ABSTRACT
The NEK1 kinase controls ciliogenesis, mitosis, and DNA repair, and NEK1 mutations cause human diseases including axial spondylometaphyseal dysplasia and amyotrophic lateral sclerosis. C21ORF2 mutations cause a similar pattern of human diseases, suggesting close functional links with NEK1 Here, we report that endogenous NEK1 and C21ORF2 form a tight complex in human cells. A C21ORF2 interaction domain "CID" at the C-terminus of NEK1 is necessary for its association with C21ORF2 in cells, and pathogenic mutations in this region disrupt the complex. AlphaFold modelling predicts an extended binding interface between a leucine-rich repeat domain in C21ORF2 and the NEK1-CID, and our model may explain why pathogenic mutations perturb the complex. We show that NEK1 mutations that inhibit kinase activity or weaken its association with C21ORF2 severely compromise ciliogenesis, and that C21ORF2, like NEK1 is required for homologous recombination. These data enhance our understanding of how the NEK1 kinase is regulated, and they shed light on NEK1-C21ORF2-associated diseases.
Subject(s)
DNA Repair , Osteochondrodysplasias , Humans , Mutation/genetics , NIMA-Related Kinase 1/genetics , Osteochondrodysplasias/genetics , PhosphorylationABSTRACT
Centrosomes are microtubule organizing centers involved in chromosome segregation, spindle orientation, cell motility and cilia formation. In recent years, they have also emerged as key modulators of asymmetric cell division. Centrosomes are composed of two centrioles that initiate duplication in S phase. The conservative nature of centriole duplication means that the two centrioles of a G1 cell are of different ages. They are also structurally different as only the older centriole carries appendages, an assembly of a subset of proteins primarily required for cilia formation. In a growing tissue, the non-motile, primary cilium acts as a mechano- and sensory organelle that influences cell behavior via modulation of signaling pathways. Here, we discuss the most recent findings about distal appendage composition and function, as well as cell cycle-specific regulation and their implications in various diseases.
Subject(s)
Centrioles , Centrosome , Centrosome/metabolism , Centrioles/genetics , Centrioles/metabolism , Cell Cycle , Organelles , Microtubule-Organizing CenterABSTRACT
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/metabolismABSTRACT
The spindle position checkpoint (SPOC) is a mitotic surveillance mechanism in Saccharomyces cerevisiae that prevents cells from completing mitosis in response to spindle misalignment, thereby contributing to genomic integrity. The kinase Kin4, one of the most downstream SPOC components, is essential to stop the mitotic exit network (MEN), a signalling pathway that promotes the exit from mitosis and cell division. Previous work, however, suggested that a Kin4-independent pathway contributes to SPOC, yet the underlying mechanisms remain elusive. Here, we established the glycogen-synthase-kinase-3 (GSK-3) homologue Mck1, as a novel component that works independently of Kin4 to engage SPOC. Our data indicate that both Kin4 and Mck1 work in parallel to counteract MEN activation by the Cdc14 early anaphase release (FEAR) network. We show that Mck1's function in SPOC is mediated by the pre-replication complex protein and mitotic cyclin-dependent kinase (M-Cdk) inhibitor, Cdc6, which is degraded in a Mck1-dependent manner prior to mitosis. Moderate overproduction of Cdc6 phenocopies MCK1 deletion and causes SPOC deficiency via its N-terminal, M-Cdk inhibitory domain. Our data uncover an unprecedented role of GSK-3 kinases in coordinating spindle orientation with cell cycle progression.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomycetales , Humans , Saccharomyces cerevisiae Proteins/genetics , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Spindle Apparatus/metabolism , Saccharomycetales/metabolism , Protein Serine-Threonine Kinases , Phosphorylation , Saccharomyces cerevisiae/genetics , Mitosis , Cell Cycle Proteins/metabolism , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolismABSTRACT
The centriole is the microtubule-based backbone that ensures integrity, function, and cell cycle-dependent duplication of centrosomes. Mostly unclear mechanisms control structural integrity of centrioles. Here, we show that the centrosome protein CEP350 functions as scaffold that coordinates distal-end properties of centrioles such as length, stability, and formation of distal and subdistal appendages. CEP350 fulfills these diverse functions by ensuring centriolar localization of WDR90, recruiting the proteins CEP78 and OFD1 to the distal end of centrioles and promoting the assembly of subdistal appendages that have a role in removing the daughter-specific protein Centrobin. The CEP350-FOP complex in association with CEP78 or OFD1 controls centriole microtubule length. Centrobin safeguards centriole distal end stability, especially in the compromised CEP350-/- cells, while the CEP350-FOP-WDR90 axis secures centriole integrity. This study identifies CEP350 as a guardian of the distal-end region of centrioles without having an impact on the proximal PCM part.
