scholarly journals The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains

2001 ◽  
Vol 153 (7) ◽  
pp. 1479-1498 ◽  
Author(s):  
Goeh Jung ◽  
Kirsten Remmert ◽  
Xufeng Wu ◽  
Joanne M. Volosky ◽  
John A. Hammer
Keyword(s):  
Fusion Proteins ◽  
Leading Edge ◽  
Fluid Phase ◽  
Actin Binding ◽  
Type I ◽  
Sh3 Domains ◽  
Capping Protein ◽  
Myosin I ◽  
Mutant Phenotypes

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.

scholarly journals Capping protein binding to actin in yeast

2004 ◽  
Vol 164 (4) ◽  
pp. 567-580 ◽  
Author(s):  
Kyoungtae Kim ◽  
Atsuko Yamashita ◽  
Martin A. Wear ◽  
Yuichiro Maéda ◽  
John A. Cooper
Keyword(s):  
Actin Filaments ◽  
Physiological Function ◽  
Binding Constants ◽  
Binding Activity ◽  
Actin Binding ◽  
Null Mutant ◽  
Capping Protein ◽  
Mutant Phenotypes ◽  
Actin Capping

The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined. The CP crystal structure suggests that the COOH-terminal regions of the CP α and β subunits bind to the barbed end. Using purified recombinant mutant yeast CP, we tested this model. CP lacking both COOH-terminal regions did not bind actin. The α COOH-terminal region was more important than that of β. The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes. Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP. Actin filaments of patches appear to be nucleated first, then capped with CP. The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains

1995 ◽  
Vol 108 (12) ◽  
pp. 3775-3786 ◽  
Author(s):  
C. Ruppert ◽  
J. Godel ◽  
R.T. Muller ◽  
R. Kroschewski ◽  
J. Reinhard ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Brush Border ◽  
Subcellular Fractionation ◽  
Actin Binding ◽  
Resting Cells ◽  
Medium Density ◽  
Subcellular Targeting ◽  
Myosin I ◽  
Punctate Pattern

Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin 1. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In subcellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density. The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are sufficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper subcellular localization.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

scholarly journals LIM and SH3 protein 1 localizes to the leading edge of protruding lamellipodia and regulates axon development

2020 ◽  
Vol 31 (24) ◽  
pp. 2718-2732
Author(s):  
Stephanie L. Pollitt ◽  
Kenneth R. Myers ◽  
Jin Yoo ◽  
James Q. Zheng
Keyword(s):  
Hippocampal Neurons ◽  
Binding Protein ◽  
Leading Edge ◽  
Actin Binding ◽  
Commissural Axon ◽  
Actin Binding Protein ◽  
Axon Elongation ◽  
Axon Development

This study reports that the actin-binding protein, LIM and SH3 Protein 1 (LASP1), regulates actin-based protrusions underlying axon elongation and branching in hippocampal neurons in culture. LASP1 also plays an important role in axon development in vivo, as loss of the Drosophila homologue LASP disrupts the commissural axon development.

Interaction of Multimerin 1 with Collagens: Role in Platelet Adhesion

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2207-2207
Author(s):  
Subia Tasneem ◽  
Monika Pawlikowska ◽  
Dominique Bihan ◽  
Richard W. Farndale ◽  
Catherine P. M. Hayward
Keyword(s):  
Platelet Adhesion ◽  
Type I Collagen ◽  
Fluid Phase ◽  
Type I ◽  
Binding Assays ◽  
P Values ◽  
Binding Peptide ◽  
Collagen Iii ◽  
Adhesion Assays

