scholarly journals Aip1p Interacts with Cofilin to Disassemble Actin Filaments

1999 ◽  
Vol 145 (6) ◽  
pp. 1251-1264 ◽  
Author(s):  
Avital A. Rodal ◽  
Jonathan W. Tetreault ◽  
Pekka Lappalainen ◽  
David G. Drubin ◽  
David C. Amberg
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Actin Filaments ◽  
Ternary Complex ◽  
Yeast Cells ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Biochemical Analyses ◽  
Two Hybrid

Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid–based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches.

scholarly journals High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

1997 ◽  
Vol 137 (2) ◽  
pp. 399-416 ◽  
Author(s):  
Kathryn R. Ayscough ◽  
Joel Stryker ◽  
Navin Pokala ◽  
Miranda Sanders ◽  
Phil Crews ◽  
...  
Keyword(s):  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Cell Polarity ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Surface Growth ◽  
Interacting Protein ◽  
Capping Protein ◽  
Latrunculin A

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

scholarly journals Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast.

1995 ◽  
Vol 128 (4) ◽  
pp. 599-615 ◽  
Author(s):  
R Li ◽  
Y Zheng ◽  
D G Drubin
Keyword(s):  
Cell Growth ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Polarized Cell Growth ◽  
Nucleation Activity

We have established an in vitro assay for assembly of the cortical actin cytoskeleton of budding yeast cells. After permeabilization of yeast by a novel procedure designed to maintain the spatial organization of cellular constituents, exogenously added fluorescently labeled actin monomers assemble into distinct structures in a pattern that is similar to the cortical actin distribution in vivo. Actin assembly in the bud of small-budded cells requires a nucleation activity provided by protein factors that appear to be distinct from the barbed ends of endogenous actin filaments. This nucleation activity is lost in cells that lack either Sla1 or Sla2, proteins previously implicated in cortical actin cytoskeleton function, suggesting a possible role for these proteins in the nucleation reaction. The rate and the extent of actin assembly in the bud are increased in permeabilized delta cap2 cells, providing evidence that capping protein regulates the ability of the barbed ends of actin filaments to grow in yeast cells. Actin incorporation in the bud can be stimulated by treating the permeabilized cells with GTP-gamma S, and, significantly, the stimulatory effect is eliminated by a mutation in CDC42, a gene that encodes a Rho-like GTP-binding protein required for bud formation. Furthermore, the lack of actin nucleation activity in the cdc42 mutant can be complemented in vitro by a constitutively active Cdc42 protein. These results suggest that Cdc42 is closely involved in regulating actin assembly during polarized cell growth.

scholarly journals 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

2006 ◽  
Vol 17 (5) ◽  
pp. 2190-2199 ◽  
Author(s):  
Kurato Mohri ◽  
Kanako Ono ◽  
Robinson Yu ◽  
Sawako Yamashiro ◽  
Shoichiro Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Actin Depolymerizing Factor ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Cytoskeletal Dynamics ◽  
End Capping

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 β-propeller, whereas one residue is located in the C-terminal β-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.

scholarly journals The Saccharomyces cerevisiae Calponin/Transgelin Homolog Scp1 Functions with Fimbrin to Regulate Stability and Organization of the Actin Cytoskeleton

2003 ◽  
Vol 14 (7) ◽  
pp. 2617-2629 ◽  
Author(s):  
Anya Goodman ◽  
Bruce L. Goode ◽  
Paul Matsudaira ◽  
Gerald R. Fink
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Associated Proteins ◽  
Cross Links ◽  
Biochemical Analyses ◽  
Actin Patch ◽  
The One

Calponins and transgelins are members of a conserved family of actin-associated proteins widely expressed from yeast to humans. Although a role for calponin in muscle cells has been described, the biochemical activities and in vivo functions of nonmuscle calponins and transgelins are largely unknown. Herein, we have used genetic and biochemical analyses to characterize the budding yeast member of this family, Scp1, which most closely resembles transgelin and contains one calponin homology (CH) domain. We show that Scp1 is a novel component of yeast cortical actin patches and shares in vivo functions and biochemical activities with Sac6/fimbrin, the one other actin patch component that contains CH domains. Purified Scp1 binds directly to filamentous actin, cross-links actin filaments, and stabilizes filaments against disassembly. Sequences in Scp1 sufficient for actin binding and cross-linking reside in its carboxy terminus, outside the CH domain. Overexpression of SCP1 suppresses sac6Δ defects, and deletion of SCP1 enhances sac6Δ defects. Together, these data show that Scp1 and Sac6/fimbrin cooperate to stabilize and organize the yeast actin cytoskeleton.