Subject(s)
Cell Cycle Proteins , Centrioles , Microtubule-Associated Proteins , Nuclear Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centrioles/metabolism , Centrosome/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/genetics , Microtubules/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolismABSTRACT
The mitotic exit network (MEN) is a conserved signalling pathway essential for the termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Instead, we identified two essential kinases OpHcd1 and OpHcd2 (homologue candidate of ScCdc15) that are homologous to SpSid1 and SpCdc7, respectively, components of the septation initiation network (SIN) of the fission yeast Schizosaccharomyces pombe. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. Unlike Cdc15 in S. cerevisiae, the association of OpHcd1 and OpHcd2 with the yeast centrosomes (named spindle pole bodies, SPBs) is restricted to the SPB in the mother cell body. SPB localisation of OpHcd2 is regulated by the status of OpTem1 GTPase, while OpHcd1 requires the polo-like kinase OpCdc5 as well as active Tem1 to ensure the coordination of mitotic exit (ME) signalling and cell cycle progression. Our study suggests that the divergence of molecular mechanisms to control the ME-signalling pathway as well as the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.
Subject(s)
Monomeric GTP-Binding Proteins , Saccharomyces cerevisiae Proteins , Saccharomycetales , Schizosaccharomyces , Humans , Mitosis , Monomeric GTP-Binding Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomycetales/genetics , Saccharomycetales/metabolism , Schizosaccharomyces/metabolism , Spindle Pole Bodies/metabolismABSTRACT
The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Cyclin-Dependent Kinases , Leukemia, Myeloid, Acute , Protein Kinase Inhibitors , Sulfonamides , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , CDC2 Protein Kinase/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Synergism , Hedgehog Proteins/metabolism , Hedgehog Proteins/therapeutic use , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/therapeutic use , Sulfonamides/pharmacology , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Primary cilia are antenna-like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady-state to promote signalling is largely unknown. Here, we define that mammalian primary cilia axonemes are formed by proximal segment (PS) and distal segment (DS) delineated by tubulin polyglutamylation-rich and -poor regions, respectively. The analysis of proximal/distal segmentation indicated that perturbations leading to cilia over-elongation influenced PS or DS length with a different impact on cilia behaviour. We identified septins as novel repressors of DS growth. We show that septins control the localisation of MKS3 and CEP290 required for a functional transition zone (TZ), and the cilia tip accumulation of the microtubule-capping kinesin KIF7, a cilia-growth inhibitor. Live-cell imaging and analysis of sonic-hedgehog (SHH) signalling activation established that DS over-extension increased cilia ectocytosis events and decreased SHH activation. Our data underlines the importance of understanding cilia segmentation for length control and cilia-dependent signalling.
Subject(s)
Cilia/metabolism , Septins/metabolism , Animals , Antigens, Neoplasm/metabolism , Cell Cycle Proteins/metabolism , Cell Line , Cytoskeletal Proteins/metabolism , Hedgehog Proteins/metabolism , Humans , Kinesins/metabolism , Membrane Proteins/metabolism , Mice , Microtubules/metabolism , Retinal Pigment Epithelium/cytology , Signal TransductionABSTRACT
Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.
Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Targeting/methods , Polymerase Chain Reaction/methods , Alleles , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , Cell Line , Cells, Cultured , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Genes, Reporter , High-Throughput Nucleotide Sequencing , Homologous Recombination , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Oligonucleotides/genetics , RNA, Guide, Kinetoplastida/genetics , TransfectionABSTRACT
Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.
Subject(s)
Centrioles/enzymology , Cilia/enzymology , Epithelial Cells/enzymology , Mitosis , NIMA-Related Kinases/metabolism , Retinal Pigment Epithelium/enzymology , Animals , Binding Sites , Cell Line , Centrioles/genetics , Cilia/genetics , Female , G2 Phase Cell Cycle Checkpoints , Hematopoietic Stem Cells/enzymology , Humans , Mammary Glands, Animal/cytology , Mammary Glands, Animal/enzymology , Mice , Microtubule Proteins/genetics , Microtubule Proteins/metabolism , NIMA-Related Kinases/genetics , Protein Binding , Retinal Pigment Epithelium/cytology , Signal Transduction , Time FactorsABSTRACT
One of the first steps in mitotic spindle assembly is the dissolution of the centrosome linker followed by centrosome separation driven by EG5, a tetrameric plus-end-directed member of the kinesin-5 family. However, even in the absence of the centrosome linker, the two centrosomes are kept together by an ill-defined microtubule-dependent mechanism. Here we show that KIFC3, a minus-end-directed kinesin-14, provides microtubule-based centrosome cohesion. KIFC3 forms a homotetramer that pulls the two centrosomes together via a specific microtubule network. At mitotic onset, KIFC3 activity becomes the main driving force of centrosome cohesion to prevent premature spindle formation after linker dissolution as it counteracts the increasing EG5-driven pushing forces. KIFC3 is eventually inactivated by NEver in mitosis-related Kinase 2 (NEK2) to enable EG5-driven bipolar spindle assembly. We further show that persistent centrosome cohesion in mitosis leads to chromosome mis-segregation. Our findings reveal a mechanism of spindle assembly that is evolutionary conserved from yeast to humans.
