scholarly journals Mobility of the gradient tracking machine in mating yeast depends on Bud1 inactivation and actin-independent vesicle delivery

2021 ◽  
Author(s):  
Xin Wang ◽  
David Stone
Keyword(s):  
Cell Cycle ◽  
Mating Type ◽  
Positive Feedback ◽  
Yeast Cells ◽  
Polarized Growth ◽  
Opposite Mating Type ◽  
Gradient Sensing ◽  
Feedback Mechanisms ◽  
Ras Gtpase ◽  
Sensing Model

Abstract The mating of budding yeast depends on chemotropism, a fundamental cellular process. Haploid yeast cells of opposite mating type signal their positions to one another through the secretion of mating pheromones. We have proposed a deterministic gradient sensing model that explains how these cells orient toward their mating partners. Using the cell-cycle determined default polarity site (DS), cells assemble a gradient tracking machine (GTM) composed of signaling, polarity, and trafficking proteins. After assembly, the GTM redistributes up the gradient, aligns with the pheromone source, and triggers polarized growth toward the partner. Because strong positive feedback mechanisms drive polarized growth at the DS, it is unclear how the GTM is released for tracking after its assembly is complete. What prevents the GTM from triggering polarized growth at the DS? Here we describe two mechanisms that enable tracking. First, the Ras GTPase Bud1 must be inactivated to release the GTM. Second, actin-independent – but not actin-dependent – vesicle delivery must be targeted upgradient to effect GTM redistribution.

scholarly journals Phosphorylated Gβ is a directional cue during yeast gradient tracking

2021 ◽  
Vol 14 (682) ◽  
pp. eabf4710
Author(s):  
Rashida Abdul-Ganiyu ◽  
Leon A. Venegas ◽  
Xin Wang ◽  
Charles Puerner ◽  
Robert A. Arkowitz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Yeast Cells ◽  
Mutant Form ◽  
Final Position ◽  
Opposite Mating Type ◽  
Gradient Sensing ◽  
Growth Site ◽  
Mating Efficiency ◽  
Sensing Model ◽  
Tracking Direction

Budding yeast cells interpret shallow pheromone gradients from cells of the opposite mating type, polarize their growth toward the pheromone source, and fuse at the chemotropic growth site. We previously proposed a deterministic, gradient-sensing model that explains how yeast cells switch from the intrinsically positioned default polarity site (DS) to the gradient-aligned chemotropic site (CS) at the plasma membrane. Because phosphorylation of the mating-specific Gβ subunit is thought to be important for this process, we developed a biosensor that bound to phosphorylated but not unphosphorylated Gβ and monitored its spatiotemporal dynamics to test key predictions of our gradient-sensing model. In mating cells, the biosensor colocalized with both Gβ and receptor reporters at the DS and then tracked with them to the CS. The biosensor concentrated on the leading side of the tracking Gβ and receptor peaks and was the first to arrive and stop tracking at the CS. Our data showed that the concentrated localization of phosphorylated Gβ correlated with the tracking direction and final position of the G protein and receptor, consistent with the idea that gradient-regulated phosphorylation and dephosphorylation of Gβ contributes to gradient sensing. Cells expressing a nonphosphorylatable mutant form of Gβ exhibited defects in gradient tracking, orientation toward mating partners, and mating efficiency.

scholarly journals Mating yeast cells use an intrinsic polarity site to assemble a pheromone-gradient tracking machine

2019 ◽  
Vol 218 (11) ◽  
pp. 3730-3752 ◽  
Author(s):  
Xin Wang ◽  
Wei Tian ◽  
Bryan T. Banh ◽  
Bethanie-Michelle Statler ◽  
Jie Liang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
G Protein ◽  
Yeast Cells ◽  
Mating Types ◽  
Gradient Sensing ◽  
Feedback Mechanisms ◽  
Growth Site ◽  
Endocytic Machinery ◽  
Mating Projection

