Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1Δ dma2Δ cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase.

Download Full-text

Modeling disease-correlated TUBA1A mutation in budding yeast reveals a molecular basis for tubulin dysfunction

10.1101/2020.04.13.039982 ◽

2020 ◽

Author(s):

E. Denarier ◽

K.H. Ecklund ◽

G. Berthier ◽

A. Favier ◽

S. Gory ◽

...

Keyword(s):

Mammalian Cells ◽

Synthetic Lethality ◽

Budding Yeast ◽

Negative Regulator ◽

Spindle Positioning ◽

Neuronal Progenitor ◽

Spindle Poles ◽

Evolutionary Spectrum ◽

Tubulin Isotype ◽

Dependent Transport

AbstractMalformations of cortical development (MCD) of the human brain are a likely consequence of defective neuronal migration, and/or proliferation of neuronal progenitor cells, both of which are dictated in part by microtubule-dependent transport of various cargoes, including the mitotic spindle. Throughout the evolutionary spectrum, proper spindle positioning depends on cortically anchored dynein motors that exert forces on astral microtubules emanating from spindle poles. A single heterozygous amino acid change, G436R, in the conserved TUBA1A α-tubulin gene was reported to account for MCD in patients. The mechanism by which this mutation disrupts microtubule function in the developing cerebral cortex is not understood. Studying the consequence of tubulin mutations in mammalian cells is challenging partly because of the large number of α-tubulin isotypes expressed. To overcome this challenge, we have generated a budding yeast strain expressing the mutated tubulin (Tub1G437R in yeast) as one of the main sources of α-tubulin (in addition to Tub3, another α-tubulin isotype in this organism). Although viability of the yeast was unimpaired by this mutation, they became reliant on Tub3, as was apparent by the synthetic lethality of this mutant in combination with tub3Δ. We find that Tub1G437R assembles into microtubules that support normal G1 activity, but lead to enhanced dynein-dependent nuclear migration phenotypes during G2/M, and a consequential disruption of spindle positioning. We find that this mutation impairs the interaction between She1 – a negative regulator of dynein – and microtubules, as was apparent from a yeast two-hybrid assay, a co-sedimentation assay, and from live cell imaging. We conclude that a weaker interaction between She1 and Tub1G437R-containing microtubules results in enhanced dynein activity, ultimately leading to the spindle positioning defect. Our results provide the first evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation, and sheds light on a mechanism that may be causative of neurodevelopmental diseases.

Download Full-text

Computational modelling of mitotic exit in budding yeast: the role of separase and Cdc14 endocycles

Journal of The Royal Society Interface ◽

10.1098/rsif.2010.0649 ◽

2011 ◽

Vol 8 (61) ◽

pp. 1128-1141 ◽

Cited By ~ 20

Author(s):

P. K. Vinod ◽

Paula Freire ◽

Ahmed Rattani ◽

Andrea Ciliberto ◽

Frank Uhlmann ◽

...

Keyword(s):

Regulatory Networks ◽

Budding Yeast ◽

Cell Cycle Control ◽

Computational Modelling ◽

Eukaryotic Cell ◽

Mitotic Exit ◽

Intuitive Reasoning ◽

Systematic Analysis ◽

Systems View

The operating principles of complex regulatory networks are best understood with the help of mathematical modelling rather than by intuitive reasoning. Hereby, we study the dynamics of the mitotic exit (ME) control system in budding yeast by further developing the Queralt's model. A comprehensive systems view of the network regulating ME is provided based on classical experiments in the literature. In this picture, Cdc20–APC is a critical node controlling both cyclin (Clb2 and Clb5) and phosphatase (Cdc14) branches of the regulatory network. On the basis of experimental situations ranging from single to quintuple mutants, the kinetic parameters of the network are estimated. Numerical analysis of the model quantifies the dependence of ME control on the proteolytic and non-proteolytic functions of separase. We show that the requirement of the non-proteolytic function of separase for ME depends on cyclin-dependent kinase activity. The model is also used for the systematic analysis of the recently discovered Cdc14 endocycles. The significance of Cdc14 endocycles in eukaryotic cell cycle control is discussed as well.

Download Full-text

Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5

Current Biology ◽

10.1016/s0960-9822(01)00228-7 ◽

2001 ◽

Vol 11 (10) ◽

pp. 784-788 ◽

Cited By ~ 104

Author(s):

Sarah E. Lee ◽

Lisa M. Frenz ◽

Nicholas J. Wells ◽

Anthony L. Johnson ◽

Leland H. Johnston

Keyword(s):

Budding Yeast ◽

Mitotic Exit ◽

Mitotic Exit Network

Download Full-text

A Defect of Kap104 Alleviates the Requirement of Mitotic Exit Network Gene Functions in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/162.4.1545 ◽

2002 ◽

Vol 162 (4) ◽

pp. 1545-1556

Author(s):

Kazuhide Asakawa ◽

Akio Toh-e

Keyword(s):

