scholarly journals Iqg1p links spatial and secretion landmarks to polarity and cytokinesis

2002 ◽  
Vol 159 (4) ◽  
pp. 601-611 ◽  
Author(s):  
Mahasin A. Osman ◽  
James B. Konopka ◽  
Richard A. Cerione
Keyword(s):  
Actin Cytoskeleton ◽  
Site Selection ◽  
Protein Trafficking ◽  
Yeast Cells ◽  
Electron Microscopic ◽  
Binding Partner ◽  
Microscopic Studies ◽  
Division Axis ◽  
Bud Site ◽  
Two Hybrid

Cytokinesis requires the polarization of the actin cytoskeleton, the secretion machinery, and the correct positioning of the division axis. Budding yeast cells commit to their cytokinesis plane by choosing a bud site and polarizing their growth. Iqg1p (Cyk1p) was previously implicated in cytokinesis (Epp and Chant, 1997; Lippincott and Li, 1998; Osman and Cerione, 1998), as well as in the establishment of polarity and protein trafficking (Osman and Cerione, 1998). To better understand how Iqg1p influences these processes, we performed a two-hybrid screen and identified the spatial landmark Bud4p as a binding partner. Iqg1p can be coimmunoprecipitated with Bud4p, and Bud4p requires Iqg1p for its proper localization. Iqg1p also appears to specify axial bud-site selection and mediates the proper localization of the septin, Cdc12p, as well as binds and helps localize the secretion landmark, Sec3p. The double mutants iqg1Δsec3Δ and bud4Δsec3Δ display defects in polarity, budding pattern and cytokinesis, and electron microscopic studies reveal that these cells have aberrant septal deposition. Taken together, these findings suggest that Iqg1p recruits landmark proteins to form a targeting patch that coordinates axial budding with cytokinesis.

scholarly journals A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

1997 ◽  
Vol 136 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Shirley Yang ◽  
Kathryn R. Ayscough ◽  
David G. Drubin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Site Selection ◽  
Mitotic Checkpoint ◽  
Cell Cortex ◽  
Spatial Cues ◽  
Genes Encoding ◽  
Mother And Daughter ◽  
Mother Cells ◽  
Bud Site

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.

scholarly journals A Cdc24p-Far1p-Gβγ Protein Complex Required for Yeast Orientation during Mating

1999 ◽  
Vol 144 (6) ◽  
pp. 1187-1202 ◽  
Author(s):  
Aljoscha Nern ◽  
Robert A. Arkowitz
Keyword(s):  
Site Selection ◽  
Morphological Changes ◽  
External Signal ◽  
Polarized Growth ◽  
The Third ◽  
Pairwise Interactions ◽  
Complex Functions ◽  
A Site ◽  
Bud Site ◽  
Two Hybrid

Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During mating, haploid Saccharomyces cerevisiae cells orient their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the bud site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here we show that a two- hybrid interaction between Cdc24p and Gβ requires Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and Gβ. Binding experiments demonstrate that Cdc24p, Far1p, and Gβ form a complex in which pairwise interactions can occur in the absence of the third protein. Cdc24p localizes to sites of polarized growth suggesting that this complex is localized. In the absence of CDC24-FAR1-mediated chemotropism, a bud site selection protein, Bud1p/Rsr1p, is essential for morphological changes in response to pheromone. These results suggest that formation of a Cdc24p-Far1p-Gβγ complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal.

scholarly journals Gic2p May Link Activated Cdc42p to Components Involved in Actin Polarization, Including Bni1p and Bud6p (Aip3p)

2000 ◽  
Vol 20 (17) ◽  
pp. 6244-6258 ◽  
Author(s):  
Malika Jaquenoud ◽  
Matthias Peter
Keyword(s):  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Single Amino Acid ◽  
Polarized Growth ◽  
Hybrid Analysis ◽  
Actin Organization ◽  
Bud Emergence ◽  
Bud Site ◽  
Two Hybrid

ABSTRACT Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2pW23A) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.

scholarly journals Interaction between bud-site selection and polarity-establishment machineries in budding yeast