Abstract Abstract 2207 Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein stored in platelets and endothelium that binds to activated platelets, endothelial cells and the extracellular matrix after agonist stimulation. MMRN1 supports platelet adhesion by von Willebrand factor (VWF) dependent and independent mechanisms, and also increases platelet adhesion to Horm collagen. Mice deficient in Mmrn1/Snca (α-synuclein) showed defective platelet adhesion to collagen in vitro and in vivo which was corrected by MMRN1. The ability of MMRN1 to support platelet adhesion in vivo and enhance platelet adhesion to collagen ex vivo, led us to explore the molecular basis of MMRN1 interactions with collagen. Solid phase binding assays were used to test MMRN1 binding to human fibril forming collagen (types I, II, III and V) and to types IV and VI collagen. Binding assays were also used to map the MMRN1 binding sites on collagen using collagen peptide toolkits III and II (which has significant similarity to type I collagen). Static adhesion assays were used to test selected collagen peptides for platelet adhesion. Platelet adhesion assays at high shear rates 1500s−1, were used to test the adhesion of washed platelets from normal and VWF-deficient subjects to type I collagen pre-treated or in fluid phase with: BSA (negative control), MMRN1, VWF or their combination. Human collagens types I, II, III and VI (p-values <0.001) but not types IV or V (p-values = 0.84 and 0.09, respectively) supported MMRN1 binding. Peptide toolkits binding studies indicated that MMRN1 bound to a single site on collagen III (peptide III-38) and to two sites on collagen II, with peptide II-9 showing much stronger binding than peptide II-44. Like the VWF binding peptide III-23 (which did not overlap the MMRN1 binding site), peptide III-38 supported platelet adhesion in combination with GFOGER, the peptide with high affinity for platelet α2β1. The possibility that MMRN1 binds to collagen at sites distinct from VWF was supported by the observations that pre-treatment of collagen I matrices, with the combination of MMRN1 and VWF, increased platelet adhesion more than MMRN1 or VWF alone (p-values< 0.001). Moreover, adhesion deficit of VWF-deficient platelets on collagen type I matrix pre-treated with a combination of MMRN1 and VWF was corrected by adding fluid phase VWF but not MMRN1 (p-values < 0.0001). Taken together, our data indicates that MMRN1 binds to different forms of human collagen that support platelet adhesion. As MMRN1 binds to sites on collagen distinct from VWF or integrin α2β1, it may be important for maximizing platelet adhesion at sites of vascular injury. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.

1996 ◽  
Vol 133 (2) ◽  
pp. 305-323 ◽  
Author(s):  
G Jung ◽  
X Wu ◽  
J A Hammer
Keyword(s):  
Constitutive Expression ◽  
Leading Edge ◽  
Fluid Phase ◽  
Absolute Amount ◽  
Tail Domain ◽  
Myosin I ◽  
Cortical Regions ◽  
Simple Notion ◽  
Kinetics Of ◽  
Doubling Times

Dictyostelium cells that lack the myoB isoform were previously shown to exhibit reduced efficiencies of phagocytosis and chemotactic aggregation ("streaming") and to crawl at about half the speed of wild-type cells. Of the four other Dictyostelium myosin I isoforms identified to date, myoC and myoD are the most similar to myoB in terms of tail domain sequence. Furthermore, we show here that myoC, like myoB and myoD, is concentrated in actin-rich cortical regions like the leading edge of migrating cells. To look for evidence of functional overlap between these isoforms, we analyzed myoB, myoC, and myoD single mutants, myoB/myoD double mutants, and myoB/myoC/myoD triple mutants, which were created using a combination of gene targeting techniques and constitutive expression of antisense RNA. With regard to the speed of locomoting, aggregation-stage cells, of the three single mutants, only the myoB mutant was significantly slower. Moreover, double and triple mutants were only slightly slower than the myoB single mutant. Consistent with this, the protein level of myoB alone rises dramatically during early development, suggesting that a special demand is placed on this one isoform when cells become highly motile. We also found, however, that the absolute amount of myoB protein in aggregation-stage cells is much higher than that for myoC and myoD, suggesting that what appears to be a case of nonoverlapping function could be the result of large differences in the amounts of functionally overlapping isoforms. Streaming assays also suggest that myoC plays a significant role in some aspect of motility other than cell speed. With regard to phagocytosis, both myoB and myoC single mutants exhibited significant reductions in initial rate, suggesting that these two isoforms perform nonredundant roles in supporting the phagocytic process. In triple mutants these defects were not additive, however. Finally, because double and triple mutants exhibited significant and progressive decreases in doubling times, we also measured the kinetics of fluid phase endocytic flux (uptake, transit time, efflux). Not only do all three isoforms contribute to this process, but their contributions are synergistic. While these results, when taken together, refute the simple notion that these three "classic" myosin I isoforms perform exclusively identical functions, they do reveal that all three share in supporting at least one cellular process (endocytosis), and they identify several other processes (motility, streaming, and phagocytosis) that are supported to a significant extent by either individual isoforms or various combinations of them.