High Temperature ◽

Cell Cycle Progression ◽

Budding Yeast ◽

Mitotic Exit ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Β Protein ◽

Responsible Gene ◽

Early Anaphase ◽

Mrna Binding

Abstract A subgroup of the karyopherin β (also called importin β) protein that includes budding yeast Kap104 and human transportin/karyopherin β2 is reported to function as a receptor for the transport of mRNA-binding proteins into the nucleus. We identified KAP104 as a responsible gene for a suppressor mutation of cdc15-2. We found that the kap104-E604K mutation suppressed the temperature-sensitive growth of cdc15-2 cells by promoting the exit from mitosis and suppressed the temperature sensitivity of various mitoticexit mutations. The cytokinesis defect of these mitotic-exit mutants was not suppressed by kap104-E604K. Furthermore, the kap104-E604K mutation delays entry into DNA synthesis even at a permissive temperature. In cdc15-2 kap104-E604K cells, SWI5 and SIC1, but not CDH1, became essential at a high temperature, suggesting that the kap104-E604K mutation promotes mitotic exit via the Swi5-Sic1 pathway. Interestingly, SPO12, which is involved in the release of Cdc14 from the nucleolus during early anaphase, also became essential in cdc15-2 kap104-E604K cells at a high temperature. The kap104-E604K mutation caused a partial delocalization of Cdc14 from the nucleolus during interphase. This delocalization of Cdc14 was suppressed by the deletion of SPO12. These results suggest that a mutation in Kap104 stimulates exit from mitosis through the activation of Cdc14 and implies a novel role for Kap104 in cell-cycle progression in budding yeast.

Download Full-text

A Mathematical Model of Mitotic Exit in Budding Yeast: The Role of Polo Kinase

PLoS ONE ◽

10.1371/journal.pone.0030810 ◽

2012 ◽

Vol 7 (2) ◽

pp. e30810 ◽

Cited By ~ 7

Author(s):

Baris Hancioglu ◽

John J. Tyson

Keyword(s):

Mathematical Model ◽

Budding Yeast ◽

Mitotic Exit ◽

Polo Kinase

Download Full-text

Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

Molecular Biology of the Cell ◽

10.1091/mbc.e14-08-1330 ◽

2015 ◽

Vol 26 (7) ◽

pp. 1286-1295 ◽

Cited By ~ 12

Author(s):

Francisco Lázaro-Diéguez ◽

Iaroslav Ispolatov ◽

Anne Müsch

Keyword(s):

Mitotic Spindle ◽

Cell Shape ◽

Spindle Assembly Checkpoint ◽

Mdck Cells ◽

Spindle Pole ◽

Spindle Orientation ◽

Cell Cortex ◽

Spindle Positioning ◽

Spindle Poles ◽

Spindle Position

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.

Download Full-text

The Differential Roles of Budding Yeast Tem1p, Cdc15p, and Bub2p Protein Dynamics in Mitotic Exit

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0708 ◽

2004 ◽

Vol 15 (4) ◽

pp. 1519-1532 ◽

Cited By ~ 63

Author(s):

Jeffrey N. Molk ◽

Scott C. Schuyler ◽

Jenny Y. Liu ◽

James G. Evans ◽

E. D. Salmon ◽

...

Keyword(s):

Budding Yeast ◽

Protein Localization ◽

Spindle Pole Body ◽

Spindle Pole ◽

Mitotic Exit ◽

Yeast Saccharomyces Cerevisiae ◽

Late Anaphase ◽

Gfp Fluorescence ◽

Mitotic Exit Network

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.

Download Full-text

Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

Molecular Biology of the Cell ◽

10.1091/mbc.e12-06-0458 ◽

2013 ◽

Vol 24 (7) ◽

pp. 901-913 ◽

Cited By ~ 37

Author(s):

Zhen Zheng ◽

Qingwen Wan ◽

Jing Liu ◽

Huabin Zhu ◽

Xiaogang Chu ◽

...

Keyword(s):

Mammalian Cells ◽

Fluorescence Recovery After Photobleaching ◽

Cytoplasmic Dynein ◽

Cell Cortex ◽

Mitotic Apparatus ◽

Spindle Positioning ◽

Spindle Poles ◽

Astral Microtubule ◽

Mammalian Homologue ◽

Cortical Localization

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.

Download Full-text

Lipid droplets maintain lipid homeostasis during anaphase for efficient cell separation in budding yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e16-02-0106 ◽

2016 ◽

Vol 27 (15) ◽

pp. 2368-2380 ◽

Cited By ~ 19

Author(s):

Po-Lin Yang ◽

Tzu-Han Hsu ◽

Chao-Wen Wang ◽

Rey-Huei Chen

Keyword(s):

Cell Separation ◽

Neutral Lipids ◽

Budding Yeast ◽

Acid Synthesis ◽

Mitotic Exit ◽

Lipid Homeostasis ◽

Physiological Conditions ◽

Division Site ◽

Quadruple Mutant ◽

Membrane Bound

The neutral lipids steryl ester and triacylglycerol (TAG) are stored in the membrane-bound organelle lipid droplet (LD) in essentially all eukaryotic cells. It is unclear what physiological conditions require the mobilization or storage of these lipids. Here, we study the budding yeast mutant are1Δ are2Δ dga1Δ lro1Δ, which cannot synthesize the neutral lipids and therefore lacks LDs. This quadruple mutant is delayed at cell separation upon release from mitotic arrest. The cells have abnormal septa, unstable septin assembly during cytokinesis, and prolonged exocytosis at the division site at the end of cytokinesis. Lipidomic analysis shows a marked increase of diacylglycerol (DAG) and phosphatidic acid, the precursors for TAG, in the mutant during mitotic exit. The cytokinesis and separation defects are rescued by adding phospholipid precursors or inhibiting fatty acid synthesis, which both reduce DAG levels. Our results suggest that converting excess lipids to neutral lipids for storage during mitotic exit is important for proper execution of cytokinesis and efficient cell separation.

Download Full-text