The mating of budding yeast depends on chemotropism, a fundamental cellular process. The two yeast mating types secrete peptide pheromones that bind to GPCRs on cells of the opposite type. Cells find and contact a partner by determining the direction of the pheromone source and polarizing their growth toward it. Actin-directed secretion to the chemotropic growth site (CS) generates a mating projection. When pheromone-stimulated cells are unable to sense a gradient, they form mating projections where they would have budded in the next cell cycle, at a position called the default polarity site (DS). Numerous models have been proposed to explain yeast gradient sensing, but none address how cells reliably switch from the intrinsically determined DS to the gradient-aligned CS, despite a weak spatial signal. Here we demonstrate that, in mating cells, the initially uniform receptor and G protein first polarize to the DS, then redistribute along the plasma membrane until they reach the CS. Our data indicate that signaling, polarity, and trafficking proteins localize to the DS during assembly of what we call the gradient tracking machine (GTM). Differential activation of the receptor triggers feedback mechanisms that bias exocytosis upgradient and endocytosis downgradient, thus enabling redistribution of the GTM toward the pheromone source. The GTM stabilizes when the receptor peak centers at the CS and the endocytic machinery surrounds it. A computational model simulates GTM tracking and stabilization and correctly predicts that its assembly at a single site contributes to mating fidelity.

Conjugation in Ustilago violacea. I. Morphology

1974 ◽  
Vol 20 (2) ◽  
pp. 187-191 ◽  
Author(s):  
N. H. Poon ◽  
J. Martin ◽  
A. W. Day
Keyword(s):  
Cell Cycle ◽  
Mating Type ◽  
Electron Micrographs ◽  
Cell Walls ◽  
Plasma Membranes ◽  
Smut Fungus ◽  
Type Ii ◽  
Opposite Mating Type ◽  
Ustilago Violacea ◽  
Anther Smut

Conjugation in the anther smut fungus, Ustilago violacea, is described and five stages are characterized viz. (i) intimate pairing of cells of opposite mating type; (ii) development of bumps from each cell at the point of pairing. The cell walls of opposing pegs are fused, but the plasma membranes are not yet affected; (iii) elongation of the bumps into pegs; (iv) dissolution of the walls and plasma membranes of opposing pegs at the point of contact, and the formation of a tube; (v) elongation of the tube to the mature mating configuration (about 5 μm). Electron micrographs and Nomarski interference contrast micrographs of this sequence are illustrated. The assembly of the conjugation tube begins as early as 1.5 h after the cells are mixed on mating medium and is completed in about 45 min. Even in asynchronous populations there is a burst of synchronous mating, followed by later asynchronous mating. Observations on the ability to mate of unbudded and budded cells support the evidence from cell cycle work that allele a1 mates only in the G1 phase (unbudded) while allele a2 is competent to mate during all phases.

scholarly journals Saccharomyces cerevisiae Cdc42p Localizes to Cellular Membranes and Clusters at Sites of Polarized Growth

2002 ◽  
Vol 1 (3) ◽  
pp. 458-468 ◽  
Author(s):  
Tamara J. Richman ◽  
Mathew M. Sawyer ◽  
Douglas I. Johnson
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Fluorescent Protein ◽  
Neck Region ◽  
Time Lapse ◽  
Yeast Cells ◽  
Membrane Localization ◽  
Cell Periphery ◽  
Polarized Growth ◽  
Bud Neck

ABSTRACT The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.

scholarly journals Pheromones Are Essential for Male Fertility and Sufficient To Direct Chemotropic Polarized Growth of Trichogynes during Mating in Neurospora crassa

2006 ◽  
Vol 5 (3) ◽  
pp. 544-554 ◽  
Author(s):  
Hyojeong Kim ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
Mating Type ◽  
Male Fertility ◽  
Mate Recognition ◽  
Mating Types ◽  
Polarized Growth ◽  
Opposite Mating Type ◽  
Cognate Receptor ◽  
Female Specific ◽  
Self Stimulation

ABSTRACT Neurospora crassa is a self-sterile filamentous fungus with two mating types, mat A and mat a. Its mating involves chemotropic polarized growth of female-specific hyphae (trichogynes) toward male cells of the opposite mating type in a process involving pheromones and receptors. mat A cells express the ccg-4 pheromone and the pre-1 receptor, while mat a strains produce mRNA for the pheromone mfa-1 and the pre-2 receptor; MFA-1 and CCG-4 are the predicted ligands for PRE-1 and PRE-2, respectively. In this study, we generated Δccg-4 and Δmfa-1 mutants and engineered a mat a strain to coexpress ccg-4 and its receptor, pre-2. As males, Δccg-4 mat A and Δmfa-1 mat a mutants were unable to attract mat a and mat A trichogynes, respectively, and consequently failed to initiate fruiting body (perithecial) development or produce meiotic spores (ascospores). In contrast, Δccg-4 mat a and Δmfa-1 mat A mutants exhibited normal chemotropic attraction and male fertility. Δccg-4 Δmfa-1 double mutants displayed defective chemotropism and male sterility in both mating types. Heterologous expression of ccg-4 enabled mat a males to attract mat a trichogynes, although subsequent perithecial differentiation did not occur. Expression of ccg-4 and pre-2 in the same strain triggered self-stimulation, resulting in formation of barren perithecia with no ascospores. Our results indicate that CCG-4 and MFA-1 are required for mating-type-specific male fertility and that pheromones (and receptors) are initial determinants for sexual identity during mate recognition. Furthermore, a self-attraction signal can be transmitted within a strain that expresses a pheromone and its cognate receptor.

