RESUMO
To fulfill their actual cellular role, individual microtubules become functionally specialized through a broad range of mechanisms. The 'search and capture' model posits that microtubule dynamics and functions are specified by cellular targets that they capture (i.e., a posteriori), independently of the microtubule-organizing center (MTOC) they emerge from. However, work in budding yeast indicates that MTOCs may impart a functional identity to the microtubules they nucleate, a priori. Key effectors in this process are microtubule plus-end tracking proteins (+TIPs), which track microtubule tips to regulate their dynamics and facilitate their targeted interactions. In this review, we discuss potential mechanisms of a priori microtubule specialization, focusing on recent findings indicating that +TIP networks may undergo liquid biomolecular condensation in different cell types.
Assuntos
Proteínas Associadas aos Microtúbulos , Microtúbulos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMO
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)/genéticaRESUMO
The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into condensates that recruit α- and ß-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and ß-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Centríolos/metabolismo , Centrossomo/metabolismo , Dineínas/metabolismo , Fuso Acromático/metabolismo , Xenopus laevis/metabolismoRESUMO
The signal transducer and activator of transcription 3 (Stat3) is activated upon phosphorylation at Y705 (pStat3) and serves the dual function of signal transduction and transcription activation. Our previous study suggested that pStat3 is functional during oocyte maturation when transcription is silenced. Therefore, we speculated that pStat3 serves other functions. Immunocytochemical analysis revealed that pStat3 emerges at microtubule asters and spindle and is subsequently localized at the spindle poles along with pericentrin during mouse oocyte maturation. Both Stat3 and pStat3 proteins were detected in conditionally knocked out Stat3-/- mouse oocytes. pStat3 localization was the same in Stat3+/+ and Stat3-/-oocytes, and oocyte maturation proceeded normally, suggesting that pStat3 was still functional. Furthermore, the treatment of oocytes with the Stat3-specific inhibitors stattic and BP-1-102 or anti-pStat3 antibody led to significantly abnormal spindle assembly and chromosome mislocation in a dose-dependent manner, and pStat3 was either absent or improperly localized in these oocytes. Moreover, the development of pre-implantation stage embryos derived from inhibitor-treated oocytes was significantly hampered following in vitro fertilization. These findings indicate a novel function of pStat3 in spindle assembly.
Assuntos
Oócitos/metabolismo , Fator de Transcrição STAT3/metabolismo , Fuso Acromático/metabolismo , Animais , Antígenos , Inibidores da Aromatase/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inibidores do Crescimento/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Fosforilação , Fator de Transcrição STAT3/genética , TranscriptomaRESUMO
Meiotic oocytes lack classic centrosomes; therefore, bipolar spindle assembly depends on the clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. The bipolar spindle is an essential cellular component that ensures accurate chromosome segregation during anaphase. If the spindle does not form properly, it can result in aneuploidy or cell death. However, the molecular mechanism by which the bipolar spindle is established is not yet fully understood. Tumor suppressor p53-binding protein 1 (TP53BP1) is known to mediate the DNA damage response. Several recent studies have indicated that TP53BP1 has noncanonical roles in processes, such as spindle formation; however, the role of TP53BP1 in oocyte meiosis is currently unclear. Our results show that TP53BP1 knockdown affects spindle bipolarity and chromatin alignment by altering MTOC stability during oocyte maturation. TP53BP1 was localized in the cytoplasm and displayed an irregular cloud pattern around the spindle/chromosome region. TP53BP1 was also required for the correct localization of MTOCs into the two spindle poles during pro-meiosis I. TP53BP1 deletion altered the MTOC-localized Aurora Kinase A. TP53BP1 knockdown caused the microtubules to detach from the kinetochores and increased the rate of aneuploidy. Taken together, our data show that TP53BP1 plays crucial roles in chromosome stability and spindle bipolarity during meiotic maturation.
