Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 116
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Cell Sci ; 135(24)2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36524422

RESUMO

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.


Assuntos
Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces , Humanos , Schizosaccharomyces/metabolismo , Saccharomyces cerevisiae/metabolismo , Microscopia , Proteínas de Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo
2.
Biol Open ; 11(11)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36259662

RESUMO

Spc110 is an essential component of the spindle pole body (SPB), the yeast equivalent of the centrosome, that recruits the γ-tubulin complex to the nuclear side of the SPB to produce the microtubules that form the mitotic spindle. Here, we identified phosphosites S11 and S36 in maternally originated Spc110 and explored their functions in vivo. Yeast expressing non-phosphorylatable Spc110S11A had a distinct spindle phenotype characterised by higher levels of α-tubulin, which was frequently asymmetrically distributed between the two SPBs. Furthermore, expression of the double mutant Spc110S11AS36A had a delayed cell cycle progression. Specifically, the final steps of mitosis were delayed in Spc110S11AS36A cells, including expression and degradation of the mitotic cyclin Clb2, disassembling the mitotic spindle and re-localizing Cdc14 to the nucleoli, resulting in late mitotic exit and entry in G1. Thus, we propose that Spc110 phosphorylation at S11 and S36 is required to regulate timely cell cycle progression in budding yeast. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Centrossomo/metabolismo , Corpos Polares do Fuso/metabolismo , Fuso Acromático/metabolismo , Mitose , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo
3.
EMBO J ; 41(21): e112107, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36125182

RESUMO

Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.


Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular , Centríolos , Animais , Humanos , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo
4.
Front Immunol ; 13: 907636, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35967419

RESUMO

Tumor-associated macrophages (TAMs) are involved in the growth of prostate cancer (PrC), while the molecular mechanisms underlying the interactive crosstalk between TAM and PrC cells remain largely unknown. Platelet-derived growth factor (PDGF) is known to promote mesenchymal stromal cell chemotaxis to the tumor microenvironment. Recently, activation of spindle pole body component 25 (SPC25) has been shown to promote PrC cell proliferation and is associated with PrC stemness. Here, the relationship between SPC25 and PDGF in the crosstalk between TAM and PrC was investigated. Significant increases in both PDGF and SPC25 levels were detected in PrC specimens compared to paired adjacent normal prostate tissues. A significant correlation was detected between PDGF and SPC25 levels in PrC specimens and cell lines. SPC25 increased PDGF production and tumor cell growth in cultured PrC cells and in xenotransplantation. Mechanistically, SPC25 appeared to activate PDGF in PrC likely through Early Growth Response 1 (Egr1), while the secreted PDGF signaled to TAM through PDGFR on macrophages and polarized macrophages, which, in turn, induced the growth of PrC cells likely through their production and secretion of transforming growth factor ß1 (TGFß1). Thus, our data suggest that SPC25 triggers the crosstalk between TAM and PrC cells via SPC25/PDGF/PDGFR/TGFß1 receptor signaling to enhance PrC growth.


Assuntos
Proteínas Associadas aos Microtúbulos , Fator de Crescimento Derivado de Plaquetas , Próstata , Neoplasias da Próstata , Corpos Polares do Fuso , Macrófagos Associados a Tumor , Linhagem Celular Tumoral , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Receptor Cross-Talk/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Corpos Polares do Fuso/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor/metabolismo
5.
Fungal Genet Biol ; 162: 103729, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35944835

RESUMO

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.


Assuntos
Proteínas de Transporte , Proteínas Fúngicas , Proteínas Associadas aos Microtúbulos , Neurospora crassa , Benomilo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Corpos Polares do Fuso/metabolismo , Tubulina (Proteína)/genética
6.
Cells ; 11(9)2022 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-35563825

RESUMO

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.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Schizosaccharomyces , Humanos , Mitose , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Schizosaccharomyces/metabolismo , Corpos Polares do Fuso/metabolismo
7.
Life Sci Alliance ; 5(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35354597

RESUMO

The number, distribution, and composition of nuclear pore complexes (NPCs) in the nuclear envelope varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization are controlled, we analyzed the NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi, and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion near SPBs. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the nuclear envelope influences local NPC distribution.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Corpos Polares do Fuso/metabolismo
8.
FASEB J ; 36(2): e22086, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35028983