Subject(s)
Centrosome/metabolism , Kinesins/metabolism , Microtubules/metabolism , Spindle Apparatus/metabolism , Chromosome Segregation/physiology , HeLa Cells , Humans , Kinesins/genetics , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitosis , NIMA-Related Kinases/metabolismABSTRACT
CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14APD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.
Subject(s)
Actin-Related Protein 2/genetics , Cilia/genetics , Neuropeptides/genetics , Phosphoric Monoester Hydrolases/genetics , Actins/genetics , Cell Line , Cell Movement/genetics , Centrosome/metabolism , Cilia/metabolism , Cyclin-Dependent Kinase 5/genetics , Endocytosis/genetics , Gene Expression Regulation, Developmental/genetics , Humans , Microtubules/genetics , Mutation , Myosin Heavy Chains/genetics , Myosin Type V/genetics , Phosphorylation , Protein Binding , Protein Tyrosine Phosphatases , Proteome/geneticsABSTRACT
Cilia perform essential signalling functions during development and tissue homeostasis. A key event in ciliogenesis occurs when the distal appendages of the mother centriole form a platform that docks ciliary vesicles and removes CP110-Cep97 inhibitory complexes. Here, we analysed the role of LRRC45 in appendage formation and ciliogenesis. We show that the core appendage proteins Cep83 and SCLT1 recruit LRRC45 to the mother centriole. Once there, LRRC45 recruits the keratin-binding protein FBF1. The association of LRRC45 with the basal body of primary and motile cilia in both differentiated and stem cells reveals a broad function in ciliogenesis. In contrast to the appendage components Cep164 and Cep123, LRRC45 was not essential for either docking of early ciliary vesicles or for removal of CP110. Rather, LRRC45 promotes cilia biogenesis in CP110-uncapped centrioles by organising centriolar satellites, establishing the transition zone and promoting the docking of Rab8 GTPase-positive vesicles. We propose that, instead of acting solely as a platform to recruit early vesicles, centriole appendages form discrete scaffolds of cooperating proteins that execute specific functions that promote the initial steps of ciliogenesis.
Subject(s)
Axoneme/metabolism , Carrier Proteins/genetics , Cilia/metabolism , Membrane Proteins/genetics , Carrier Proteins/metabolism , Humans , Membrane Proteins/metabolismABSTRACT
Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Spermatids/enzymology , Animals , Cell Differentiation/physiology , Fertility/physiology , Male , Mice , Mice, Knockout , Microtubules/metabolism , Protein Serine-Threonine Kinases/genetics , Spermatocytes/cytology , Spermatocytes/enzymology , Spermatogenesis/physiology , Spermatozoa/enzymology , Testis/enzymologyABSTRACT
Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha.
Subject(s)
Cell Cycle Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomycetales/metabolism , Spindle Pole Bodies/metabolism , Microscopy, Electron , Saccharomycetales/ultrastructureABSTRACT
Cell cycle-dependent morphogenesis of unicellular organisms depends on the spatiotemporal control of cell polarity. Rho GTPases are the major players that guide cells through structural reorganizations such as cytokinesis (Rho1 dependent) and polarity establishment (Cdc42 dependent). In budding yeast, the protein Gps1 plays a pivotal role in both processes. Gps1 resides at the bud neck to maintain Rho1 localization and restrict Cdc42 activity during cytokinesis. Here we analyze how Gps1 is recruited to the bud neck during the cell cycle. We show that different regions of Gps1 and the septin-associated kinase Gin4 are involved in maintaining Gps1 at the bud neck from late G1 phase until midanaphase. From midanaphase, the targeting function of Gin4 is taken over by the bud neck-associated protein Nba1. Our data show that Gps1 is targeted to the cell division site in a biphasic manner, via Gin4 and Nba1, to control the spatiotemporal activation of Rho1 and inhibition of Cdc42.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Cycle/physiology , Cell Cycle Proteins/metabolism , Cell Division/physiology , Cytokinesis , Mitosis , Protein Kinases/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Septins/metabolism , Signal Transduction , rho GTP-Binding Proteins/metabolismABSTRACT
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/metabolismABSTRACT
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/ultrastructureABSTRACT
In mitotic exit, proteins that were highly phosphorylated are sequentially targeted by the phosphatase PP2A-B55, but what underlies substrate selection is unclear. In this issue, Cundell et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201606033) identify the determinants of PP2A-B55's dephosphorylation program, thereby influencing spindle disassembly, nuclear envelope reformation, and cytokinesis.
Subject(s)
Mitosis , Protein Phosphatase 2/metabolism , HeLa Cells , Humans , Models, Biological , Phosphorylation , Spindle Apparatus/metabolism , Substrate SpecificityABSTRACT
The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control.