scholarly journals BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae.

1996 ◽  
Vol 132 (1) ◽  
pp. 137-151 ◽  
Author(s):  
G Mondésert ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Actin Cytoskeleton ◽  
Dependent Manner ◽  
Polarized Growth ◽  
Checkpoint Control ◽  
Wild Type ◽  
Ellipsoidal Shape ◽  
Bud Emergence ◽  
Morphogenesis Checkpoint

The ellipsoidal shape of the yeast Saccharomyces cerevisiae is the result of successive isotropic/apical growth switches that are regulated in a cell cycle-dependent manner. It is thought that growth polarity is governed by the remodeling of the actin cytoskeleton that is itself under the control of the cell cycle machinery. The cell cycle and the morphogenesis cycle are tightly coupled and it has been recently suggested that a morphogenesis/polarity checkpoint control monitors bud emergence in order to maintain the coupling of these two events (Lew, D. J., and S. I. Reed. 1995. J. Cell Biol. 129:739-749). During a screen based on the inability of cells impaired in the budding process to survive when the morphogenesis checkpoint control is abolished, we identified and characterized BED1, a new gene that is required for efficient budding. Cells carrying a disrupted allele of BED1 no longer have the wild-type ellipsoidal shape characteristic of S. cerevisiae, are larger than wild-type cells, are deficient in bud emergence, and depend upon an intact morphogenesis checkpoint control to survive. These cells show defects in polarized growth despite the fact that the actin cytoskeleton appears normal. Our results suggest that Bed1 is a type II membrane protein localized in the endoplasmic reticulum. BED1 is significantly homologous to gma12+, a S. pombe gene coding for an alpha-1,2,-galactosyltransferase, suggesting that glycosylation of specific proteins or lipids could be important for signaling in the switch to polarized growth and in bud emergence.

scholarly journals A Monitor for Bud Emergence in the Yeast Morphogenesis Checkpoint

2003 ◽  
Vol 14 (8) ◽  
pp. 3280-3291 ◽  
Author(s):  
Chandra L. Theesfeld ◽  
Trevin R. Zyla ◽  
Elaine G.S. Bardes ◽  
Daniel J. Lew
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Actin Cytoskeleton ◽  
Budding Yeast ◽  
Bud Formation ◽  
Bud Emergence ◽  
Checkpoint Pathway ◽  
Morphogenesis Checkpoint ◽  
External Signals ◽  
Biochemical Signals

Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation.

scholarly journals Pak-Family Kinases Regulate Cell and Actin Polarization Throughout the Cell Cycle of Saccharomyces cerevisiae

1999 ◽  
Vol 147 (4) ◽  
pp. 845-856 ◽  
Author(s):  
Stephen P. Holly ◽  
Kendall J. Blumer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Yeast Cell ◽  
Fluorescent Protein ◽  
Surface Growth ◽  
Yeast Cell Cycle ◽  
Cortical Actin ◽  
Bud Emergence

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho–guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4–green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.

scholarly journals Identification of Genes Controlling Growth Polarity in the Budding Yeast Saccharomyces cerevisiae: A Possible Role of N-Glycosylation and Involvement of the Exocyst Complex

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 421-434 ◽  
Author(s):  
G Mondésert ◽  
D J Clarke ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Cell Cycle Progression ◽  
Cellular Systems ◽  
Surface Growth ◽  
Polarized Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycle Progression

The regulation of secretion polarity and cell surface growth during the cell cycle is critical for proper morphogenesis and viability of Saccharomyces cerevisiae. A shift from isotropic cell surface growth to polarized growth is necessary for bud emergence and a repolarization of secretion to the bud neck is necessary for cell separation. Although alterations in the actin cytoskeleton have been implicated in these changes in secretion polarity, clearly other cellular systems involved in secretion are likely to be targets of cell cycle regulation. To investigate mechanisms coupling cell cycle progression to changes in secretion polarity in parallel with and downstream of regulation of actin polarization, we implemented a screen for mutants defective specifically in polarized growth but with normal actin cytoskeleton structure. These mutants fell into three classes: those partially defective in N-glycosylation, those linked to specific defects in the exocyst, and a third class neither defective in glycosylation nor linked to the exocyst. These results raise the possibility that changes in N-linked glycosylation may be involved in a signal linking cell cycle progression and secretion polarity and that the exocyst may have regulatory functions in coupling the secretory machinery to the polarized actin cytoskeleton.