scholarly journals Cdc24, the source of active Cdc42, transiently interacts with septins to create a positive feedback between septin assembly and bud site formation in yeast cells

2019 ◽  
Author(s):  
Julian Chollet ◽  
Alexander Dünkler ◽  
Anne Bäuerle ◽  
Laura Vivero-Pol ◽  
Thomas Gronemeyer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Positive Feedback ◽  
Daughter Cell ◽  
Yeast Cells ◽  
Site Formation ◽  
Critical Events ◽  
High Concentration ◽  
Assembly Pathway ◽  
The Stability ◽  
Bud Site

AbstractYeast cells select at the beginning of each cell cycle the position of their new bud. The recruitment of the septins to this prospective bud site (PBS) is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. The septin-rods follow hereby the high concentration of Cdc42GTP that is generated by the focused location of its GEF Cdc24. We show that Cdc24 not only activates Cdc42 but temporarily interacts shortly before budding with Cdc11, the subunit that caps septin rods at its both ends. Mutations in Cdc24 that reduce the affinity to Cdc11 impair septin assembly and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize into the ring, Cdc24 transfers to its center and directs from there the further outgrowth of the membrane.

scholarly journals Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase

2019 ◽  
Author(s):  
Iker Lamas ◽  
Laura Merlini ◽  
Aleksandar Vještica ◽  
Vincent Vincenzetti ◽  
Sophie G Martin
Keyword(s):  
Plasma Membrane ◽  
Positive Feedback ◽  
Active Zone ◽  
Feedback Regulation ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Yeast Cells ◽  
Ras Gtpase ◽  
Dependent Protein ◽  
Protein Recruitment

AbstractThe small GTPase Cdc42 is critical for cell polarization. Scaffold-mediated positive feedback regulation was proposed to mediate symmetry-breaking to a single active zone in budding yeast cells. In rod-shaped fission yeast S. pombe cells, active Cdc42-GTP localizes to both cell poles, where it promotes bipolar growth. Here, we implement the CRY2-CIBN optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the GEF Scd1, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is completely bypassed and positive feedback restored by an engineered interaction between the GEF and a Cdc42 effector, the Pak1 kinase. Remarkably, such re-wired cells are viable and grow in a bipolar manner even when lacking otherwise essential Cdc42 activators. Interestingly, these cells reveal that Ras1 GTPase plays a dual role in localizing and activating the GEF, thus potentiating the feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, is minimally required for rod-shape formation.

Physical monitoring of mating type switching in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2342-2349 ◽  
Author(s):  
B Connolly ◽  
C I White ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mating Type ◽  
G1 Phase ◽  
Opposite Mating Type ◽  
Synthesis Inhibitor ◽  
Mating Type Switching ◽  
G2 Phase ◽  
Kinetics Of ◽  
Ho Gene

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

scholarly journals Physical monitoring of mating type switching in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (6) ◽  
pp. 2342-2349 ◽  
Author(s):  
B Connolly ◽  
C I White ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mating Type ◽  
G1 Phase ◽  
Opposite Mating Type ◽  
Synthesis Inhibitor ◽  
Mating Type Switching ◽  
G2 Phase ◽  
Kinetics Of ◽  
Ho Gene

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

scholarly journals Checkpoint Adaptation Precedes Spontaneous and Damage-Induced Genomic Instability in Yeast

2001 ◽  
Vol 21 (5) ◽  
pp. 1710-1718 ◽  
Author(s):  
David J. Galgoczy ◽  
David P. Toczyski
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Genomic Instability ◽  
Cell Cycle Progression ◽  
Repair Process ◽  
Yeast Cells ◽  
Chromosome Loss ◽  
Checkpoint Adaptation ◽  
Block Cell Cycle Progression ◽  
Break Induced Replication

ABSTRACT Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents. We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest. Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entrée into some forms of genomic instability. Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants. This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest. Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair.

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