scholarly journals Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.

1992 ◽  
Vol 118 (3) ◽  
pp. 561-571 ◽  
Author(s):  
S Chowdhury ◽  
K W Smith ◽  
M C Gustin
Keyword(s):  
Osmotic Stress ◽  
Actin Filament ◽  
Physiological Process ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rhodamine Phalloidin ◽  
Diploid Cells

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

scholarly journals Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ.

1991 ◽  
Vol 115 (6) ◽  
pp. 1629-1638 ◽  
Author(s):  
E L Bearer
Keyword(s):  
Actin Filament ◽  
Direct Observation ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Capping Protein ◽  
Calcium Dependent ◽  
Definition Of

Dynamic behavior of actin filaments in cells is the basis of many different cellular activities. Remodeling of the actin filament network involves polymerization and depolymerization of the filaments. Proteins that regulate these behaviors include proteins that sever and/or cap actin filaments. This report presents direct observation of severing of fluorescently-labeled actin filaments. Coverslips coated with gelsolin, a multi-domain, calcium-dependent capping and severing protein, bound rhodamine-phalloidin-saturated filaments along their length in the presence of EGTA. Upon addition of calcium, attached filaments bent as they broke. Actophorin, a low molecular weight, monomer sequestering, calcium-independent severing protein did not sever phalloidin-saturated filaments. Both gCap 39, a gelsolin-like, calcium-dependent capping protein that does not sever filaments, and CapZ, a heterodimeric, non-calcium-dependent capping protein, bound the filaments by one end to the coverslip. Visualization of individual filaments also revealed severing activity present in mixtures of actin-binding proteins isolated by filamentous actin affinity chromatography from early Drosophila embryos. This activity was different from either gelsolin or actophorin because it was not inhibited by phalloidin, but was calcium independent. The results of these studies provide new information about the molecular mechanisms of severing and capping by well-characterized proteins as well as definition of a novel type of severing activity.

scholarly journals The Novel Adaptor Protein, Mti1p, and Vrp1p, a Homolog of Wiskott-Aldrich Syndrome Protein-Interacting Protein (WIP), May Antagonistically Regulate Type I Myosins in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (3) ◽  
pp. 923-934
Author(s):  
Junko Mochida ◽  
Takaharu Yamamoto ◽  
Konomi Fujimura-Kamada ◽  
Kazuma Tanaka
Keyword(s):  
Adaptor Protein ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Null Mutation ◽  
Type I ◽  
Cortical Actin ◽  
Tail Region ◽  
Interacting Protein ◽  
Wiskott Aldrich Syndrome ◽  
Syndrome Protein

Abstract Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like structures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.

scholarly journals Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding

2008 ◽  
Vol 183 (5) ◽  
pp. 865-879 ◽  
Author(s):  
Christian Frantz ◽  
Gabriela Barreiro ◽  
Laura Dominguez ◽  
Xiaoming Chen ◽  
Robert Eddy ◽  
...  
Keyword(s):  
Actin Filament ◽  
Structural Changes ◽  
Ph Sensor ◽  
Ph Sensitive ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Dynamics ◽  
Ph Dependent ◽  
Dynamics Simulations

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H+ efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

scholarly journals Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin.

1991 ◽  
Vol 113 (5) ◽  
pp. 1057-1067 ◽  
Author(s):  
M L Vitale ◽  
A Rodríguez Del Castillo ◽  
L Tchakarov ◽  
J M Trifaró
Keyword(s):  
Actin Filament ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Catecholamine Secretion ◽  
Cortical Region ◽  
Cortical Surface ◽  
Filamentous Actin ◽  
Cell Stimulation ◽  
Rhodamine Phalloidin ◽  
Filament Networks

Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

Purification and characterization of a nonpolymerizing long-pitch actin dimer

2006 ◽  
Vol 84 (5) ◽  
pp. 695-702 ◽  
Author(s):  
Braden Sweeting ◽  
John F. Dawson
Keyword(s):  
Critical Concentration ◽  
Dnase I ◽  
Actin Binding ◽  
Wild Type ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Fold Reduction ◽  
Crystallization Conditions ◽  
Self Association ◽  
Pointed End

