scholarly journals SFI1 promotes centriole duplication by recruiting USP9X to stabilize the microcephaly protein STIL

2019 ◽  
Vol 218 (7) ◽  
pp. 2185-2197 ◽  
Author(s):  
Andrew Kodani ◽  
Tyler Moyer ◽  
Allen Chen ◽  
Andrew Holland ◽  
Christopher A. Walsh ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Mammalian Cells ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
S Phase ◽  
Loss Of Function ◽  
Centrosomal Protein ◽  
Centriole Duplication ◽  
Pole Body ◽  
Mammalian Homologue

In mammals, centrioles participate in brain development, and human mutations affecting centriole duplication cause microcephaly. Here, we identify a role for the mammalian homologue of yeast SFI1, involved in the duplication of the yeast spindle pole body, as a critical regulator of centriole duplication in mammalian cells. Mammalian SFI1 interacts with USP9X, a deubiquitylase associated with human syndromic mental retardation. SFI1 localizes USP9X to the centrosome during S phase to deubiquitylate STIL, a critical regulator of centriole duplication. USP9X-mediated deubiquitylation protects STIL from degradation. Consistent with a role for USP9X in stabilizing STIL, cells from patients with USP9X loss-of-function mutations have reduced STIL levels. Together, these results demonstrate that SFI1 is a centrosomal protein that localizes USP9X to the centrosome to stabilize STIL and promote centriole duplication. We propose that the USP9X protection of STIL to facilitate centriole duplication underlies roles of both proteins in human neurodevelopment.

scholarly journals The Mammalian γ-Tubulin Complex Contains Homologues of the Yeast Spindle Pole Body Components Spc97p and Spc98p

1998 ◽  
Vol 141 (3) ◽  
pp. 663-674 ◽  
Author(s):  
Steven M. Murphy ◽  
Lenore Urbani ◽  
Tim Stearns
Keyword(s):  
Mammalian Cells ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Organizing Centers ◽  
Molecular Weights ◽  
Microtubule Polymerization ◽  
Pole Body ◽  
Human Proteins ◽  
Body Components ◽  
Cytoplasmic Complex

γ-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. γ-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic γ-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human γ-tubulin were made. The epitope-tagged γ-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic γ-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the γ-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with γ-tubulin at the centrosome, cosediment with γ-tubulin in sucrose gradients, and coimmunoprecipitate with γ-tubulin, indicating that they are part of the γ-tubulin complex. The conservation of a complex involving γ-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.

Identification of an Spc110p-related protein in vertebrates

1997 ◽  
Vol 110 (20) ◽  
pp. 2533-2545 ◽  
Author(s):  
A.M. Tassin ◽  
C. Celati ◽  
M. Paintrand ◽  
M. Bornens
Keyword(s):  
Mammalian Cells ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Partial Purification ◽  
Related Protein ◽  
Microtubule Nucleation ◽  
Pole Body ◽  
Egg Extract ◽  
Ring Complex ◽  
Gamma Tubulin

Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.

scholarly journals The S. pombe mitotic regulator Cut12 promotes spindle pole body activation and integration into the nuclear envelope

2009 ◽  
Vol 185 (5) ◽  
pp. 875-888 ◽  
Author(s):  
Victor A. Tallada ◽  
Kenji Tanaka ◽  
Mitsuhiro Yanagida ◽  
Iain M. Hagan
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Cell Cycle Control ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Loss Of Function ◽  
Pole Body ◽  
Cytoplasmic Structure ◽  
Monopolar Spindle ◽  
Nuclear Component

The fission yeast spindle pole body (SPB) comprises a cytoplasmic structure that is separated from an ill-defined nuclear component by the nuclear envelope. Upon mitotic commitment, the nuclear envelope separating these domains disperses as the two SPBs integrate into a hole that forms in the nuclear envelope. The SPB component Cut12 is linked to cell cycle control, as dominant cut12.s11 mutations suppress the mitotic commitment defect of cdc25.22 cells and elevated Cdc25 levels suppress the monopolar spindle phenotype of cut12.1 loss of function mutations. We show that the cut12.1 monopolar phenotype arises from a failure to activate and integrate the new SPB into the nuclear envelope. The activation of the old SPB was frequently delayed, and its integration into the nuclear envelope was defective, resulting in leakage of the nucleoplasm into the cytoplasm through large gaps in the nuclear envelope. We propose that these activation/integration defects arise from a local deficiency in mitosis-promoting factor activation at the new SPB.

scholarly journals Identification and localization of a novel, cytoskeletal, centrosome-associated protein in PtK2 cells.