scholarly journals Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

2012 ◽  
Vol 444 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Keefe T. Chan ◽  
David W. Roadcap ◽  
Nicholas Holoweckyj ◽  
James E. Bear
Keyword(s):  
Binding Site ◽  
Leading Edge ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Unique Region ◽  
Equivalent Residue ◽  
Filament Networks ◽  
Actin Binding Site

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

Enolase exists in the fluid phase of cytoplasm in 3T3 cells

1989 ◽  
Vol 94 (2) ◽  
pp. 333-342
Author(s):  
L. Pagliaro ◽  
K. Kerr ◽  
D.L. Taylor
Keyword(s):  
Diffusion Coefficient ◽  
Fluid Phase ◽  
Glycolytic Enzyme ◽  
Binding Activity ◽  
Actin Binding ◽  
Filamentous Actin ◽  
3T3 Cells ◽  
Ratio Imaging

We have investigated the intracellular distribution and mobility of the glycolytic enzyme enolase, using functional fluorescent analogs labeled with the succinimidyl esters of carboxyfluorescein (F1-enolase) and carboxytetramethylrhodamine (Rh-enolase) In contrast to aldolase, neither native enolase nor labeled enolase gelled filamentous actin (F-actin), as measured by falling-ball viscometry, indicating a lack of interaction between enolase and F-actin. Fluorescence redistribution after photo-bleaching (FRAP) measurements of the diffusion coefficient (D) of F1-enolase in aqueous solutions gave a value of D37,aq = 6.08 × 10(−7) cm2s-1, and no immobile fraction, consistent with a native molecular weight of 90,000. These values were not significantly different with Rh-enolase, or in the presence of F-actin, 2-phosphoglycerate or F-actin-aldolase gels, demonstrating that neither F1-enolase nor Rh-enolase binds to F-actin or aldolase in vitro. FRAP measurements of F1- and Rh-enolase microinjected into living Swiss 3T3 cells revealed spatial differences in the diffusion coefficient, but not the mobile fraction. In the perinuclear cytoplasm, we measured an apparent diffusion coefficient of 1.1 × 10(−7) cm2s-1, compared to 7.1 × 10(−8) cm2s-1 in the peripheral cytoplasm, with approximately 100% mobility of F1- or Rh-enolase in both regions. Imaging of cells co-injected with Rh-enolase and size-fractionated FITC-dextran (FD-90) revealed that Rh-enolase entered the nucleus, while FD-90 was excluded. Ratio imaging showed a relatively high nuclear ratio of Rh-enolase/FD-90, and a uniform cytoplasmic ratio, with no indication of increased concentration of enolase around stress fibers. These data demonstrate that Rh- and F1-enolase do not bind to F-actin in vitro, and are 100% mobile in vivo. Together with our recent finding that a significant fraction of aldolase binds to F-actin in vitro and is immobile in vivo, these data suggest a correlation between actin-binding activity and cytoplasmic mobility of glycolytic enzymes.

scholarly journals In Vivo Importance of Actin Nucleotide Exchange Catalyzed by Profilin

2000 ◽  
Vol 150 (4) ◽  
pp. 895-904 ◽  
Author(s):  
Amy K. Wolven ◽  
Lisa D. Belmont ◽  
Nicole M. Mahoney ◽  
Steven C. Almo ◽  
David G. Drubin
Keyword(s):  
Point Mutations ◽  
Fluid Phase ◽  
Intrinsic Rate ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
Cytoskeleton Organization

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP2 and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin–cofilin generated during filament disassembly.

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