Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells

2001 ◽  
Vol 114 (18) ◽  
pp. 3273-3284 ◽  
Author(s):  
Masatoshi Eda ◽  
Shigenobu Yonemura ◽  
Takayuki Kato ◽  
Naoki Watanabe ◽  
Toshimasa Ishizaki ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Green Fluorescence Protein ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Cortical Actin ◽  
Interphase Cells ◽  
C3 Exoenzyme ◽  
Dividing Cells ◽  
Fluorescence Protein ◽  
Citron Kinase

Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)-fusion, we monitored its localization in cell cycle. We have found: (1) Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase; (2) Rho colocalizes with Citron-K in the cortex of ana- to telophase cells and the two proteins are concentrated in the cleavage furrow and to the midbody; (3) inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, but prevented its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, we expressed active Val14RhoA in interphase cells expressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to the ventral cortex of interphase cells, where it formed band-like structures in a complex with Rho. This structure was localized at the same plane as actin stress fibers, and they exclude each other. Disruption of F-actin abolished the band and dispersed the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells.

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.

scholarly journals EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (8) ◽  
pp. 4294-4304 ◽  
Author(s):  
H Y Tang ◽  
A Munn ◽  
M Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Growth Factor Receptor ◽  
Cell Cortex ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Cortical Actin ◽  
Kinase Substrate ◽  
Physiological Relevance ◽  
Eh Domain

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

scholarly journals Bee1, a Yeast Protein with Homology to Wiscott-Aldrich Syndrome Protein, Is Critical for the Assembly of Cortical Actin Cytoskeleton

1997 ◽  
Vol 136 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Rong Li
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Function ◽  
Actin Filaments ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Yeast Protein ◽  
Wiskott Aldrich Syndrome ◽  
Striking Change ◽  
And Function ◽  
Syndrome Protein

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.

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 Cleavage furrow: timing of emergence of contractile ring actin filaments and establishment of the contractile ring by filament bundling in sea urchin eggs

1994 ◽  
Vol 107 (7) ◽  
pp. 1853-1862 ◽  
Author(s):  
I. Mabuchi
Keyword(s):  
Sea Urchin ◽  
Binding Sites ◽  
Actin Filaments ◽  
Early Stage ◽  
Cortical Layer ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Cortical Actin ◽  
Contractile Ring ◽  
Rhodamine Phalloidin

Cleavage furrow formation at the first cell division of sea urchin and sand dollar eggs was investigated in detail by fluorescence staining of actin filaments with rhodamine-phalloidin of either whole eggs or isolated egg cortices. Cortical actin filaments were clustered at anaphase and then the clusters became fibrillar at the end of anaphase. The timing when the contractile ring actin filaments appear was precisely determined in the course of mitosis: accumulation of the contractile ring actin filaments at the equatorial cell cortex is first noticed at the beginning of telophase (shortly before furrow formation), when the chromosomal vesicles are fusing with each other. The accumulated actin filaments were not well organized at the early stage but were organized into parallel bundles as the furrowing progressed. The bundles were finally fused into a tightly packed filament belt. Wheat germ agglutinin (WGA)-binding sites were distributed on the surface of the egg in a manner similar to the actin filaments after anaphase. The WGA-binding sites became accumulated in the contractile ring together with the contractile ring actin filaments, indicating an intimate relationship between these sites and actin filament-anchoring sites on the plasma membrane. Myosin also appeared in the contractile ring together with the actin filaments. The ‘cleavage stimulus’, a signal hypothesized by Rappaport (reviewed by R. Rappaport (1986) Int. Rev. Cytol. 105, 245–281) was suggested to induce aggregation or bundling of the actin filaments in the cortical layer.

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.

Myosin II-independent F-actin flow contributes to cell locomotion in dictyostelium

1999 ◽  
Vol 112 (6) ◽  
pp. 877-886 ◽  
Author(s):  
Y. Fukui ◽  
T. Kitanishi-Yumura ◽  
S. Yumura
Keyword(s):  
Cell Body ◽  
Myosin Ii ◽  
Cleavage Furrow ◽  
Organizational Changes ◽  
Cortical Actin ◽  
Charge Coupled Device ◽  
Cell Locomotion ◽  
Intensity Changes ◽  
Dividing Cells ◽  
Temporal And Spatial

While the treadmilling and retrograde flow of F-actin are believed to be responsible for the protrusion of leading edges, little is known about the mechanism that brings the posterior cell body forward. To elucidate the mechanism for global cell locomotion, we examined the organizational changes of filamentous (F-) actin in live Dictyostelium discoideum. We labeled F-actin with a trace amount of fluorescent phalloidin and analyzed its dynamics in nearly two-dimensional cells by using a sensitive, high-resolution charge-coupled device. We optically resolved a cyclic mode of tightening and loosening of fibrous cortical F-actin and quantitated its flow by measuring temporal and spatial intensity changes. The rate of F-actin flow was evaluated with respect to migration velocity and morphometric changes. In migrating monopodial cells, the cortical F-actin encircling the posterior cell body gradually accumulated into the tail end at a speed of 0.35 microm/minute. We show qualitatively and quantitatively that the F-actin flow is closely associated with cell migration. Similarly, in dividing cells, the cortical F-actin accumulated into the cleavage furrow. Although five times slower than the wild type, the F-actin also flows rearward in migrating mhcA- cells demonstrating that myosin II (‘conventional’ myosin) is not absolutely required for the observed dynamics of F-actin. Yet consistent with the reported transportation of ConA-beads, the direction of observed F-actin flow in Dictyostelium is conceptually opposite from a barbed-end binding to the plasma membrane. This study suggests that the posterior end of the cell has a unique motif that tugs the cortical actin layer rearward by means of a mechanism independent from myosin II; this mechanism may be also involved in cleavage furrow formation.

scholarly journals Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

2011 ◽  
Vol 301 (1) ◽  
pp. C31-C43 ◽  
Author(s):  
Winnie Waichi Shum ◽  
Nicolas Da Silva ◽  
Clémence Belleannée ◽  
Mary McKee ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Apical Membrane ◽  
Green Fluorescence Protein ◽  
Cortical Actin ◽  
Clear Cells ◽  
Luminal Perfusion ◽  
Intracellular Signaling Cascade ◽  
Fluorescence Protein ◽  
And Storage

Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H+-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of “resting” clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10–30 μM) and HA1077 (30 μM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 μg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 μM) or HA1077 (30 μM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.

scholarly journals Novel Proteins Linking the Actin Cytoskeleton to the Endocytic Machinery in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (10) ◽  
pp. 3646-3661 ◽  
Author(s):  
H. Dewar ◽  
D. T. Warren ◽  
F. C. Gardiner ◽  
C. G. Gourlay ◽  
N. Satish ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Elevated Temperatures ◽  
Adaptor Protein ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

scholarly journals Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

2009 ◽  
Vol 29 (10) ◽  
pp. 2816-2827 ◽  
Author(s):  
Masaru Mitsushima ◽  
Fumiko Toyoshima ◽  
Eisuke Nishida
Keyword(s):  
Actin Cytoskeleton ◽  
Small Gtpases ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Adherent Cells ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Independent Manner ◽  
Oriented Parallel

ABSTRACT The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. βPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-βPix-actin pathway for this mechanism.

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