scholarly journals Opposing Roles for Actin in Cdc42p Polarization

2005 ◽  
Vol 16 (3) ◽  
pp. 1296-1304 ◽  
Author(s):  
Javier E. Irazoqui ◽  
Audrey S. Howell ◽  
Chandra L. Theesfeld ◽  
Daniel J. Lew
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Polarity ◽  
Budding Yeast ◽  
Cortical Actin ◽  
Independent Manner ◽  
Actin Depolymerization ◽  
Cell Biol ◽  
Actin Cables ◽  
Unexpected Difference

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399–416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.

scholarly journals Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

1997 ◽  
Vol 8 (3) ◽  
pp. 533-545 ◽  
Author(s):  
T Harder ◽  
R Kellner ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Independent Manner ◽  
Low Concentrations ◽  
Early Endosomes ◽  
Associated Proteins ◽  
Cytoskeletal Elements

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

SRO9, a Multicopy Suppressor of the Bud Gpowth Defect in the Saccharomyces cerevisiae rho3-Deficient Cells, Shows Strong Genetic Interactions With Tropomyosin Genes, Suggesting Its Role in Organization of the Actin Cytoskeleton

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1003-1016
Author(s):  
Mitsuhiro Kagami ◽  
Akio Toh-e ◽  
Yasushi Matsui
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Rna Binding ◽  
Small Gtpase ◽  
Growth Defect ◽  
Cortical Actin ◽  
Cytoplasmic Factor ◽  
Multicopy Suppressor ◽  
Actin Cables

RHO3 encodes a Rho-type small GTPase in the yeast Saccharomyces cerevisiae and is involved in the proper organization of the actin cytoskeleton required for bud growth. SRO9 (YCL37c) was isolated as a multicopy suppressor of a rho3Δ mutation. An Sro9p domain required for function is similar to a domain in the La protein (an RNA-binding protein). Disruption of SRO9 did not affect vegetative growth, even with the simultaneous disruption of an SRO9 homologue, SRO99. However, sro9Δ was synthetically lethal with a disruption of TPM1, which encodes tropomyosin; sro9Δ tpm1Δ cells did not distribute cortical actin patches properly and lysed. We isolated TPM2, the other gene for tropomyosin, as a multicopy suppressor of a tpm1Δ sro9Δ double mutant. Genetic analysis suggests that TPM2 is functionally related to TPM1 and that tropomyosin is important but not essential for cell growth. Overexpression of SRO9 suppressed the growth defect in tpm1Δ tpm2Δ cells, disappearance of cables of actin filaments in both rho3Δ cells and tpm1Δ cells, and temperature sensitivity of actin mutant cells (act1-1 cells), suggesting that Sro9p has a function that overlaps or is related to tropomyosin function. Unlike tropomyosin, Sro9p does not colocalize with actin cables but is diffusely cytoplasmic. These results suggest that Sro9p is a new cytoplasmic factor involved in the organization of actin filaments.

scholarly journals Polarization of cell growth in yeast

2000 ◽  
Vol 113 (4) ◽  
pp. 571-585 ◽  
Author(s):  
D. Pruyne ◽  
A. Bretscher
Keyword(s):  
Actin Cytoskeleton ◽  
Structural Basis ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Class V ◽  
Precise Control ◽  
Dependent Protein ◽  
Actin Cables ◽  
Protein Kinase Signaling ◽  
Structural Determinant

The actin cytoskeleton provides the structural basis for cell polarity in Saccharomyces cerevisiae as well as most other eukaryotes. In Part I of this two-part commentary, presented in the previous issue of Journal of Cell Science, we discussed the basis by which yeast establishes and maintains different states of polarity through Ρ GTPases and cyclin-dependent protein kinase signaling. Here we discuss how, in response to those signals, the actin cytoskeleton guides growth of the yeast cell. A polarized array of actin cables at the cell cortex is the primary structural determinant of polarity. Motors such as class V myosins use this array to transport secretory vesicles, mRNA and organelles towards growth sites, where they are anchored by a cap of cytoskeletal and regulatory proteins. Cortical actin patches enhance and maintain this polarity, probably through endocytic recycling, which allows reuse of materials and prevents continued growth at old sites. The dynamic arrangement of targeting and recycling provides flexibility for the precise control of morphogenesis.

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.

Cell adhesion and the actin cytoskeleton of the enveloping layer in the zebrafish embryo during epiboly

1999 ◽  
Vol 77 (6) ◽  
pp. 527-542 ◽  
Author(s):  
Sara E Zalik ◽  
Ewa Lewandowski ◽  
Zvi Kam ◽  
Benjamin Geiger
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Zebrafish Embryo ◽  
Cultured Cells ◽  
Kinase Inhibitor ◽  
Embryonic Cells ◽  
Cortical Actin ◽  
Actin Cables

