scholarly journals The Phosphatidylinositol 4,5-Biphosphate and TORC2 Binding Proteins Slm1 and Slm2 Function in Sphingolipid Regulation

2006 ◽  
Vol 26 (15) ◽  
pp. 5861-5875 ◽  
Author(s):  
Mitsuaki Tabuchi ◽  
Anjon Audhya ◽  
Ainslie B. Parsons ◽  
Charles Boone ◽  
Scott D. Emr
Keyword(s):  
Actin Cytoskeleton ◽  
Essential Role ◽  
Synthetic Lethality ◽  
Sphingolipid Metabolism ◽  
Actin Organization ◽  
Cytoskeleton Organization ◽  
Calcium Calmodulin Dependent ◽  
Actin Cytoskeleton Organization ◽  
Phosphatidylinositol 4

ABSTRACT The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the essential role of Stt4-mediated PI4P production, we performed a genetic screen using the stt4 ts mutation to identify candidate regulators and effectors of PI4P. From this analysis, we identified several genes that have been previously implicated in lipid metabolism. In particular, we observed synthetic lethality when both sphingolipid and PI4P synthesis were modestly diminished. Consistent with these data, we show that the previously characterized phosphoinositide effectors, Slm1 and Slm2, which regulate actin organization, are also necessary for normal sphingolipid metabolism, at least in part through regulation of the calcium/calmodulin-dependent phosphatase calcineurin, which binds directly to both proteins. Additionally, we identify Isc1, an inositol phosphosphingolipid phospholipase C, as an additional target of Slm1 and Slm2 negative regulation. Together, our data suggest that Slm1 and Slm2 define a molecular link between phosphoinositide and sphingolipid signaling and thereby regulate actin cytoskeleton organization.

The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana

Development ◽  
1999 ◽  
Vol 126 (24) ◽  
pp. 5559-5568 ◽  
Author(s):  
J. Mathur ◽  
P. Spielhofer ◽  
B. Kost ◽  
N. Chua
Keyword(s):  
Arabidopsis Thaliana ◽  
Actin Cytoskeleton ◽  
Spatial Patterning ◽  
Actin Microfilaments ◽  
Cell Morphogenesis ◽  
Actin Organization ◽  
Cytoskeleton Organization ◽  
Branch Formation ◽  
Actin Cytoskeleton Organization ◽  
Trichome Cell

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the ‘distorted’ class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.

scholarly journals RhoE Regulates Actin Cytoskeleton Organization and Cell Migration

1998 ◽  
Vol 18 (8) ◽  
pp. 4761-4771 ◽  
Author(s):  
Rosa M. Guasch ◽  
Peter Scambler ◽  
Gareth E. Jones ◽  
Anne J. Ridley
Keyword(s):  
Actin Cytoskeleton ◽  
Mdck Cells ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Complete Disappearance ◽  
Actin Organization ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization ◽  
Rho Family ◽  
As Stress

ABSTRACT The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.

scholarly journals The MAP Kinase Slt2 Is Involved in Vacuolar Function and Actin Remodeling in Saccharomyces cerevisiae Mutants Affected by Endogenous Oxidative Stress

2013 ◽  
Vol 79 (20) ◽  
pp. 6459-6471 ◽  
Author(s):  
Nuria Pujol-Carrion ◽  
Mima I. Petkova ◽  
Luis Serrano ◽  
Maria Angeles de la Torre-Ruiz
Keyword(s):  
Oxidative Stress ◽  
Actin Cytoskeleton ◽  
Iron Homeostasis ◽  
Synthetic Lethality ◽  
Actin Dynamics ◽  
Triple Mutant ◽  
Cytoskeleton Organization ◽  
Cellular Models ◽  
Vacuole Fusion ◽  
Actin Cytoskeleton Organization