scholarly journals Skp1p and the F-Box Protein Rcy1p Form a Non-SCF Complex Involved in Recycling of the SNARE Snc1p in Yeast

2001 ◽  
Vol 21 (9) ◽  
pp. 3105-3117 ◽  
Author(s):  
Jean-Marc Galan ◽  
Andreas Wiederkehr ◽  
Jae Hong Seol ◽  
Rosine Haguenauer-Tsapis ◽  
Raymond J. Deshaies ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Actin Cytoskeleton ◽  
Dependent Manner ◽  
Polarized Growth ◽  
Core Complex ◽  
Direct Role ◽  
Scf Complex ◽  
F Box Protein

ABSTRACT Skp1p–cullin–F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p inSaccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

NIPA Checkpoint Control In Oncogenic Transformation Is Dependent on p53

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4178-4178
Author(s):  
Anna Lena Illert ◽  
Hiroyuki Kawaguchi ◽  
Corinna Albers ◽  
Melanie Sickinger ◽  
Ulrich Keller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin B1 ◽  
Conditional Knockout ◽  
Substantial Contribution ◽  
Dependent Manner ◽  
Checkpoint Control ◽  
Oncogenic Transformation ◽  
Wild Type ◽  
Focus Formation ◽  
Wild Type Mefs

Abstract Abstract 4178 The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding NIPA. NIPA-deficient animals are viable, but sterile due to a block of spermatogenesis. Moreover, our studies demonstrate that loss of NIPA has no substantive effect on the physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, NIPA checkpoint control can be unmasked by oncogenic transformation by c-Myc. Here we show that transformed focus formation assays revealed highly significant differences in c-Myc-induced transformation in NIPA-deficient and wild-type MEFs. c-Myc transduction caused a pronounced upregulation of cyclin-B in NIPA-null MEFs, which was completely reversible by ectopic NIPA expression. The increased cyclin-B1 expression after c-Myc transduction in the absence of NIPA has considerable functional consequences for the cells: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced in comparison to wild-type MEFs (24.6% vs. 100%). Moreover, c-Myc expression caused 12.8% apoptotic subG1 cells in wild-type MEFs, whereas Nipa-/- MEFs were more affected by c-Myc-induced apoptosis (22.45%). Next, we sought to know, whether increased apoptosis in Nipa-deficient c-Myc transduced MEFs is dependent on a functional p53-Axis. Therefore, Nipa-wildtype and knockout MEFs were first infected with a retroviral Supernatant encoding for a p53Mir- and thereafter with the oncogene c-Myc. Interestingly the effect of Nipa knockout on c-Myc-mediated oncogenic transformation was totally abolished by the knock-down of p53. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53 in comparison to wildtype cells, suggesting the importance of functional p53 in Nipa-induced cell death. Taken together, our data demonstrate that NIPA is required for efficient c-Myc transformation in a p53-dependent manner. Moreover, our results highlight the functional importance of the NIPA-p53 axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Haspin Modulates the G2/M Transition Delay in Response to Polarization Failures in Budding Yeast

2021 ◽  
Vol 8 ◽  
Author(s):  
Martina Galli ◽  
Laura Diani ◽  
Roberto Quadri ◽  
Alessandro Nespoli ◽  
Elena Galati ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
M Cell ◽  
Checkpoint Activation ◽  
Bud Emergence ◽  
Morphogenesis Checkpoint ◽  
Transition Delay ◽  
Bud Site ◽  
Surveillance Mechanism

Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of Saccharomyces cerevisiae cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In alk1Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in Saccharomyces cerevisiae, where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition.

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 22 (20) ◽  
pp. 6946-6948 ◽  
Author(s):  
Joanna Kamińska ◽  
Beata Gajewska ◽  
Anita K. Hopper ◽  
Teresa ˙Zołądek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Growth Defects ◽  
Genes Encoding ◽  
Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

scholarly journals DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

1994 ◽  
Vol 125 (3) ◽  
pp. 625-638 ◽  
Author(s):  
J Lukas ◽  
H Müller ◽  
J Bartkova ◽  
D Spitkovsky ◽  
A A Kjerulff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Cyclin D1 ◽  
Cell Lines ◽  
T Antigen ◽  
Regulatory Function ◽  
Retinoblastoma Gene ◽  
Checkpoint Control ◽  
Wild Type ◽  
In Cells

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

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