2013 ◽  
Vol 368 (1629) ◽  
pp. 20130006 ◽  
Author(s):  
Chi-Fang Wu ◽  
Natasha S. Savage ◽  
Daniel J. Lew
Keyword(s):  
Cell Cycle ◽  
Site Selection ◽  
Time Lapse ◽  
Yeast Cells ◽  
Bud Emergence ◽  
Ras Family ◽  
Polarity Establishment ◽  
Diploid Cells ◽  
Bud Site ◽  
Time Lapse Imaging

Saccharomyces cerevisiae yeast cells polarize in order to form a single bud in each cell cycle. Distinct patterns of bud-site selection are observed in haploid and diploid cells. Genetic approaches have identified the molecular machinery responsible for positioning the bud site: during bud formation, specific locations are marked with immobile landmark proteins. In the next cell cycle, landmarks act through the Ras-family GTPase Rsr1 to promote local activation of the conserved Rho-family GTPase, Cdc42. Additional Cdc42 accumulates by positive feedback, creating a concentrated patch of GTP-Cdc42, which polarizes the cytoskeleton to promote bud emergence. Using time-lapse imaging and mathematical modelling, we examined the process of bud-site establishment. Imaging reveals unexpected effects of the bud-site-selection system on the dynamics of polarity establishment, raising new questions about how that system may operate. We found that polarity factors sometimes accumulate at more than one site among the landmark-specified locations, and we suggest that competition between clusters of polarity factors determines the final location of the Cdc42 cluster. Modelling indicated that temporally constant landmark-localized Rsr1 would weaken or block competition, yielding more than one polarity site. Instead, we suggest that polarity factors recruit Rsr1, effectively sequestering it from other locations and thereby terminating landmark activity.

scholarly journals The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1376-1390 ◽  
Author(s):  
G C Chen ◽  
L Zheng ◽  
C S Chan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Selection ◽  
Yeast Cells ◽  
Gtpase Activating Protein ◽  
Lim Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Lim Domains ◽  
Bud Site

Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.

scholarly journals Distinct Domains of Yeast Cortical Tag Proteins Bud8p and Bud9p Confer Polar Localization and Functionality

2007 ◽  
Vol 18 (9) ◽  
pp. 3323-3339 ◽  
Author(s):  
Anne-Brit Krappmann ◽  
Naimeh Taheri ◽  
Melanie Heinrich ◽  
Hans-Ulrich Mösch
Keyword(s):  
Site Selection ◽  
Yeast Cells ◽  
Guanosine Triphosphate ◽  
Polar Transport ◽  
Polar Localization ◽  
Exchange Factor ◽  
Guanosine Diphosphate ◽  
Median Part ◽  
Systematic Structure ◽  
Bud Site

In Saccharomyces cerevisiae, diploid yeast cells follow a bipolar budding program, which depends on the two transmembrane glycoproteins Bud8p and Bud9p that potentially act as cortical tags to mark the cell poles. Here, we have performed systematic structure-function analyses of Bud8p and Bud9p to identify functional domains. We find that polar transport of Bud8p and Bud9p does not depend on N-terminal sequences but instead on sequences in the median part of the proteins and on the C-terminal parts that contain the transmembrane domains. We show that the guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factor Bud5p, which is essential for bud site selection and physically interacts with Bud8p, also interacts with Bud9p. Regions of Bud8p and Bud9p predicted to reside in the extracellular space are likely to confer interaction with the N-terminal region of Bud5p, implicating indirect interactions between the cortical tags and the GDP/GTP exchange factor. Finally, we have identified regions of Bud8p and Bud9p that are required for interaction with the cortical tag protein Rax1p. In summary, our study suggests that Bud8p and Bud9p carry distinct domains for delivery of the proteins to the cell poles, for interaction with the general budding machinery and for association with other cortical tag proteins.