Atomic resolution structures of filamentous actin have not been obtained owing to the self-association of actin under crystallization conditions. Obtaining short filamentous actin complexes of defined lengths is therefore a highly desirable goal. Here we report the production and isolation of a long-pitch actin dimer employing chemical crosslinking between wild-type actin and Q41C/C374A mutant actin. The Q41C/C374A mutant actin possessed altered polymerization properties, with a 2-fold reduction in the rate of elongation and an increased critical concentration relative to wild-type actin. The Q41C/C374A mutant actin also displayed an increase in the IC50 for DNase I, a pointed-end actin-binding protein. The long-pitch dimer was bound by DNase I to prevent polymerization and purified. It was found that each actin dimer is bound by 2 DNase I molecules, 1 likely bound to each of the actin protomers. The long-pitch dimer bound by DNase I did not form short F actin structures, as assessed by the binding of rhodamine–phalloidin.

scholarly journals αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

2013 ◽  
Vol 24 (23) ◽  
pp. 3710-3720 ◽  
Author(s):  
Scott D. Hansen ◽  
Adam V. Kwiatkowski ◽  
Chung-Yueh Ouyang ◽  
HongJun Liu ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Structure ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Underlying Mechanisms ◽  
Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

Actin filaments regulate epithelial Na+ channel activity

1991 ◽  
Vol 261 (5) ◽  
pp. C882-C888 ◽  
Author(s):  
H. F. Cantiello ◽  
J. L. Stow ◽  
A. G. Prat ◽  
D. A. Ausiello
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Functional Role ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Cortical Actin ◽  
Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

scholarly journals TetraThymosinβ Is Required for Actin Dynamics in Caenorhabditis elegans and Acts via Functionally Different Actin-binding Repeats

2004 ◽  
Vol 15 (10) ◽  
pp. 4735-4748 ◽  
Author(s):  
Marleen Van Troys ◽  
Kanako Ono ◽  
Daisy Dewitte ◽  
Veronique Jonckheere ◽  
Natalie De Ruyck ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Actin Filament ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Actin Binding ◽  
In Vitro Assays ◽  
Filamentous Actin ◽  
Lethal Mutant ◽  
Interaction Sites

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinβ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinβ has four repeats, each related to the actin monomer-sequestering protein thymosinβ 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinβ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinβ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinβ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinβ is of crucial importance at specific developmental stages requiring actin polymerization.

scholarly journals The BUD4 protein of yeast, required for axial budding, is localized to the mother/BUD neck in a cell cycle-dependent manner.

1996 ◽  
Vol 134 (2) ◽  
pp. 413-427 ◽  
Author(s):  
S L Sanders ◽  
I Herskowitz
Keyword(s):  
Cell Cycle ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Alpha Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Neck ◽  
A Cell ◽  
Cycle Dependent ◽  
Diploid Cells

A and alpha cells of the yeast Saccharomyces cerevisiae exhibit an axial budding pattern, whereas a/alpha diploid cells exhibit a bipolar pattern. Mutations in BUD3, BUD4, and AXL1 cause a and alpha cells to exhibit the bipolar pattern, indicating that these genes are necessary to specify the axial budding pattern (Chant, J., and I. Herskowitz. 1991. Cell. 65:1203-1212; Fujita, A., C. Oka, Y. Arikawa, T. Katagi, A. Tonouchi, S. Kuhara, and Y. Misumi. 1994. Nature (Lond.). 372:567-570). We cloned and sequenced BUD4, which codes for a large, novel protein (Bud4p) with a potential GTP-binding motif. Bud4p is expressed and localized to the mother/bud neck in all cell types. Most mitotic cells contain two apparent rings of Bud4 immunoreactive staining, as observed for Bud3p (Chant, J., M. Mischke, E. Mitchell, I. Herskowitz, and J.R. Pringle. 1995. J. Cell Biol. 129: 767-778). Early G1 cells contain a single ring of Bud4p immunoreactive staining, whereas cells at START and in S phase lack these rings. The level of Bud4p is also regulated in a cell cycle-dependent manner. Bud4p is inefficiently localized in bud3 mutants and after a temperature shift of a temperature-sensitive mutant, cdc12, defective in the neck filaments. These observations suggest that Bud4p and Bud3p cooperate to recognize a spatial landmark (the neck filaments) during mitosis and support the hypothesis that they subsequently become a landmark for establishing the axial budding pattern in G1.

scholarly journals Cdc50p, a Conserved Endosomal Membrane Protein, Controls Polarized Growth in Saccharomyces cerevisiae

2003 ◽  
Vol 14 (2) ◽  
pp. 730-747 ◽  
Author(s):  
Kenjiro Misu ◽  
Konomi Fujimura-Kamada ◽  
Takashi Ueda ◽  
Akihiko Nakano ◽  
Hiroyuki Katoh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Transmembrane Protein ◽  
Small Gtpase ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Endosomal Membrane ◽  
Actin Cables ◽  
Vacuolar Protein

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in thevps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.

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