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 Roles of Bud-Site-Selection Proteins during Haploid Invasive Growth in Yeast

2002 ◽  
Vol 13 (9) ◽  
pp. 2990-3004 ◽  
Author(s):  
Paul J. Cullen ◽  
George F. Sprague
Keyword(s):  
Site Selection ◽  
Growth Response ◽  
Invasive Growth ◽  
Glucose Limitation ◽  
Glucose Depletion ◽  
Distal Pole ◽  
Bud Emergence ◽  
Diploid Cells ◽  
Bud Site ◽  
Site Marker

In haploid strains of Saccharomyces cerevisiae, glucose depletion causes invasive growth, a foraging response that requires a change in budding pattern from axial to unipolar-distal. To begin to address how glucose influences budding pattern in the haploid cell, we examined the roles of bud-site-selection proteins in invasive growth. We found that proteins required for bipolar budding in diploid cells were required for haploid invasive growth. In particular, the Bud8p protein, which marks and directs bud emergence to the distal pole of diploid cells, was localized to the distal pole of haploid cells. In response to glucose limitation, Bud8p was required for the localization of the incipient bud site marker Bud2p to the distal pole. Three of the four known proteins required for axial budding, Bud3p, Bud4p, and Axl2p, were expressed and localized appropriately in glucose-limiting conditions. However, a fourth axial budding determinant, Axl1p, was absent in filamentous cells, and its abundance was controlled by glucose availability and the protein kinase Snf1p. In thebud8 mutant in glucose-limiting conditions, apical growth and bud site selection were uncoupled processes. Finally, we report that diploid cells starved for glucose also initiate the filamentous growth response.

scholarly journals Regulation of Bud Emergence by a MAPK Pathway

2019 ◽  
Author(s):  
Aditi Prabhakar ◽  
Jacky Chow ◽  
Alan J. Siegel ◽  
Paul J. Cullen
Keyword(s):  
Cell Cycle ◽  
Symmetry Breaking ◽  
Rho Gtpase ◽  
Mapk Pathway ◽  
Time Lapse ◽  
Filamentous Growth ◽  
Effector Proteins ◽  
Critical Determinant ◽  
Bud Emergence ◽  
Polarity Establishment

ABSTRACTAll cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in yeast, where the Rho GTPase Cdc42p regulates symmetry breaking at bud sites and the establishment of polarity by interacting with effector proteins. The prevailing view of bud emergence does not account for regulation by extrinsic cues or signal transduction pathways. Here, we show that the MAPK pathway that controls filamentous growth (fMAPK pathway), which also requires Cdc42p and the effector p21 activated kinase (PAK) Ste20p, regulates bud emergence under nutrient-limiting conditions that favor filamentous/invasive growth. The fMAPK pathway regulated the expression of polarity targets that included the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and highest at a period in the cell cycle that coincided with bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced growth at multiple sites that resulted from multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

scholarly journals Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Methods ◽  
2018 ◽  
Vol 133 ◽  
pp. 81-90 ◽  
Author(s):  
Katja M. Piltti ◽  
Brian J. Cummings ◽  
Krystal Carta ◽  
Ayla Manughian-Peter ◽  
Colleen L. Worne ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Neural Stem Cells ◽  
Single Cell ◽  
Cell Tracking ◽  
Time Lapse ◽  
Live Cell ◽  
Time Lapse Imaging

scholarly journals Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Diego Velázquez ◽  
Marcel Albacar ◽  
Chunyi Zhang ◽  
Carlos Calafí ◽  
María López-Malo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Protein Phosphatase ◽  
Mitotic Cell ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cellular Targets ◽  
Major Mechanism ◽  
Bud Emergence ◽  
Phosphorylation Pattern

Abstract Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

scholarly journals Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroaki Shimono ◽  
Atsushi Kaida ◽  
Hisao Homma ◽  
Hitomi Nojima ◽  
Yusuke Onozato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Time Lapse ◽  
S Phase ◽  
G1 Phase ◽  
M Phase ◽  
G2 Phase ◽  
The Difference ◽  
Time Lapse Imaging ◽  
First Time