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 Polarized Growth Controls Cell Shape and Bipolar Bud Site Selection in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (14) ◽  
pp. 5235-5247 ◽  
Author(s):  
Yi-Jun Sheu ◽  
Yves Barral ◽  
Michael Snyder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Selection ◽  
Critical Role ◽  
Apical Growth ◽  
Yeast Cells ◽  
Cell Cycle Regulators ◽  
Polarized Growth ◽  
Growth Phases ◽  
Division Site ◽  
Bud Site

ABSTRACT We examined the relationship between polarized growth and division site selection, two fundamental processes important for proper development of eukaryotes. Diploid Saccharomyces cerevisiaecells exhibit an ellipsoidal shape and a specific division pattern (a bipolar budding pattern). We found that the polarity genesSPA2, PEA2, BUD6, andBNI1 participate in a crucial step of bud morphogenesis, apical growth. Deleting these genes results in round cells and diminishes bud elongation in mutants that exhibit pronounced apical growth. Examination of distribution of the polarized secretion marker Sec4 demonstrates that spa2Δ, pea2Δ,bud6Δ, and bni1Δ mutants fail to concentrate Sec4 at the bud tip during apical growth and at the division site during repolarization just prior to cytokinesis. Moreover, cell surface expansion is not confined to the distal tip of the bud in these mutants. In addition, we found that the p21-activated kinase homologue Ste20 is also important for both apical growth and bipolar bud site selection. We further examined how the duration of polarized growth affects bipolar bud site selection by using mutations in cell cycle regulators that control the timing of growth phases. Thegrr1Δ mutation enhances apical growth by stabilizing G1 cyclins and increases the distal-pole budding in diploids. Prolonging polarized growth phases by disrupting the G2/M cyclin gene CLB2 enhances the accuracy of bud site selection in wild-type, spa2Δ, andste20Δ cells, whereas shortening the polarized growth phases by deleting SWE1 decreases the fidelity of bipolar budding. This study reports the identification of components required for apical growth and demonstrates the critical role of polarized growth in bipolar bud site selection. We propose that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes.

THE INTEGRITY OF THE CELL WALL DURING BUD FORMATION IN YEASTS

1965 ◽  
Vol 11 (3) ◽  
pp. 447-452 ◽  
Author(s):  
Dan O. McClary ◽  
Wilbert D. Bowers Jr.
Keyword(s):  
Cell Wall ◽  
Mother Cell ◽  
Budding Yeast ◽  
Dark Field ◽  
Yeast Cells ◽  
Constant Thickness ◽  
Full Thickness ◽  
Electron Microscopic ◽  
Bud Formation ◽  
Microscopic Studies

Dark-field and electron microscopic studies of budding yeast cells have shown an extension of a wall of full thickness, rather than a break in the wall, when the bud emerges. The bud appears as a minute bulge and grows steadily, not explosively, during which time both it and the mother cell are enclosed within a single wall. The wall maintains essentially a constant thickness throughout the growth of the bud, and at maturity both the wall and the cytoplasm of the two cells are separated by a cleavage wall which is laid down between them.

Electron microscopic studies on ultrathin frozen sections of lactating mouse mammary gland

1978 ◽  
Vol 36 (2) ◽  
pp. 46-47
Author(s):  
Jan Zarzycki ◽  
Joseph Szroeder
Keyword(s):  
Mammary Gland ◽  
Protein Denaturation ◽  
Chemical Constituents ◽  
Freeze Substitution ◽  
Electron Microscopic Examination ◽  
Mouse Mammary Gland ◽  
Electron Microscopic ◽  
Preparation Methods ◽  
Microscopic Studies ◽  
Ultrathin Frozen Sections

The mammary gland ultrastructure in various functional states is the object of our investigations. The material prepared for electron microscopic examination by the conventional chemical methods has several limitations, the most important are the protein denaturation processes and the loss of large amounts of chemical constituents from the cells. In relevance to this,one can't be sure about a degree the observed images are adequate to the realy ultrastructure of a living cell. To avoid the disadvantages of the chemical preparation methods,some autors worked out alternative physical methods based on tissue freezing / freeze-drying, freeze-substitution, freeze-eatching techniqs/; actually the technique of cryoultraraicrotomy,i,e.cutting ultrathin sections from deep frozen specimens is assented as a complete alternative method. According to the limitations of the routine plastic embbeding methods we were interested to analize the mammary gland ultrastructure during lactation by the cryoultramicrotomy method.

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