As the zebrafish embryo undergoes gastrulation and epiboly, the cells of the enveloping layer (EVL) expand, covering the entire yolk cell. During the epiboly process, the EVL cells move as a coherent layer, remaining tightly attached to each other and to the underlying yolk syncytial layer (YSL). In view of the central role of the actin cytoskeleton, in both cell motility and cell cell adhesion, we have labeled these cells in situ with fluorescent phalloidin and anti-actin antibodies. We show that, throughout their migration, the EVL cells retain a conspicuous cortical actin cytoskeletal belt coinciding with cell surface cadherins. At the margins approaching the YSL, the EVL cells extend, from their apicolateral domains, actin-rich filopodial protrusions devoid of detectable cadherin. We have studied the role of the actin cytoskeleton in the maintenance of EVL cohesion during epiboly. Cytochalasin treatment of embryos induces EVL dissociation accompanied by general detachment of the rest of the embryonic cells. In the dissociating EVL cells, the cortical actin belt undergoes fragmentation with the formation of actin aggregates; cadherins, on the other hand, remain evenly distributed at the junctional cell surface. Removal of Ca2+ by ethyleneglycolbis (amino-ethyl-ether)-tetraacetic acid (EGTA) treatment also induces cell dissociation without visible disruption of the cortical actin belt. The protein kinase inhibitor (1-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H-7), which blocks acto-myosin contractility and disrupts actin cables in cultured cells, also potentiates cytochalasin-induced dissociation and promotes the projection of numerous actin-rich lamellipodial extensions. The fact that EVL cells produce microspike-like structures towards the YSL and are capable of lamellipodial activity lend further support to the suggestion (R.W. Keller and J.P. Trinkaus. 1987. Dev. Biol. 120: 12-24) that the EVL cells are not passively mobilized on the expanding YSL but actively participate in epiboly.Key words: actin, adhesion, cadherin, cytochalasin, embryo, zebrafish.

scholarly journals Regulating polarity by directing traffic: Cdc42 prevents adherens junctions from Crumblin' aPart

2008 ◽  
Vol 183 (6) ◽  
pp. 971-974 ◽  
Author(s):  
Mara C. Duncan ◽  
Mark Peifer
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Cell Polarity ◽  
Adherens Junctions ◽  
Vesicular Trafficking ◽  
Cell Junction ◽  
Par Proteins ◽  
Cell Biol ◽  
Embryonic Morphogenesis ◽  
Junction Proteins

The GTPase Cdc42 was among the original genes identified with roles in cell polarity, and interest in its cellular roles from yeast to humans remains high. Cdc42 is a well-known regulator of the actin cytoskeleton, but also plays important roles in vesicular trafficking. In this issue, Harris and Tepass (Harris, K.P, and U. Tepass. 2008. J. Cell. Biol. 183:1129–1143) provide new insights into how Cdc42 and Par proteins work together to modulate cell adhesion and polarity during embryonic morphogenesis by regulating the traffic of key cell junction proteins.

scholarly journals An actin nucleation complex catalyzes filament formation at sites of exocytosis

2019 ◽  
Author(s):  
Oliver Glomb ◽  
Yehui Wu ◽  
Lucia Rieger ◽  
Diana Rüthnick ◽  
Medhanie Mulaw ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Yeast Cells ◽  
Filament Formation ◽  
Cortical Actin ◽  
Peroxisomal Membrane ◽  
Directed Movement ◽  
Vesicle Docking ◽  
Actin Structure ◽  
Local Enrichment ◽  
Actin Cables

AbstractDue to the local enrichment of factors that influence its formation, dynamics, and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells post-Golgi vesicles ride on long actin cables to the bud tip. The scaffold proteins Boi1 and Boi2 participate in tethering and docking these vesicles to the plasma membrane. Here we show that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the physical connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation in the bud, reduces the directed movement of the vesicles to the tip, and shortens their tethering time at the cortex. Artificially transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2 is sufficient to induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusing with the membrane.

scholarly journals Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

2005 ◽  
Vol 16 (1) ◽  
pp. 372-384 ◽  
Author(s):  
Avital A. Rodal ◽  
Lukasz Kozubowski ◽  
Bruce L. Goode ◽  
David G. Drubin ◽  
John H. Hartwig
Keyword(s):  
Cell Division ◽  
High Resolution ◽  
Actin Cytoskeleton ◽  
Yeast Cell ◽  
Budding Yeast ◽  
Model Organism ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Actin Patch ◽  
Order Structures

Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells.

scholarly journals Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

2006 ◽  
Vol 175 (6) ◽  
pp. 957-969 ◽  
Author(s):  
Thomas M. Huckaba ◽  
Thomas Lipkin ◽  
Liza A. Pon
Keyword(s):  
Actin Polymerization ◽  
Budding Yeast ◽  
Type Ii ◽  
Retrograde Flow ◽  
Cortical Actin ◽  
Actin Networks ◽  
Actin Cable ◽  
Intracellular Movement ◽  
Actin Cables ◽  
Type Ii Myosin

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.

scholarly journals A Role for Actin, Cdc1p, and Myo2p in the Inheritance of Late Golgi Elements in Saccharomyces cerevisiae

2001 ◽  
Vol 153 (1) ◽  
pp. 47-62 ◽  
Author(s):  
Olivia W. Rossanese ◽  
Catherine A. Reinke ◽  
Brooke J. Bevis ◽  
Adam T. Hammond ◽  
Irina B. Sears ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fluorescence Microscopy ◽  
Actin Cytoskeleton ◽  
Polarized Growth ◽  
Independent Manner ◽  
Multiple Alleles ◽  
Actin Cables ◽  
Type V

In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud. The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance. Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner. We propose that early Golgi elements arise from ER membranes that are present in the bud. These two pathways of Golgi inheritance in S. cerevisiae resemble Golgi inheritance pathways in vertebrate cells.

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