scholarly journals Role of Inn1 and its interactions with Hof1 and Cyk3 in promoting cleavage furrow and septum formation in S. cerevisiae

2009 ◽  
Vol 185 (6) ◽  
pp. 995-1012 ◽  
Author(s):  
Ryuichi Nishihama ◽  
Jennifer H. Schreiter ◽  
Masayuki Onishi ◽  
Elizabeth A. Vallen ◽  
Julia Hanna ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Exit ◽  
Chitin Synthase ◽  
Cleavage Furrow ◽  
Sh3 Domains ◽  
C2 Domains ◽  
Division Site ◽  
Primary Septum ◽  
N Terminus

Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Δ cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow.

scholarly journals Role of the Hof1–Cyk3 interaction in cleavage-furrow ingression and primary-septum formation during yeast cytokinesis

2018 ◽  
Vol 29 (5) ◽  
pp. 597-609 ◽  
Author(s):  
Meng Wang ◽  
Ryuichi Nishihama ◽  
Masayuki Onishi ◽  
John R. Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Significance ◽  
Normal Rate ◽  
Cleavage Furrow ◽  
Type Ii ◽  
Septum Formation ◽  
Primary Septum ◽  
Direct Binding ◽  
Type Ii Myosin

In Saccharomyces cerevisiae, it is well established that Hof1, Cyk3, and Inn1 contribute to septum formation and cytokinesis. Because hof1∆ and cyk3∆ single mutants have relatively mild defects but hof1∆ cyk3∆ double mutants are nearly dead, it has been hypothesized that these proteins contribute to parallel pathways. However, there is also evidence that they interact physically. In this study, we examined this interaction and its functional significance in detail. Our data indicate that the interaction 1) is mediated by a direct binding of the Hof1 SH3 domain to a proline-rich motif in Cyk3; 2) occurs specifically at the time of cytokinesis but is independent of the (hyper)phosphorylation of both proteins that occurs at about the same time; 3) is dispensable for the normal localization of both proteins; 4) is essential for normal primary-septum formation and a normal rate of cleavage-furrow ingression; and 5) becomes critical for growth when either Inn1 or the type II myosin Myo1 (a key component of the contractile actomyosin ring) is absent. The similarity in phenotype between cyk3∆ mutants and mutants specifically lacking the Hof1–Cyk3 interaction suggests that the interaction is particularly important for Cyk3 function, but it may be important for Hof1 function as well.

scholarly journals Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II

2010 ◽  
Vol 191 (7) ◽  
pp. 1333-1350 ◽  
Author(s):  
Xiaodong Fang ◽  
Jianying Luo ◽  
Ryuichi Nishihama ◽  
Carsten Wloka ◽  
Christopher Dravis ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Trafficking ◽  
Myosin Ii ◽  
Force Generation ◽  
Extracellular Matrix Remodeling ◽  
Cleavage Furrow ◽  
Matrix Remodeling ◽  
Division Site ◽  
Targeting Signals

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR). However, how myosin II is targeted to the division site and promotes AMR assembly, and how the AMR coordinates with membrane trafficking during cytokinesis, remains poorly understood. Here we show that Myo1 is a two-headed myosin II in Saccharomyces cerevisiae, and that Myo1 localizes to the division site via two distinct targeting signals in its tail that act sequentially during the cell cycle. Before cytokinesis, Myo1 localization depends on the septin-binding protein Bni5. During cytokinesis, Myo1 localization depends on the IQGAP Iqg1. We also show that the Myo1 tail is sufficient for promoting the assembly of a “headless” AMR, which guides membrane deposition and extracellular matrix remodeling at the division site. Our study establishes a biphasic targeting mechanism for myosin II and highlights an underappreciated role of the AMR in cytokinesis beyond force generation.

scholarly journals Mitotic exit kinase Dbf2 directly phosphorylates chitin synthase Chs2 to regulate cytokinesis in budding yeast

2012 ◽  
Vol 23 (13) ◽  
pp. 2445-2456 ◽  
Author(s):  
Younghoon Oh ◽  
Kuang-Jung Chang ◽  
Peter Orlean ◽  
Carsten Wloka ◽  
Raymond Deshaies ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Cell Cycle Control ◽  
Mitotic Exit ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ecm Remodeling ◽  
Division Site ◽  
Primary Septum

