scholarly journals Nucleation of polar actin filament assembly by a positively charged surface.

1979 ◽  
Vol 80 (2) ◽  
pp. 499-504 ◽  
Author(s):  
S S Brown ◽  
J A Spudich
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Charged Surface ◽  
Actin Assembly ◽  
Filamentous Form ◽  
Filament Assembly ◽  
Biochemical Assays ◽  
Positively Charged

Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Polymerization of actin by positively charged liposomes.

1988 ◽  
Vol 106 (4) ◽  
pp. 1221-1227 ◽  
Author(s):  
A Laliberte ◽  
C Gicquaud
Keyword(s):  
Electron Microscopy ◽  
Direct Contact ◽  
Atp Hydrolysis ◽  
Actin Assembly ◽  
Liposome Membrane ◽  
Low Salt ◽  
Spectrofluorimetric Analysis ◽  
Positively Charged

By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.

scholarly journals Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array

2009 ◽  
Vol 184 (2) ◽  
pp. 269-280 ◽  
Author(s):  
Christopher J. Staiger ◽  
Michael B. Sheahan ◽  
Parul Khurana ◽  
Xia Wang ◽  
David W. McCurdy ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Stochastic Dynamics ◽  
Actin Dynamics ◽  
Epifluorescence Microscopy ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Filament Dynamics ◽  
Cortical Array

Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 µm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

scholarly journals Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Scott D Hansen ◽  
R Dyche Mullins
Keyword(s):  
Axon Guidance ◽  
Actin Filaments ◽  
Network Formation ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Polymerase Activity ◽  
Actin Assembly ◽  
Filament Assembly

Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

scholarly journals Activation of the Arp2/3 Complex by the Actin Filament Binding Protein Abp1p

2001 ◽  
Vol 153 (3) ◽  
pp. 627-634 ◽  
Author(s):  
Bruce L. Goode ◽  
Avital A. Rodal ◽  
Georjana Barnes ◽  
David G. Drubin
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Assembly ◽  
Related Protein ◽  
Genetic Studies ◽  
Actin Networks ◽  
Specific Step

The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex. Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins. We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro. Genetic studies indicate that this activity is important for Abp1p functions in vivo. In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers. Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro. We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation. Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis. Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis.

The capactins, a class of proteins that cap the ends of actin filaments

1982 ◽  
Vol 299 (1095) ◽  
pp. 263-273 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Preliminary Evidence ◽  
The Other ◽  
Cellular Regulation ◽  
Filament Assembly ◽  
Filament Bundles

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the ‘capactins’. On the basis of their effect on actin filament assembly and interaction in vitro , we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.

scholarly journals Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the limulus acrosomal process

1983 ◽  
Vol 96 (4) ◽  
pp. 1097-1107 ◽  
Author(s):  
EM Bonder ◽  
MS Mooseker
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Initial Rate ◽  
Potent Inhibitor ◽  
Actin Assembly ◽  
Electron Microscopic ◽  
Filament Assembly ◽  
Large Numbers ◽  
End Capping ◽  
Dependent Interaction

We have re-examined the Ca(++)-dependent interaction of an intestinal microvillar 95- kdalton protein (MV-95K) and actin using the isolated acrosomal process bundles from limulus sperm. Making use of the processes as nuclei for assembling actin filaments, we quantitatively and qualitatively examined MV-95K's effect on filament assembly and on F- actin, both in the presence and in the absence of Ca(++). The acrosomal processes are particularly advantageous for this approach because they nucleate large numbers of filaments, they are extremely stable, and their morphology can be used to determine the polarity of any nucleated filaments. When filament nucleation was initiated in the presence of MV-95K and the absence of Ca(++), there was biased filament assembly from the bundle ends. The calculated elongation rates from both the barbed and pointed filament ends were virtually indistinguishable from control preparations. In the presence of Ca(++), MV-95K completely inhibited filament assembly from the barbed filament end without affecting the initial rate of assembly from the pointed filament end. The inhibition of assembly results from MV-95K binding to and capping the barbed filament end, thereby preventing monomer addition. This indicates that, while MV-95K is a potent nucleator of actin assembly, it is also a potent inhibitor of actin filament elongation. To examine the effects of MV-95K on F-actin in the presence of Ca(++), we developed an assay where MV-95K is added to filaments previously assembled from acrosomal processes without causing filament breakage during mixing. These results clearly demonstrated that rapid filament shortening by MV-95K results through a mechanism of disrupting intrafilament monomer-monomer interactions. Finally, we show that tropomyosin-containing actin filaments are insensitive to cutting, but not to capping, by MV-95K in the presence of Ca(++).

