Zyxin-VASP interactions alter actin regulatory activity in zyxin-VASP complexes

Cell-cell and cell-substrate adhesions are sites of dramatic actin rearrangements and where actin-membrane connections are tightly regulated. Zyxin-VASP complexes localize to sites of cell-cell and cell-substrate adhesion and function to regulate actin dynamics and actin-membrane connections at these sites. To accomplish these functions, zyxin recruits VASP to cellular sites via proline-rich binding sites near zyxin’s amino terminus. While the prevailing thought has been that zyxin simply acts as a scaffold protein for VASP binding, the identification of a LIM domain-VASP interaction could complicate this view. Here we assess how zyxin-VASP binding through both the proline rich motifs and the LIM domains alters specific VASP functions. We find that neither individual interaction alters VASP’s actin regulatory activities. In contrast, however, we find that full-length zyxin dramatically reduces VASPmediated actin bundling and actin assembly. Taken together, these results suggest a model where zyxin-VASP complexes occur in complex organizations with suppressed actin regulatory activity.     

Paramyxovirus membrane protein enhances antibody production to new antigenic determinants in the actin molecule: a model for virus-induced autoimmunity.

Paramyxovirus membrane (M) protein specifically binds to cellular actin but not to bovine serum albumin or myoglobin, as determined by affinity chromatography and enzyme-linked immunosorbent assay. The binding site for M protein on actin is different from the binding sites for antiactin antibodies. The interaction of M protein with actin resulted in production of antibodies to several new antigenic sites on the actin molecule. Five rabbits immunized with actin alone produced antibodies against the N-terminal sequence (residues 1 to 39). Another five rabbits immunized with a mixture of M protein and actin produced antibodies against a C-terminal fragment and a central region as well as the N-terminal fragment. By immunoblotting with proteolytic fragments of actin, the new antigenic sites were located between amino acid residues 40 to 113, 114 to 226, and 227 to 375. Antisera taken from some patients with recent measles virus infections demonstrated antiactin antibodies to sites other than the N-terminal fragment of actin (amino acids 1 to 39). The interaction of paramyxovirus M protein with actin and the subsequent production of antibodies to new antigenic sites may serve as a model for one of the mechanisms of virus-induced autoimmunity.     

Studies on the topographical localization of the binding sites for substrate and for actin on the enzymes, glyceraldehydephosphate dehydrogenase and phosphofructokinase

The effect of proteolysis on the catalytic activity and the binding capacity for actin has been studied in the case of both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase (PFK). With both of these enzymes, the differential response of these two parameters is interpreted as an indication of the distinct topographical separation of the active sites and binding sites. These results have been discussed in relation to the positioning of the catalytic and binding sites on these enzymes, the nature of their interaction with actin, their relative stability in cellular situations and the phenomenon of enzyme ambiguity.     

Cortactin branches out: roles in regulating protrusive actin dynamics

Since its discovery in the early 1990's, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures.     

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.     

Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.     

IQGAP1 regulates Salmonella invasion through interactions with actin, Rac1, and Cdc42

To infect host cells, Salmonella utilizes an intricate system to manipulate the actin cytoskeleton and promote bacterial uptake. Proteins injected into the host cell by Salmonella activate the Rho GTPases, Rac1 and Cdc42, to induce actin polymerization. Following uptake, a different set of proteins inactivates Rac1 and Cdc42, returning the cytoskeleton to normal. Although the signaling pathways allowing Salmonella to invade host cells are beginning to be understood, many of the contributing factors remain to be elucidated. IQGAP1 is a multidomain protein that influences numerous cellular functions, including modulation of Rac1/Cdc42 signaling and actin polymerization. Here, we report that IQGAP1 regulates Salmonella invasion. Through its interaction with actin, IQGAP1 co-localizes with Rac1, Cdc42, and actin at sites of bacterial uptake, whereas infection promotes the interaction of IQGAP1 with both Rac1 and Cdc42. Knockdown of IQGAP1 significantly reduces Salmonella invasion and abrogates activation of Cdc42 and Rac1 by Salmonella. Overexpression of IQGAP1 significantly increases the ability of Salmonella to enter host cells and required interaction with both actin and Cdc42/Rac1. Together, these data identify IQGAP1 as a novel regulator of Salmonella invasion.     