scholarly journals Zn2+-induced reversible dissociation of subunit Rpn10/p54 of the Drosophila 26 S proteasome

2005 ◽  
Vol 391 (2) ◽  
pp. 301-310 ◽  
Author(s):  
Petra Kiss ◽  
Áron Szabó ◽  
Éva Hunyadi-Gulyás ◽  
Katalin F. Medzihradszky ◽  
Zoltán Lipinszki ◽  
...  
Keyword(s):  
Atp Depletion ◽  
Yeast Cells ◽  
Peptidase Activity ◽  
Interacting Protein ◽  
Functional Changes ◽  
Reversible Dissociation ◽  
Physiological Relevance ◽  
Two Hybrid ◽  
Chemical Cross Linking

In the presence of Zn2+, the Drosophila 26 S proteasome disassembles into RP (regulatory particle) and CP (catalytic particle), this process being accompanied by the dissociation of subunit Rpn10/p54, the ubiquitin receptor subunit of the proteasome. The dissociation of Rpn10/p54 induces extensive rearrangements within the lid subcomplex of the RP, while the structure of the ATPase ring of the base subcomplex seems to be maintained. As a consequence of the dissociation of the RP, the peptidase activity of the 26 S proteasome is lost. The Zn2+-induced structural and functional changes are fully reversible; removal of Zn2+ is followed by reassociation of subunit Rpn10/p54 to the RP, reassembly of the 26 S proteasome and resumption of the peptidase activity. After the Zn2+-induced dissociation, Rpn10/p54 interacts with a set of non-proteasomal proteins. Hsp82 (heat-shock protein 82) has been identified by MS as the main Rpn10/p54-interacting protein, suggesting its role in the reassembly of the 26 S proteasome after Zn2+ removal. The physiological relevance of another Rpn10/p54-interacting protein, the Smt3 SUMO (small ubiquitin-related modifier-1)-activating enzyme, detected by chemical cross-linking, has been confirmed by yeast two-hybrid analysis. Besides the Smt3 SUMO-activating enzyme, the Ubc9 SUMO-conjugating enzyme also exhibited in vivo interaction with the 5′-half of Rpn10/p54 in yeast cells. The mechanism of 26 S proteasome disassembly after ATP depletion is clearly different from that induced by Zn2+. Rpn10/p54 is permanently RP-bound during the ATP-dependent assembly–disassembly cycle, but during the Zn2+ cycle it reversibly shuttles between the RP-bound and free states.

XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1), which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins

1999 ◽  
Vol 112 (10) ◽  
pp. 1553-1565 ◽  
Author(s):  
K. Okada ◽  
T. Obinata ◽  
H. Abe
Keyword(s):  
Actin Filaments ◽  
Electron Microscopic Observation ◽  
Affinity Column ◽  
Predicted Amino Acid Sequence ◽  
Cortical Actin ◽  
Electron Microscopic ◽  
Cell Stage ◽  
Interacting Protein ◽  
In Vivo Effects