RESUMO

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of human kidney cancer with a high probability of metastasis. To understand the molecular processing essential for ccRCC tumorigenicity, we conducted an integrative in silico analysis of The Cancer Genome Atlas (TCGA) ccRCC dataset and clustered randomly interspersed short palindromic repeats (CRISPR) screening dataset of ccRCC cell lines from Depmap. We identified spindle pole body component 24 homolog (SPC24) as an essential gene for ccRCC cell lines with prognostic significance in the TCGA database. Targeting SPC24 by CRISPR/Cas9-mediated gene knockout attenuated ccRCC proliferation, metastasis, and in vivo tumor growth. Furthermore, we found that SPC24 regulates metastasis genes expression in a SRY-box transcription factor 2 (SOX2)-dependent manner. The anti-proliferative effects of SPC24 knockout were strengthened with SOX2 knockdown. Collectively, our findings suggest SPC24 has a pivotal function in promoting ccRCC progression, providing a new insight for the treatment of ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Fatores de Transcrição SOXB1/genética , Corpos Polares do Fuso/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/genética , Fatores de Transcrição/genética
9.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34747981

RESUMO

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.


Assuntos
Antígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Homeostase , Meiose , Ploidias , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Corpos Polares do Fuso/metabolismo , Cromossomos Fúngicos/metabolismo , Esporos Fúngicos/metabolismo , Zigoto/citologia
10.
FEBS Lett ; 595(22): 2781-2792, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34674264

RESUMO

The timing of cytokinesis relative to other mitotic events in the fission yeast Schizosaccharomyces pombe is controlled by the septation initiation network (SIN). During a mitotic checkpoint, the SIN is inhibited by the E3 ubiquitin ligase Dma1 to prevent chromosome mis-segregation. Dma1 dynamically localizes to spindle pole bodies (SPBs) and the contractile ring (CR) during mitosis, though its role at the CR is unknown. Here, we examined whether Dma1 phosphorylation affects its localization or function. We found that preventing Dma1 phosphorylation by substituting the six phosphosites with alanines diminished its CR localization but did not affect its mitotic checkpoint function. These studies reinforce the conclusion that Dma1 localization to the SPB is key to its role in the mitotic checkpoint.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Corpos Polares do Fuso/metabolismo , Proteínas de Ciclo Celular/genética , Fosforilação , Transporte Proteico , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética
11.
J Cell Sci ; 134(16)2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34346498

RESUMO

Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles, the existence of which implies that microtubule spindles self-assemble without the participation of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we recently reported the formation of self-assembled microtubule arrays, which were able to segregate chromosomes, in fission yeast mutants, in which the contribution of the spindle pole body (SPB; the centrosome equivalent in yeast) was specifically blocked during meiosis. Here, we demonstrate that this unexpected microtubule formation represents a bona fide type of acentrosomal spindle. Moreover, a comparative analysis of these self-assembled spindles and the canonical SPB-dependent spindle reveals similarities and differences; for example, both spindles have a similar polarity, but the location of the γ-tubulin complex differs. We also show that the robustness of self-assembled spindles can be reinforced by eliminating kinesin-8 family members, whereas kinesin-8 mutants have an adverse impact on SPB-dependent spindles. Hence, we consider that reinforced self-assembled spindles in yeast will help to clarify the molecular mechanisms behind acentrosomal meiosis, a crucial step towards better understanding gametogenesis.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Feminino , Humanos , Cinesinas/genética , Meiose , Microtúbulos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fuso Acromático/genética , Corpos Polares do Fuso
12.
J Cell Sci ; 134(16)2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34328180

RESUMO

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) have been described in many cell types. The functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans, additional MTOCs have been discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates the activities of the SPB outer plaque and the sMTOC. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest that the polo-like kinase is a regulator of MTOC activities and acts as a scaffolding unit through interaction with γ-TuRC receptors.


Assuntos
Aspergillus nidulans , Centro Organizador dos Microtúbulos , Animais , Aspergillus nidulans/genética , Centrossomo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos , Fuso Acromático , Corpos Polares do Fuso , Tubulina (Proteína)
13.
Methods Mol Biol ; 2329: 277-289, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085230

RESUMO

Photoactivated localization microscopy (PALM), one of the super resolution microscopy methods improving the resolution limit to 20 nm, allows the detection of single molecules in complex protein structures in living cells. Microtubule-organizing centres (MTOCs) are large, multisubunit protein complexes, required for microtubule polymerization. The prominent MTOC in higher eukaryotes is the centrosome, and its functional ortholog in fungi is the spindle-pole body (SPB). There is ample evidence that besides centrosomes other MTOCs are important in eukaryotic cells. The filamentous ascomycetous fungus Aspergillus nidulans is a model organism, with hyphae consisting of multinucleate compartments separated by septa. In A. nidulans, besides the SPBs, a second type of MTOCs was discovered at septa (called septal MTOCs, sMTOC). All the MTOC components appear as big dots at SPBs and sMTOCs when tagged with a fluorescent protein and observed with conventional fluorescence microscopy due to the diffraction barrier. In this chapter, we describe the application of PALM in quantifying the numbers of individual proteins at both MTOC sites in A. nidulans and provide evidence that the composition of MTOCs is highly dynamic and dramatically changes during the cell cycle.