AbstractIn this study, we examined the fluctuation in radioresponse of HeLa cells during the cell cycle. For this purpose, we used HeLa cells expressing two types of fluorescent ubiquitination-based cell cycle indicators (Fucci), HeLa-Fucci (CA)2 and HeLa-Fucci (SA), and combined this approach with the micronucleus (MN) assay to assess radioresponse. The Fucci system distinguishes cell cycle phases based on the colour of fluorescence and cell morphology under live conditions. Time-lapse imaging allowed us to further identify sub-positions within the G1 and S phases at the time of irradiation by two independent means, and to quantitate the number of MNs by following each cell through M phase until the next G1 phase. Notably, we found that radioresponse was low in late G1 phase, but rapidly increased in early S phase. It then decreased until late S phase and increased in G2 phase. For the first time, we demonstrated the unique fluctuation of radioresponse by the MN assay during the cell cycle in HeLa cells. We discuss the difference between previous clonogenic experiments using M phase-synchronised cell populations and ours, as well as the clinical implications of the present findings.

295 THE SHAPE OF PORCINE NEURAL PROGENITOR CELL CELLULAR GENEALOGIES REVEALED BY TIME-LAPSE IMAGING

2011 ◽  
Vol 23 (1) ◽  
pp. 245
Author(s):  
I. Faerge ◽  
A. Egeskov-Madsen ◽  
P. Holm
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Cycle Length ◽  
Individual Cell ◽  
Time Lapse ◽  
Cell Type ◽  
Epidermal Growth ◽  
Cellular Development ◽  
Time Lapse Imaging ◽  
Cell Cycle Length

Porcine neural progenitor cells (pNPC) derived from embryonic stem cells are capable of self-renewal and differentiation into neural and glia lineages, rendering them promising candidates for cell-based therapy of neurodegenerative diseases in a large animal biomedical model. A prerequisite for the successful future therapeutic use of pNPC is a comprehensive characterisation and understanding of the neurogenic process. This is important for learning how to direct cell fates into required proportions of the cell type wanted for the specific brain disease to be treated, and it is crucial for avoiding uncontrolled cell proliferation leading to fatal tumour formations. Time-lapse analysis is a powerful tool to obtain live cell characterisation by analysing individual cell fate. Information on cellular development, division, and differentiation can be composed into a pedigree-like structure denoted as cellular genealogy giving an overview of the proliferation profile of a cell culture and the duration of each cell cycle (Al-Kofani et al. 2006). The aim of the study was to construct cellular genealogies of pNPC and differentiated neural lineages, respectively, by time-lapse imaging to evaluate the effect of external variables observed by changes in the topology of the cellular genealogy. Porcine NPC were derived from epiblast cells isolated from day-9 porcine blastocysts and cultured in DMEM/12, Pen/strep, B27 and N2 with basic fibroblast growth factor and epidermal growth factor, and differentiation was obtained by withdrawal of basic fibroblast growth factor and epidermal growth factor. The state of cellular development of undifferentiated and differentiated pNPC was verified immunohistochemically by the presence of SOX2, NESTIN, TUJI, and GFAB (Rasmussen et al. 2010). The time-lapse images were captured by a Nikon Biostation with a 10× resolution under phase contrast in a humidified chamber at 38°C with 5% CO2, 5% O2, and 90% N2. For each sequence, images were captured at intervals of 10 min in 16 frames. Sequences 1, 2, and 3 constituted passage 15 pNPC, passage 4 pNPC, and presumably differentiated cells, respectively. For each sequence, cell cycle length was calculated after manual tracking of selected cells. The cell cycle length of pNPC is shown in Table 1. Based on these data, cellular genealogies characteristic of each individual cell type have been constructed. Table 1.Cell cycle length of porcine neutral progenitor cells (pNPC) before and after differentiation

scholarly journals Role of Bud3p in producing the axial budding pattern of yeast.

