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1.
Open Biol ; 13(9): 230125, 2023 09.
Article in English | MEDLINE | ID: mdl-37751887

ABSTRACT

Coordination of mitotic exit with chromosome segregation is key for successful mitosis. Mitotic exit in budding yeast is executed by the mitotic exit network (MEN), which is negatively regulated by the spindle position checkpoint (SPOC). SPOC kinase Kin4 is crucial for SPOC activation in response to spindle positioning defects. Here, we report that the lysosomal signalling lipid phosphatidylinositol-3,5-bisphosphate (PI3,5P2) has an unanticipated role in the timely execution of mitotic exit. We show that the lack of PI3,5P2 causes a delay in mitotic exit, whereas elevated levels of PI3,5P2 accelerates mitotic exit in mitotic exit defective cells. Our data indicate that PI3,5P2 promotes mitotic exit in part through impairment of Kin4. This process is largely dependent on the known PI3,5P2 effector protein Atg18. Our work thus uncovers a novel link between PI3,5P2 and mitotic exit.


Subject(s)
Mitosis , Signal Transduction , Humans , Chromosome Segregation , M Phase Cell Cycle Checkpoints , Lipids
2.
Mol Biol Cell ; 34(2): ar11, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36542480

ABSTRACT

Faithful chromosome segregation in budding yeast requires correct positioning of the mitotic spindle along the mother to daughter cell polarity axis. When the anaphase spindle is not correctly positioned, a surveillance mechanism, named as the spindle position checkpoint (SPOC), prevents the progression out of mitosis until correct spindle positioning is achieved. How SPOC works on a molecular level is not well understood. Here we performed a genome-wide genetic screen to search for components required for SPOC. We identified the SWR1 chromatin-remodeling complex (SWR1-C) among several novel factors that are essential for SPOC integrity. Cells lacking SWR1-C were able to activate SPOC upon spindle misorientation but underwent mitotic slippage upon prolonged SPOC arrest. This mitotic slippage required the Cdc14-early anaphase release pathway and other factors including the SAGA (Spt-Ada-Gcn5 acetyltransferase) histone acetyltransferase complex, proteasome components and the mitotic cyclin-dependent kinase inhibitor Sic1. Together, our data establish a novel link between SWR1-C chromatin remodeling and robust checkpoint arrest in late anaphase.


Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Chromatin Assembly and Disassembly , Mitosis , M Phase Cell Cycle Checkpoints , Spindle Apparatus/metabolism , Cell Cycle Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Adenosine Triphosphatases/metabolism
3.
Elife ; 102021 10 11.
Article in English | MEDLINE | ID: mdl-34633288

ABSTRACT

Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the spindle position checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5, and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.


Subject(s)
Cell Polarity , M Phase Cell Cycle Checkpoints/physiology , Mitosis , Protein Phosphatase 1/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/physiology , Spindle Apparatus/physiology , Protein Phosphatase 1/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism
4.
Nat Commun ; 8: 14129, 2017 01 24.
Article in English | MEDLINE | ID: mdl-28117323

ABSTRACT

The spatiotemporal control of mitotic exit is crucial for faithful chromosome segregation during mitosis. In budding yeast, the mitotic exit network (MEN) drives cells out of mitosis, whereas the spindle position checkpoint (SPOC) blocks MEN activity when the anaphase spindle is mispositioned. How the SPOC operates at a molecular level remains unclear. Here, we report novel insights into how mitotic signalling pathways orchestrate chromosome segregation in time and space. We establish that the key function of the central SPOC kinase, Kin4, is to counterbalance MEN activation by the cdc fourteen early anaphase release (FEAR) network in the mother cell compartment. Remarkably, Kin4 becomes dispensable for SPOC function in the absence of FEAR. Cells lacking both FEAR and Kin4 show that FEAR contributes to mitotic exit through regulation of the SPOC component Bfa1 and the MEN kinase Cdc15. Furthermore, we uncover controls that specifically promote mitotic exit in the daughter cell compartment.


Subject(s)
Genes, Fungal/physiology , Mitosis/physiology , Signal Transduction/physiology , Spindle Apparatus/physiology , Chromosome Segregation/physiology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
5.
Methods Mol Biol ; 1505: 167-182, 2017.
Article in English | MEDLINE | ID: mdl-27826864

ABSTRACT

Fluorescence recovery after photobleaching (FRAP) is a powerful technique to study in vivo binding and diffusion dynamics of fluorescently labeled proteins. In this chapter, we describe how to determine spindle pole body (SPB) binding dynamics of mitotic exit network (MEN) and spindle position checkpoint (SPOC) proteins using FRAP microscopy. Procedures presented here include the growth of the yeast cultures, sample preparation, image acquisition and analysis.