scholarly journals Variable autoinhibition among deafness-associated variants of Diaphanous 1 (DIAPH1)

2021 ◽  
Author(s):  
Rabina Lakha ◽  
Angela M. Montero ◽  
Tayyaba Jabeen ◽  
Christina C. Costeas ◽  
Jia Ma ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Molecular Mechanisms ◽  
Actin Assembly ◽  
Thermal Stabilities ◽  
Filament Assembly ◽  
Biochemical Assays ◽  
Quaternary Structures ◽  
Mutant Forms

AbstractOne of the earliest mapped human deafness genes, DIAPH1, encodes the formin DIAPH1. To date, at least three distinct mutations in the C-terminal domains and two additional mutations in the N-terminal region are associated with autosomal dominant hearing loss. The underlying molecular mechanisms are not known, and the role of formins in the inner ear is not well understood. In this study we use biochemical assays to test the hypotheses that autoinhibition and/or actin assembly activities are disrupted by DFNA1 mutations. Our results indicate that C-terminal mutant forms of DIAPH1 are functional in vitro and promote actin filament assembly. Similarly, N-terminal mutants are well-folded and have quaternary structures and thermal stabilities similar to the WT protein. The strength of the autoinhibitory interactions varies widely among mutants, with the ttaa, A265S and I530S mutations having an affinity similar to WT and the 1213x and Δag mutations completely abolishing autoinhibition. These data indicate that, in some cases, hearing loss may be linked to reduced inhibition of actin assembly.

scholarly journals Twinfilin, a molecular mailman for actin monomers

2002 ◽  
Vol 115 (5) ◽  
pp. 881-886 ◽  
Author(s):  
Sandra Palmgren ◽  
Maria Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Drosophila Melanogaster ◽  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Capping Protein ◽  
Filament Assembly

Twinfilin is a ubiquitous actin-monomer-binding protein that is composed of two ADF-homology domains. It forms a 1:1 complex with ADP-actin-monomers,inhibits nucleotide exchange on actin monomers and prevents assembly of the monomer into filaments. The two ADF-H domains in twinfilin probably have 3D structures similar to those of the ADF/cofilin proteins and overlapping actin-binding sites. Twinfilin also interacts with PtdIns(4,5)P2, which inhibits its actin-monomer-sequestering activity in vitro. Mutations in the twinfilin gene result in defects in the bipolar budding pattern in S. cerevisiae and in a rough eye phenotype and aberrant bristle morphology in Drosophila melanogaster. These phenotypes are caused by the uncontrolled polymerization of actin filaments in the absence of twinfilin. Studies on budding yeast suggest that twinfilin contributes to actin filament turnover by localizing actin monomers, in their `inactive'ADP-form, to the sites of rapid filament assembly. This is mediated through direct interactions between twinfilin and capping protein. Therefore,twinfilin might serve as a link between rapid actin filament depolymerization and assembly in cells.

scholarly journals Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks.

1981 ◽  
Vol 91 (3) ◽  
pp. 695-705 ◽  
Author(s):  
J V Small
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Leading Edge ◽  
Cultured Fibroblasts ◽  
Cell Biol ◽  
Filament Bundles

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

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