Dynein binds to beta-catenin and may tether microtubules at adherens junctions

Interactions between microtubule and actin networks are thought to be crucial for mechanical and signalling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions. Here, we report that dynein is localized to the cortex at adherens junctions in cultured epithelial cells and that this localization is sensitive to drugs that disrupt the actin cytoskeleton. Dynein is recruited to developing contacts between cells, where it localizes with the junctional proteins beta-catenin and E-cadherin. Microtubules project towards these early contacts and we hypothesize that dynein captures and tethers microtubules at these sites. Dynein immunoprecipitates with beta-catenin, and biochemical analysis shows that dynein binds directly to beta-catenin. Overexpression of beta-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing beta-catenin, the centrosome becomes disorganized and microtubules no longer appear to be anchored at the cortex. These results identify a novel role for cytoplasmic dynein in capturing and tethering microtubules at adherens junctions, thus mediating cross-talk between actin and microtubule networks at the cell cortex.     

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α_1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β₃ subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.     

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.     

Evidence for an interaction between the membrane protein of a paramyxovirus and actin.

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.     

Localization and identification of actin structures involved in the filamin-actin interaction.

The interface between gizzard filamin and skeletal muscle actin was located on the actin monomer. Conserved sequences 105-120 and 360-372, in the actin subdomain 1 near the myosin binding sites, were involved in this interaction. The corresponding peptides for these sequences were each found to bind filamin and compete in the actin-filamin interaction. When these two peptides were used together in the presence of filamin and filamentous actin, they dissociated sedimentable complexes formed by these two proteins.     

Conversion of mechanical force into biochemical signaling

Physical forces play important roles in regulating cell proliferation, differentiation, and death by activating intracellular signal transduction pathways. How cells sense mechanical stimulation, however, is largely unknown. Most studies focus on cellular membrane proteins such as ion channels, integrins, and receptors for growth factors as mechanosensory units. Here we show that mechanical stretch-induced c-Src protein tyrosine kinase activation is mediated through the actin filament-associated protein (AFAP). Distributed along the actin filaments, AFAP can directly active c-Src through binding to its Src homology 3 and/or 2 domains. Mutations at these specific binding sites on AFAP blocked mechanical stretch-induced c-Src activation. Therefore, mechanical force can be transmitted along the cytoskeleton, and interaction between cytoskeletal associated proteins and enzymes related to signal transduction may convert physical forces into biochemical reactions. Cytoskeleton deformation-induced protein-protein interaction via specific binding sites may represent a novel intracellular mechanism for cells to sense mechanical stimulation.     

Cellular actin is affected by interaction with Candida albicans

Attachment of Candida albicans, an important opportunistic pathogen, to host tissues is an initial step in the development of the infection. The events occurring in the fungal and in the host cells after interaction are poorly understood. In this study we concentrated on the events occurring in the mammalian cells after the interaction with Candida, with emphasis on the cytoskeleton actin. Human cell line cells (HEp2) were exposed to C. albicans or C. albicans-secreted material (culture filtrate) (actin-rearranging Candida-secreted factor, arcsf). The HEp2 cells were examined for cellular changes using confocal laser microscopy (CLSM), transmission and scanning electron microscopy (TEM and SEM). The CLSM studies, using fluorescein isothiocyanate-labeled C. albicans and rhodamine phalloidin actin staining, revealed yeasts adhering to the HEp2 cells or internalized into the cells, with actin surrounding the fungi. Furthermore, actin rearrangement from filamentous network to actin aggregates was noticed. Interaction between the HEp2 cells and C. albicans could be demonstrated also by SEM and TEM after a 2-4-h exposure of the cells to the fungus. Yeasts and hyphae were found attaching to the surface and within the cells. CLSM studies revealed that exposure of HEp2 cells to arcsf was also followed by cellular actin rearrangement, reduced membrane ruffling and decreased cellular motility. The effect was dose- and time-dependent. All these data indicate that the interaction of Candida with HEp2 cells involves signaling events and affects the cellular actin.     

Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton

Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro. Little is known about MAP2 regulation or its interaction with the cytoskeleton in vivo. Here we investigate the in vivo function of three specific sites of phosphorylation on MAP2. cAMP-dependent protein kinase activity disrupts the MAP2-microtubule interaction in living HeLa cells and promotes MAP2c localization to peripheral membrane ruffles enriched in actin. cAMP-dependent protein kinase phosphorylates serines within three KXGS motifs, one within each tubulin-binding repeat. These highly conserved motifs are also found in homologous proteins tau and MAP4. Phosphorylation at two of these sites was detected in brain tissue. Constitutive phosphorylation at these sites was mimicked by single, double, and triple mutations to glutamic acid. Biochemical and microscopy-based assays indicated that mutation of a single residue was adequate to disrupt the MAP2-microtubule interaction in HeLa cells. Double or triple point mutation promoted MAP2c localization to the actin cytoskeleton. Specific association between MAP2c and the actin cytoskeleton was demonstrated by retention of MAP2c-actin colocalization after detergent extraction. Specific phosphorylation states may enhance the interaction of MAP2 with the actin cytoskeleton, thereby providing a regulated mechanism for MAP2 function within distinct cytoskeletal domains.     

Enhancement of actin-depolymerizing factor/cofilin-dependent actin disassembly by actin-interacting protein 1 is required for organized actin filament assembly in the Caenorhabditis elegans body wall muscle

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal beta-propeller, whereas one residue is located in the C-terminal beta-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.     

The binding of Ca2+ to actin monomer is monitored by the fluorescence of actin-bound auramine O.

The fluorescence of the cation auramine O was substantially enhanced by the presence of actin monomer. Titrations of this fluorescence enhancement indicated that actin monomer had two auramine O binding sites, each with a dissociation constant of approx. 20 microM. Calcium ions had no effect on the number of actin monomer-bound auramine O molecules or on the dissociation constant for that interaction. However, calcium ions increased the maximum change of fluorescence that occurs when actin monomer was fully saturated with auramine O. This effect of calcium was saturable and yielded a Ca2+ dissociation constant of 1.6 mM. It was concluded that auramine O bound to sites on actin monomer and independently monitored the binding of Ca2+ ion(s) to other site(s) on actin monomer. Further, the magnitude of the Ca2+ dissociation constant suggested that this Ca2+-binding site may be representative of the multiple bivalent cation-binding sites on actin monomer which are thought to be directly involved in actin polymerization. However, the exact relationship between these sites remains unclear.     

Dynamin at the actin-membrane interface

Many important cellular processes such as phagocytosis, cell motility and endocytosis require the participation of a dynamic and interactive actin cytoskeleton that acts to deform cellular membranes. The extensive family of non-traditional myosins has been implicated in linking the cortical actin gel with the plasma membrane. Recently, however, the dynamins have also been included in these cell processes as a second family of mechanochemical enzymes that self-associate and hydrolyze nucleotides to perform 'work' while linking cellular membranes to the actin cytoskeleton. The dynamins are believed to form large helical polymers from which extend many interactive proline-rich tail domains, and these domains bind to a variety of SH3-domain-containing proteins, many of which appear to be actin-binding proteins. Recent data support the concept that the dynamin family might act as a 'polymeric contractile scaffold' at the interface between biological membranes and filamentous actin.     

Protein phosphatase-1 binding to scd5p is important for regulation of actin organization and endocytosis in yeast

SCD5, an essential gene, encodes a protein important for endocytosis and actin organization in yeast. Previous two-hybrid screens showed that Scd5p interacts with Glc7p, a yeast Ser/Thr-specific protein phosphatase-1 (PP1) that participates in a variety of cellular processes. PP1 substrate specificity in vivo is regulated by association with different regulatory or targeting subunits, many of which have a consensus PP1-binding site ((V/I)XF, with a basic residue at the -1 or -2 position). Scd5p contains two of these potential PP1-binding motifs: KVDF (amino acids 240-243) and KKVRF (amino acids 272-276). Deletion analysis mapped the PP1-binding domain to a region of Scd5p containing these motifs. Therefore, the consequence of mutating these two potential PP1-binding sites was examined. Although mutation of KVDF had no effect, alteration of KKVRF dramatically reduced Scd5p interaction with Glc7p and resulted in temperature-sensitive growth. Furthermore, this mutation caused defects in fluid phase and receptor-mediated endocytosis and actin organization. Overexpression of GLC7 suppressed the temperature-sensitive growth of the KKVRF mutant and partially rescued the actin organization phenotype. These results provide evidence that Scd5p is a PP1 targeting subunit for regulation of actin organization and endocytosis or that Scd5p is a PP1 substrate, which regulates the function of Scd5p in these processes.