Subject(s)
Cell Cycle Proteins/analysis , Fluorescence Recovery After Photobleaching/methods , Microscopy, Fluorescence/methods , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/cytology , Spindle Pole Bodies/metabolism , Cell Cycle Proteins/metabolism , M Phase Cell Cycle Checkpoints , Mitosis , Optical Imaging/methods , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Spindle Pole Bodies/ultrastructure
6.
Mol Biol Cell ; 25(14): 2143-51, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24850890

ABSTRACT

In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1-centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.


Subject(s)
Chromosomes, Fungal/metabolism , Cytoskeletal Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , 14-3-3 Proteins/physiology , M Phase Cell Cycle Checkpoints , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Saccharomyces cerevisiae/cytology , Spindle Apparatus/metabolism
7.
Mol Syst Biol ; 8: 582, 2012 May 08.
Article in English | MEDLINE | ID: mdl-22580890

ABSTRACT

The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother-to-daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase-activating protein (GAP) complex Bfa1-Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1-Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1-Bub2 and Tem1 localization at the SPBs. Based on the measured SPB-bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition.


Subject(s)
Gene Expression Regulation, Fungal , Models, Theoretical , Saccharomyces cerevisiae/genetics , Spindle Apparatus/metabolism , Systems Biology/methods , Asymmetric Cell Division , Cell Cycle , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Computer Simulation , Cytoplasm/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , GTP Phosphohydrolases/antagonists & inhibitors , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Microscopy, Fluorescence , Mitosis , Monomeric GTP-Binding Proteins/antagonists & inhibitors , Monomeric GTP-Binding Proteins/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/genetics
8.
Exp Cell Res ; 318(12): 1421-7, 2012 Jul 15.
Article in English | MEDLINE | ID: mdl-22510435

ABSTRACT

The asymmetrically dividing budding yeast relies upon the alignment of the mitotic spindle along the mother to daughter cell polarity axis for the fidelity of chromosome segregation during mitosis. In the case of spindle misalignment, a surveillance mechanism named the spindle position checkpoint (SPOC) prevents cells from exiting mitosis through the inhibition of the mitotic exit network (MEN). MEN is a signal transduction pathway that mediates mitotic exit through fully activation of the Cdk-counteracting phosphatase Cdc14. In this mini-review, we briefly describe the mechanisms leading to mitotic exit in budding yeast cells focusing on the control of MEN by the SPOC. In addition, we summarize the recent advances in the molecular understanding of SPOC regulation and discuss whether similar checkpoints may exist in higher eukaryotic cells that undergo asymmetric divisions.


Subject(s)
Chromosome Segregation/genetics , M Phase Cell Cycle Checkpoints/physiology , Saccharomycetales/genetics , Spindle Apparatus/metabolism , Animals , Chromosome Segregation/physiology , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Guanine Nucleotide Exchange Factors/physiology , Humans , M Phase Cell Cycle Checkpoints/genetics , Male , Models, Biological , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomycetales/physiology , Spindle Apparatus/genetics , Spindle Apparatus/physiology
9.
J Cell Biol ; 190(6): 975-89, 2010 Sep 20.
Article in English | MEDLINE | ID: mdl-20855503

ABSTRACT

Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.


Subject(s)
Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Spindle Apparatus/enzymology , Enzyme Activation , Gene Deletion , Metaphase , Phosphorylation , Phosphothreonine/metabolism , Protein Binding , Protein Kinases/chemistry , Protein Serine-Threonine Kinases , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry
10.
Dev Cell ; 16(1): 146-56, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19154725

ABSTRACT

In many polarized cells, the accuracy of chromosome segregation depends on the correct positioning of the mitotic spindle. In budding yeast, the spindle positioning checkpoint (SPOC) delays mitotic exit when the anaphase spindle fails to extend toward the mother-daughter axis. However it remains to be established how spindle orientation is translated to SPOC components at the yeast spindle pole bodies (SPB). Here, we used photobleaching techniques to show that the dynamics with which Bub2-Bfa1 turned over at SPBs significantly increased upon SPOC activation. A version of Bfa1 that was stably associated with SPBs rendered the cells SPOC deficient without affecting other Bub2-Bfa1 functions, demonstrating the functional importance of regulating the dynamics of Bfa1 SPB association. In addition, we established that the SPOC kinase Kin4 is the major regulator of Bfa1 residence time at SPBs. We suggest that upon SPOC activation Bfa1-Bub2 spreads throughout the cytoplasm, thereby inhibiting mitotic exit.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle/physiology , Spindle Apparatus/metabolism , Cell Cycle Proteins/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Fluorescence Recovery After Photobleaching , Microtubules/metabolism , Protein Binding , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/ultrastructure
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