1988 ◽  
Vol 107 (6) ◽  
pp. 2669-2678 ◽  
Author(s):  
A T Baron ◽  
J L Salisbury
Keyword(s):  
Mammalian Cells ◽  
Cellular Proliferation ◽  
Cell Structure ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Yeast Cells ◽  
Cell Cycle Gene ◽  
Microtubule Organizing Centers ◽  
Pole Body ◽  
Cell Biol

Antisera raised against centrin (Salisbury, J.L., A.T. Baron, B. Surek, and M. Melkonian. 1984. J. Cell Biol. 99:962-970) have been used, here, to identify a centrosome-associated protein with an Mr of 165,000. Immunocytochemistry indicates that this protein is a component of pericentriolar satellites, basal feet, and pericentriolar matrix of interphase cells. These components of pericentriolar material are, in part, composed of 3-8-nm-diam filaments, which interconnect to form a three-dimensional pericentriolar lattice. We conclude that the 165,000-Mr protein is immunologically related to centrin, and that it is a component of a novel centrosome-associated cytoskeletal filament system. Microtubule organizing centers such as the flagellar apparatus of algal cells, spindle pole body of yeast cells, and centrosome of mammalian cells are homologous structures essential for cytoplasmic organization and cellular proliferation. Molecular cloning studies have recently shown that the cell cycle gene product CDC31, required for spindle pole body duplication, shares 50% sequence homology with centrin (Huang, B., A. Mengersen, and V.D. Lee. 1988. J. Cell Biol. 107:133-140). The evolutionary conservation of centrin-related sequences and immunologic epitopes to microtubule organizing centers of divergent phylogeny suggests that a functional attribute(s) may have been conserved as well. Elucidation of a common thread between these related molecules may be fundamental to our understanding of cell structure and function.

SFI1 and centrin form a distal end complex critical for proper centriole architecture and ciliogenesis

2021 ◽  
Author(s):  
Imène B. Bouhlel ◽  
Marine. H. Laporte ◽  
Eloïse Bertiaux ◽  
Alexia Giroud ◽  
Susanne Borgers ◽  
...  
Keyword(s):  
Basal Body ◽  
Spindle Pole Body ◽  
Super Resolution ◽  
Spindle Pole ◽  
Specific Loss ◽  
Centriole Duplication ◽  
Pole Body ◽  
Core Elements ◽  
Bona Fide ◽  
And Function

AbstractOver the course of evolution, the function of the centrosome has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, as illustrated by the presence of centrioles in humans and a spindle pole body in yeast (SPB). Consistently, the composition of these two core elements has diverged greatly, with the exception of centrin, a protein known to form a complex with Sfi1 in yeast to structurally initiate SPB duplication. Even though SFI1 has been localized to human centrosomes, whether this complex exists at centrioles and whether its function has been conserved is still unclear. Here, using conventional fluorescence and super-resolution microscopies, we demonstrate that human SFI1 is a bona fide centriolar protein localizing to the very distal end of the centriole, where it associates with a pool of distal centrin. We also found that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the specific loss of the distal pool of centrin, without altering centriole duplication in human cells, in contrast to its function for SPB. Instead, we found that SFI1/centrin complexes are essential for correct centriolar architecture as well as for ciliogenesis. We propose that SFI1/centrin complexes may guide centriole growth to ensure centriole integrity and function as a basal body.

scholarly journals BIMA, a TPR-containing protein required for mitosis, localizes to the spindle pole body in Aspergillus nidulans.

1993 ◽  
Vol 120 (4) ◽  
pp. 959-968 ◽  
Author(s):  
P M Mirabito ◽  
N R Morris
Keyword(s):  
Aspergillus Nidulans ◽  
Spindle Pole Body ◽  
Apparent Molecular Weight ◽  
Spindle Pole ◽  
Nuclear Staining ◽  
Loss Of Function ◽  
Independent Manner ◽  
Pole Body ◽  
A Cell ◽  
Arrest Growth

The Aspergillus nidulans bimA gene is required for mitosis. Loss of function mutations in bimA cause cells to arrest growth with condensed chromatin and a short, metaphaselike mitotic spindle. bimA is a member of a gene family defined by a repeated motif called the Tetratrico Peptide Repeat (TPR), which is found in genes from bacteria, yeast and insects. Several yeast TPR genes are also required for mitosis, including Saccharomyces cerevisiae CDC27 and Schizosaccharomyces pombe nuc2+, which appear to be functional homologs of bimA. We have developed antisera specific to the bimA protein (BIMA) and have characterized BIMA by western blot and immunocytochemical analyses. BIMA is heterogeneous in apparent molecular weight, consisting of a major 90-kD species and at least two minor species of approximately 105 kD. The results of BIMA localization by immunofluorescence microscopy depend on the level of BIMA expression. Overexpression of BIMA, which had no deleterious affect on growth or mitosis, resulted in localization of BIMA on or throughout most nuclei. Nuclear staining was granular, and overlapped but was not completely coincident with DNA staining by DAPI. In contrast, when expressed at normal levels, BIMA colocalized with the spindle pole body (SPB). BIMA localized to the SPB in a cell cycle independent manner. These results show that BIMA is either associated with or is a component of the SPB, and they suggest that BIMA functions at the spindle poles to promote the onset of anaphase.

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