Assuntos
Aspergillus nidulans/fisiologia , Proteínas Fúngicas/análise , Centro Organizador dos Microtúbulos/metabolismo , Imagem Individual de Molécula/métodos , Ciclo Celular , Microscopia , Modelos Biológicos , Corpos Polares do Fuso/metabolismo
14.
Mol Biol Cell ; 32(16): 1487-1500, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34133218

RESUMO

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB-yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy, we show that the conserved Sad1-UNC-84 homology-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.


Assuntos
Centrômero/metabolismo , Centrossomo/metabolismo , Mitose , Membrana Nuclear/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Nucleares/metabolismo , Transporte Proteico , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Fuso Acromático/metabolismo , Corpos Polares do Fuso/metabolismo
15.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523111

RESUMO

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo , Sequência de Aminoácidos , Anáfase , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Corpos Polares do Fuso/ultraestrutura , Relação Estrutura-Atividade
16.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33464308

RESUMO

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end-directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule-pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end-directed force generation will be needed to achieve antiparallel alignment.


Assuntos
Microtúbulos/metabolismo , Mitose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Engenharia Genética , Mutação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo , Torção Mecânica
17.
Curr Opin Struct Biol ; 66: 22-31, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33113389

RESUMO

The fungal kingdom is large and diverse, representing extremes of ecology, life cycles and morphology. At a cellular level, the diversity among fungi is particularly apparent at the spindle pole body (SPB). This nuclear envelope embedded structure, which is essential for microtubule nucleation, shows dramatically different morphologies between different fungi. However, despite phenotypic diversity, many SPB components are conserved, suggesting commonalities in structure, function and duplication. Here, I review the organization of the most well-studied SPBs and describe how advances in genomics, genetics and cell biology have accelerated knowledge of SPB architecture in other fungi, providing insights into microtubule nucleation and other processes conserved across eukaryotes.


Assuntos
Proteínas Fúngicas , Corpos Polares do Fuso , Fungos , Centro Organizador dos Microtúbulos , Microtúbulos , Membrana Nuclear , Fuso Acromático
18.
Cells ; 11(1)2021 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-35011608

RESUMO

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.


Assuntos
Pontos de Checagem do Ciclo Celular , Mitose , Saccharomycetales/citologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Alelos , Anáfase , Genes Dominantes , Metáfase , Mutação/genética , Saccharomycetales/crescimento & desenvolvimento , Fuso Acromático/metabolismo , Corpos Polares do Fuso/metabolismo
19.
PLoS Genet ; 16(12): e1008911, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332348

RESUMO

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids 'rescues' SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes.


Assuntos
Centrômero/genética , Cromossomos Fúngicos/genética , Diploide , Dosagem de Genes , Centrômero/metabolismo , Cromossomos Fúngicos/metabolismo , Saccharomyces cerevisiae , Corpos Polares do Fuso/genética , Corpos Polares do Fuso/metabolismo
20.
Mol Cell ; 80(2): 311-326.e4, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32970994

RESUMO

To determine whether double-strand break (DSB) mobility enhances the physical search for an ectopic template during homology-directed repair (HDR), we tested the effects of factors that control chromatin dynamics, including cohesin loading and kinetochore anchoring. The former but not the latter is altered in response to DSBs. Loss of the nonhistone high-mobility group protein Nhp6 reduces histone occupancy and increases chromatin movement, decompaction, and ectopic HDR. The loss of nucleosome remodeler INO80-C did the opposite. To see whether enhanced HDR depends on DSB mobility or the global chromatin response, we tested the ubiquitin ligase mutant uls1Δ, which selectively impairs local but not global movement in response to a DSB. Strand invasion occurs in uls1Δ cells with wild-type kinetics, arguing that global histone depletion rather than DSB movement is rate limiting for HDR. Impaired break movement in uls1Δ correlates with elevated MRX and cohesin loading, despite normal resection and checkpoint activation.


Assuntos
Quebras de DNA de Cadeia Dupla , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Bleomicina/farmacologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/metabolismo , Histonas/metabolismo , Modelos Biológicos , Fosforilação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...