We carried out affinity column chromatography using Xenopus ADF/cofilin (XAC), identified several polypeptides in oocytes specifically bound to this column with actin, and isolated a full-length cDNA clone for a 65 kDa protein in this fraction. The predicted amino acid sequence revealed that the 65 kDa protein has seven obvious WD repeats and exhibits striking homology with yeast actin interacting protein 1 (AIP1). Thus, we designated this protein Xenopus AIP1 (XAIP1). We purified XAIP1 from Xenopus oocytes, and its interaction with actin was characterized by a pelleting assay, photometrical analysis and electron microscopy. Although XAIP1 itself cosedimented with F-actin and increased unsedimented actin to some extent, it induced a rapid, drastic disassembly of actin filaments associated with XAC. Electron microscopic observation revealed that XAIP1 severs actin filaments in the presence of XAC. To elucidate the in vivo effects of XAIP1, the purified protein was injected into blastomeres at the two-cell stage. Although the localization of XAIP1 was similar to that of XAC, at the cortical cytoskeleton and diffusely in the cytoplasm, injection of a large amount of XAIP1 arrested development and abolished the strong cortical staining of both actin and XAC. From these results, we concluded that XAIP1 regulates the dynamics of the cortical actin cytoskeleton cooperatively with XAC in eggs.

scholarly journals Cofilin, But Not Profilin, Is Required for Myosin-I-Induced Actin Polymerization and the Endocytic Uptake in Yeast

2002 ◽  
Vol 13 (11) ◽  
pp. 4074-4087 ◽  
Author(s):  
Fatima-Zahra Idrissi ◽  
Bianka L. Wolf ◽  
M. Isabel Geli
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Cortical Actin ◽  
Myosin I ◽  
Robust Evidence

Mutations in the budding yeast myosins-I (MYO3 andMYO5) cause defects in the actin cytoskeleton and in the endocytic uptake. Robust evidence also indicates that these proteins induce Arp2/3-dependent actin polymerization. Consistently, we have recently demonstrated, using fluorescence microscopy, that Myo5p is able to induce cytosol-dependent actin polymerization on the surface of Sepharose beads. Strikingly, we now observed that, at short incubation times, Myo5p induced the formation of actin foci that resembled the yeast cortical actin patches, a plasma membrane-associated structure that might be involved in the endocytic uptake. Analysis of the machinery required for the formation of the Myo5p-induced actin patches in vitro demonstrated that the Arp2/3 complex was necessary but not sufficient in the assay. In addition, we found that cofilin was directly involved in the process. Strikingly though, the cofilin requirement seemed to be independent of its ability to disassemble actin filaments and profilin, a protein that closely cooperates with cofilin to maintain a rapid actin filament turnover, was not needed in the assay. In agreement with these observations, we found that like the Arp2/3 complex and the myosins-I, cofilin was essential for the endocytic uptake in vivo, whereas profilin was dispensable.

scholarly journals Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin, a Ubiquitous Actin Monomer-sequestering Protein

1998 ◽  
Vol 142 (3) ◽  
pp. 723-733 ◽  
Author(s):  
Bruce L. Goode ◽  
David G. Drubin ◽  
Pekka Lappalainen
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Green Fluorescent

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.

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.

Understanding Biological Structures Through Exploring the Mechanical Response of Cell-Like Systems

2008 ◽  
Author(s):  
Ying Zhang ◽  
Philip R. LeDuc
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Living Cell ◽  
Cellular Response ◽  
Cancer Metastasis ◽  
Cell Structure ◽  
Polymer Physics ◽  
Wide Range

The actin cytoskeleton provides mechanical support for the cell and influences activities such as cancer metastasis and chemotaxis. While their mechanical responses have been studied in vivo and in vitro, understanding the link between these two forms remains challenging. To explore this gap and further understand cell structure, we reconstructed the cell cytoskeleton in a membrane-like spherical liposome to mimic the cellular environment; this enables an artificial “cell like” system. Through this approach, we are pursuing a path to compare in vitro mechanics from a polymer physics perspective of individual actin filaments with the in vivo mechanics of a living cell [1]. A living cell contains many organelles, which are in a highly packed environment and require significant organization to function. The actin cytoskeleton provides both structural and organizational regulation that is essential for cellular response. Here, we first encapsulated G-actin into giant unilamellar vesicles through an electroformation technique and then polymerized them into actin filaments (F-actin) within individual vesicles. To probe their conformation, we visualized these vesicles with fluorescence and laser scanning confocal microscopy. We then used a tapping mode atomic force microscopy to determine the mechanical properties of these cell-like systems. These results provide insight into a wide range of fields and studies including polymer physics, cell biology, and biotechnology.

Sign in / Sign up

Export Citation Format

Share Document