How cell cycle machinery regulates extracellular matrix (ECM) remodeling during cytokinesis remains poorly understood. In the budding yeast Saccharomyces cerevisiae, the primary septum (PS), a functional equivalent of animal ECM, is synthesized during cytokinesis by the chitin synthase Chs2. Here, we report that Dbf2, a conserved mitotic exit kinase, localizes to the division site after Chs2 and directly phosphorylates Chs2 on several residues, including Ser-217. Both phosphodeficient (chs2‑S217A) and phosphomimic (chs2‑S217D) mutations cause defects in cytokinesis, suggesting that dynamic phosphorylation–dephosphorylation of Ser-217 is critical for Chs2 function. It is striking that Chs2‑S217A constricts asymmetrically with the actomyosin ring (AMR), whereas Chs2-S217D displays little or no constriction and remains highly mobile at the division site. These data suggest that Chs2 phosphorylation by Dbf2 triggers its dissociation from the AMR during the late stage of cytokinesis. Of interest, both chs2‑S217A and chs2‑S217D mutants are robustly suppressed by increased dosage of Cyk3, a cytokinesis protein that displays Dbf2‑dependent localization and also stimulates Chs2‑mediated chitin synthesis. Thus Dbf2 regulates PS formation through at least two independent pathways: direct phosphorylation and Cyk3‑mediated activation of Chs2. Our study establishes a mechanism for direct cell cycle control of ECM remodeling during cytokinesis.

scholarly journals Phosphatase 2A Negatively Regulates Mitotic Exit in Saccharomyces cerevisiae

2006 ◽  
Vol 17 (1) ◽  
pp. 80-89 ◽  
Author(s):  
Yanchang Wang ◽  
Tuen-Yung Ng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Exit ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Catalytic Subunits ◽  
Phosphatase 2A ◽  
Negative Role

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.

scholarly journals Inhibition of Cdc42 during mitotic exit is required for cytokinesis

2013 ◽  
Vol 202 (2) ◽  
pp. 231-240 ◽  
Author(s):  
Benjamin D. Atkins ◽  
Satoshi Yoshida ◽  
Koji Saito ◽  
Chi-Fang Wu ◽  
Daniel J. Lew ◽  
...  
Keyword(s):  
Cell Cycle ◽  
General Feature ◽  
Budding Yeast ◽  
Mitotic Exit ◽  
Division Site ◽  
Polo Kinase ◽  
Cell Cycle Regulator ◽  
P21 Activated Kinase

The role of Cdc42 and its regulation during cytokinesis is not well understood. Using biochemical and imaging approaches in budding yeast, we demonstrate that Cdc42 activation peaks during the G1/S transition and during anaphase but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to down-regulate Cdc42 during mitotic exit impairs the normal localization of key cytokinesis regulators—Iqg1 and Inn1—at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase Ste20. Inhibition of Cdc42 and related Rho guanosine triphosphatases may be a general feature of cytokinesis in eukaryotes.

In budding yeast, contraction of the actomyosin ring and formation of the primary septum at cytokinesis depend on each other

2002 ◽  
Vol 115 (2) ◽  
pp. 293-302 ◽  
Author(s):  
Martin Schmidt ◽  
Blair Bowers ◽  
Archana Varma ◽  
Dong-Hyun Roh ◽  
Enrico Cabib
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Double Mutant ◽  
Chitin Synthase ◽  
Contractile Ring ◽  
Ring Contraction ◽  
Primary Septum ◽  
Common Process ◽  
Lateral Walls

Saccharomyces cerevisiae chs2 mutants are unable to synthesize primary septum chitin, and myo1 mutants cannot construct a functional contractile ring. The morphology of the two mutants, as observed by electron microscopy, is very similar. In both cases, neither an invagination of the plasma membrane, which normally results from contraction of the actomyosin ring, nor generation of a chitin disc, the primary septum, is observed. Rather, both mutants are able to complete cytokinesis by an abnormal process in which lateral walls thicken gradually and finally meet over an extended region, giving rise to a thick septum lacking the normal trilaminar structure and often enclosing lacunae. Defects in chs2 or myo1 strains were not aggravated in a double mutant, an indication that the corresponding proteins participate in a common process. In contrast, in a chs3 background the chs2 mutation is lethal and the myo1 defect is greatly worsened, suggesting that the synthesis of chitin catalyzed by chitin synthase III is necessary for the functionality of the remedial septa. Both chs2 and myo1 mutants show abnormalities in budding pattern and a decrease in the level of certain proteins associated with budding, such as Bud3p, Bud4p and Spa2p. The possible reasons for these phenotypes and for the interdependence between actomyosin ring contraction and primary septum formation are discussed.

scholarly journals Structural role of Sfi1p–centrin filaments in budding yeast spindle pole body duplication

2006 ◽  
Vol 173 (6) ◽  
pp. 867-877 ◽  
Author(s):  
Sam Li ◽  
Alan M. Sandercock ◽  
Paul Conduit ◽  
Carol V. Robinson ◽  
Roger L. Williams ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Crystal Structures ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
N Terminus ◽  
Α Helix

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–centrin complexes containing several repeats show Sfi1p as an α helix with centrins wrapped around each repeat and similar centrin–centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.

scholarly journals CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2485-2496 ◽  
Author(s):  
B Santos ◽  
A Duran ◽  
M H Valdivieso
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Fusion ◽  
Chitin Synthase ◽  
Activity Levels ◽  
Wild Type ◽  
Neurofilament Proteins ◽  
Chitin Synthesis ◽  
Mating Efficiency ◽  
Carboxy Terminal

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.

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