1995 ◽  
Vol 129 (3) ◽  
pp. 767-778 ◽  
Author(s):  
J Chant ◽  
M Mischke ◽  
E Mitchell ◽  
I Herskowitz ◽  
J R Pringle
Keyword(s):  
Cell Cycle ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Alpha Cells ◽  
Double Ring ◽  
Division Site ◽  
Bud Emergence ◽  
Mother And Daughter ◽  
Associated Proteins

Yeast cells can select bud sites in either of two distinct spatial patterns. a cells and alpha cells typically bud in an axial pattern, in which both mother and daughter cells form new buds adjacent to the preceding division site. In contrast, a/alpha cells typically bud in a bipolar pattern, in which new buds can form at either pole of the cell. The BUD3 gene is specifically required for the axial pattern of budding: mutations of BUD3 (including a deletion) affect the axial pattern but not the bipolar pattern. The sequence of BUD3 predicts a product (Bud3p) of 1635 amino acids with no strong or instructive similarities to previously known proteins. However, immunofluorescence localization of Bud3p has revealed that it assembles in an apparent double ring encircling the mother-bud neck shortly after the mitotic spindle forms. The Bud3p structure at the neck persists until cytokinesis, when it splits to yield a single ring of Bud3p marking the division site on each of the two progeny cells. These single rings remain for much of the ensuing unbudded phase and then disassemble. The Bud3p rings are indistinguishable from those of the neck filament-associated proteins (Cdc3p, Cdc10p, Cdc11p, and Cdc12p), except that the latter proteins assemble before bud emergence and remain in place for the duration of the cell cycle. Upon shift of a temperature-sensitive cdc12 mutant to restrictive temperature, localization of both Bud3p and the neck filament-associated proteins is rapidly lost. In addition, a haploid cdc11 mutant loses its axial-budding pattern upon shift to restrictive temperature. Taken together, the data suggest that Bud3p and the neck filaments are linked in a cycle in which each controls the position of the other's assembly: Bud3p assembles onto the neck filaments in one cell cycle to mark the site for axial budding (including assembly of the new ring of neck filaments) in the next cell cycle. As the expression and localization of Bud3p are similar in a, alpha, and a/alpha cells, additional regulation must exist such that Bud3p restricts the position of bud formation in a and alpha cells but not in a/alpha cells.

scholarly journals GENETIC CONTROL OF THE CELL DIVISION CYCLE IN YEAST: V. GENETIC ANALYSIS OF cdc MUTANTS

Genetics ◽  
1973 ◽  
Vol 74 (2) ◽  
pp. 267-286
Author(s):  
Leland H Hartwell ◽  
Robert K Mortimer ◽  
Joseph Culotti ◽  
Marilyn Culotti
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Nuclear Gene ◽  
Temperature Shift ◽  
Time Lapse ◽  
Cell Division Cycle ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Products ◽  
Diploid Cells

ABSTRACT One hundred and forty-eight temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae have been isolated and characterized. Complementation studies ordered these recessive mutations into 32 groups and tetrad analysis revealed that each of these groups defines a single nuclear gene. Fourteen of these genes have been located on the yeast genetic map. Functionally related cistrons are not tightly clustered. Mutations in different cistrons frequently produce different cellular and nuclear morphologies in the mutant cells following incubation at the restrictive temperature, but all the mutations in the same cistron produce essentially the same morphology. The products of these genes appear, therefore, each to function individually in a discrete step of the cell cycle and they define collectively a large number of different steps. The mutants were examined by time-lapse photomicroscopy to determine the number of cell cycles completed at the restrictive temperature before arrest. For most mutants, cells early in the cell cycle at the time of the temperature shift (before the execution point) arrest in the first cell cycle while those later in the cycle (after the execution point) arrest in the second cell cycle. Execution points for allelic mutations that exhibit first or second cycle arrest are rather similar and appear to be cistron-specific. Other mutants traverse several cycles before arrest, and its suggested that the latter type of response may reveal gene products that are temperature-sensitive for synthesis, whereas the former may be temperature-sensitive for function. The gene products that are defined by the cdc cistrons are essential for the completion of the cell cycle in haploids of a and α mating type and in a/α diploid cells. The same genes, therefore, control the cell cycle in each of these stages of the life cycle.

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