We hypothesized that Ena and Dia directly bind each other and this binding regulates their activity. Through yeast two-hybrid and GST pull-downs, we found that Ena′s EVH1 domain binds to Dia′s proline-rich FH1 domain. To test how this Dia:Ena binding interaction affects cell protrusions, we are using three parallel model sytems: in vitro actin assembly assays, Drosophila D16 cells, and Drosophila embryos. To compare protrusive behavior, we developed a software program to measure cell protrusion dynamics, including filopodia number, length, and stability, and lamellipodial protrusion. Our data reveal that while both Dia and Ena drive ectopic filopodia, these have distinct properties and dynamics. Consistent with our earlier in vivo experiments, together they act in non-additive ways. The EVH1 domain is sufficient to reduce the number of ectopic filopodia driven by activated Dia, suggesting that Dia:Ena binding is a negative regulatory mechanism. To test this hypothesis, we performed pyrene-labeled actin assembly assays and found that EVH1 reduces actin assembly by Dia′s FH1FH2 domains. We are now using SNAP-tagged versions of activated Dia and Ena in TIRF assays to visualize their interactions with actin filaments and each other to understand their mechanism of controlling actin dynamics. We also are testing this hypothesis by examining the functional interactions of Ena EVH1 mutants and Dia FH1 mutants that block Dia:Ena interactions, to address whether this negative regulatory mechanism requires direct binding. Together, these assays will provide insight into how similar actin regulators interact to tune cell behavior.
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Anillin regulates junctional integrity and RhoA activation at cell-cell junctions in the intact epithelium.
C. C. Reyes1, M. Jin2, A. L. Miller1,2; 1Graduate Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI, 2Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI
My lab is focused on studying the molecular mechanisms by which Rho family small GTPases regulate cytokinesis in the context of the intact epithelium. Understanding this process is important because successful cytokinesis is essential for proper development, and cytokinesis failure can contribute to tumor formation. Anillin is a contractile ring component that is required for cytokinesis in several model organisms. Anillin can link multiple components of the contractile ring including F-actin, Myosin-2, and Septins with the plasma membrane, and it scaffolds a number of other proteins including the small GTPase RhoA and the RhoA regulator MgcRacGAP. While Anillin is important for stable furrow positioning in isolated cultured cells, little is known about Anillin’s function during cytokinesis in multicellular organisms in vivo or whether it plays functional roles outside of cytokinesis. Here, we used Xenopus laevis embryos as a model system to examine the role of Anillin in regulating cytokinesis in the intact epithelium. We find that a population of Anillin is localized at cell-cell junctions throughout the cell cycle and is particularly enriched at cell-cell junctions of dividing cells. Anillin functionally regulates cell-cell junctions; both tight junctions and adherens junctions are disrupted in Anillin knock down embryos. Additionally, we observe increased intercellular spaces between cells in Anillin knock down embryos. When control embryos are mounted in fluorescent dextran, the dextran cannot penetrate into intercellular spaces. However, in Anillin knock down embryos, dextran can penetrate, indicating that the epithelial barrier function is compromised. Because Anillin is reported to interact with both RhoA and MgcRacGAP, we tested the effect of knocking down Anillin on RhoA activity at cell-cell junctions. Surprisingly, Anillin knock down results in increased spontaneous flares of RhoA activity at cell-cell junctions. These RhoA activity flares are prominent at cell-cell junctions in both dividing cells and non-dividing regions of the epithelium. These results reveal a novel role for Anillin in regulating cell-cell junctions likely via RhoA. We propose that Anillin is required to properly distribute cortical tension and RhoA activity in order to maintain cell-cell junctions. This function is particularly important during the process of cytokinesis in an epithelial context when the dividing cell must maintain and remodel cell-cell junctions with neighboring cells as well as manage the contractile forces and shape changes associated with cytokinesis.