ABSTRACTOxidative stress causes transient actin cytoskeleton depolarization and also provokes vacuole fragmentation in wild-type cells. Under conditions of oxidative stress induced by hydrogen peroxide, the Slt2 protein is required to repolarize the actin cytoskeleton and to promote vacuole fusion. In this study, we show thatgrx3 grx4andgrx5mutants are cellular models of endogenous oxidative stress. This stress is the result of alterations in iron homeostasis that lead to impairment of vacuolar function and also to disorganization of the actin cytoskeleton. Slt2 overexpression suppresses defects in vacuolar function and actin cytoskeleton organization in thegrx3 grx4mutant. Slt2 exerts this effect independently of the intracellular levels of reactive oxygen species (ROS) and of iron homeostasis. The deletion ofSLT2in thegrx3 grx4mutant results in synthetic lethality related to vacuolar function with substantial vacuole fragmentation. The observation that both Vps4 and Vps73 (two proteins related to vacuole sorting) suppress vacuole fragmentation and actin depolarization in thegrx3 grx4 slt2triple mutant strengthens the hypothesis that Slt2 plays a role in vacuole homeostasis related to actin dynamics. Here we show that insod1,grx5, andgrx3 grx4 slt2mutants, all of which are affected by chronic oxidative stress, the overexpression of Slt2 favors vacuole fusion through a mechanism dependent on an active actin cytoskeleton.

scholarly journals Sac1 Lipid Phosphatase and Stt4 Phosphatidylinositol 4-Kinase Regulate a Pool of Phosphatidylinositol 4-Phosphate That Functions in the Control of the Actin Cytoskeleton and Vacuole Morphology

2001 ◽  
Vol 12 (8) ◽  
pp. 2396-2411 ◽  
Author(s):  
Michelangelo Foti ◽  
Anjon Audhya ◽  
Scott D. Emr
Keyword(s):  
Actin Cytoskeleton ◽  
Temperature Sensitive ◽  
Primary Role ◽  
Cytoskeleton Organization ◽  
Phosphoinositide Phosphatase ◽  
Actin Cytoskeleton Organization ◽  
Sensitive Allele ◽  
Phosphatidylinositol 4 ◽  
Mutant Cells

Synthesis and turnover of phosphoinositides are tightly regulated processes mediated by a set of recently identified kinases and phosphatases. We analyzed the primary role of the phosphoinositide phosphatase Sac1p in Saccharomyces cerevisiae with the use of a temperature-sensitive allele of this gene. Our analysis demonstrates that inactivation of Sac1p leads to a specific increase in the cellular levels of phosphatidylinositol 4-phosphate (PtdIns(4)P), accompanied by changes in vacuole morphology and an accumulation of lipid droplets. We have found that the majority of Sac1p localizes to the endoplasmic reticulum, and this localization is crucial for the efficient turnover of PtdIns(4)P. By generating double mutant strains harboring the sac1tsallele and one of two temperature-sensitive PtdIns 4-kinase genes,stt4tsor pik1ts, we have demonstrated that the bulk of PtdIns(4)P that accumulates insac1 mutant cells is generated by the Stt4 PtdIns 4-kinase, and not Pik1p. Consistent with these findings, inactivation of Sac1p partially rescued defects associated withstt4tsbut notpik1tsmutant cells. To analyze potential overlapping functions between Sac1p and other homologous phosphoinositide phosphatases, sac1tsmutant cells lacking various other synaptojanin-like phosphatases were generated. These double and triple mutants exacerbated the accumulation of intracellular phosphoinositides and caused defects in Golgi function. Together, our results demonstrate that Sac1p primarily turns over Stt4p-generated PtdIns(4)P and that the membrane localization of Sac1p is important for its function in vivo. Regulation of this PtdIns(4)P pool appears to be crucial for the maintenance of vacuole morphology, regulation of lipid storage, Golgi function, and actin cytoskeleton organization.

scholarly journals The Functional Role of CrkII in Actin Cytoskeleton Organization and Mitogenesis

1999 ◽  
Vol 274 (5) ◽  
pp. 3001-3008 ◽  
Author(s):  
Naoki Nakashima ◽  
David W. Rose ◽  
Sen Xiao ◽  
Katsuya Egawa ◽  
Stuart S. Martin ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Functional Role ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

scholarly journals Pan1p, End3p, and Sla1p, Three Yeast Proteins Required for Normal Cortical Actin Cytoskeleton Organization, Associate with Each Other and Play Essential Roles in Cell Wall Morphogenesis

2000 ◽  
Vol 20 (1) ◽  
pp. 12-25 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Jing Xu ◽  
Mingjie Cai
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Normal Cell ◽  
Cell Cortex ◽  
Repeat Region ◽  
Cortical Actin ◽  
Cytoskeleton Organization ◽  
Eh Domain ◽  
Actin Cytoskeleton Organization ◽  
Cell Wall Morphogenesis

ABSTRACT The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Δ, andend3Δ mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.

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