Assuntos
Cromossomos de Mamíferos/metabolismo , Cinetocoros/metabolismo , Meiose , Oócitos/metabolismo , Fuso Acromático/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Aurora Quinase A/metabolismo , Citoplasma/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos ICR , Oócitos/citologiaRESUMO
Mutations in the GJB2 gene account for approximately 20-50% of all non-syndromic hereditary deafness cases. The malformed organ of Corti (OC) was observed in different Cx26-null mouse models, which was mainly caused by the developmental arrest of pillar cells (PCs). However, the mechanism of developmental abnormalities in PCs caused by Cx26 deletion is still unclear. In this study, the ultrastructure of PCs at different postnatal days was observed in Cx26-null mice. Knockout of cochlear Cx26 led to the malformed assembly of non-centrosomal microtubule-organizing centers (MTOCs) far from the centrosome rather than near the centrosome. Additionally, the microtubule (MT) arrays emitted by abnormal non-centrosomal MTOCs were significantly reduced. In addition, we found that the protein expression of calmodulin-regulated, spectrin-associated protein2 (camsap2), a microtubule minus-end targeting protein associated with the organization of non-centrosomal MTs, was decreased in juvenile PCs in the Cx26-null group. Our results indicated that the malformation of non-centrosomal MTOCs in cochlear PCs might lead to the corresponding MTs' failure to be captured and anchored in Cx26-null mice, which results in the deformity of OC. Additionally, this abnormal developmental process might be correlated with the reduced expression of camsap2 caused by Cx26 deletion in the early developmental stage.
RESUMO
Centrosomes are best known as the microtubule organizing centers (MTOCs) of eukaryotic cells. In addition to their classic role in chromosome segregation, centrosomes play diverse roles unrelated to their MTOC activity during cell proliferation and quiescence. Metazoan centrosomes and their functional doppelgängers from lower eukaryotes, the spindle pole bodies (SPBs), act as important structural platforms that orchestrate signaling events essential for cell cycle progression, cellular responses to DNA damage, sensory reception and cell homeostasis. Here, we provide a critical overview of the unconventional and often overlooked roles of centrosomes/SPBs in the life cycle of eukaryotic cells.
RESUMO
Aberrant energy metabolism and cell cycle regulation both critically contribute to malignant cell growth and both processes represent targets for anticancer therapy. It is shown here that depletion of the AAA+-ATPase thyroid hormone receptor interacting protein 13 (Trip13) results in mitotic cell death through a combined mechanism linking lipid metabolism to aberrant mitosis. Diminished Trip13 levels in hepatocellular carcinoma cells result in insulin-receptor-/Akt-pathway-dependent accumulation of lipid droplets, which act as functional acentriolar microtubule organizing centers disturbing mitotic spindle polarity. Specifically, the lipid-droplet-coating protein perilipin 2 (Plin2) is required for multipolar spindle formation, induction of DNA damage, and mitotic cell death. Plin2 expression in different tumor cells confers susceptibility to cell death induced by Trip13 depletion as well as treatment with paclitaxel, a spindle-interfering drug commonly used against different cancers. Thus, assessment of Plin2 levels enables the stratification of tumor responsiveness to mitosis-targeting drugs, including clinically approved paclitaxel and Trip13 inhibitors currently under development.
Assuntos
Insulinas , Neoplasias Hepáticas , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Humanos , Insulinas/metabolismo , Lipídeos , Proteínas Mad2/metabolismo , Paclitaxel/farmacologia , Perilipina-2 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores dos Hormônios Tireóideos/metabolismoRESUMO
Francisella tularensis is the causative agent of the infectious disease tularemia and is designated a category A bioterrorism agent. The type VI secretion system encoded by the Francisella pathogenicity island (FPI) is necessary for intracellular growth; however, the functions of FPI proteins are largely unknown. In this study, we found that the FPI protein intracellular growth locus E (IglE) showed a unique localization pattern compared to other FPI proteins. Deleting iglE from Francisella tularensis subsp. novicida (F. novicida) decreased intracellular growth. Immunoprecipitation and pull-down assays revealed that IglE was associated with ß-tubulin. Additionally, GFP-fused IglE colocalized with microtubule organizing centers (MTOCs) in 293T cells. The iglE deletion mutant was transferred with dynein toward MTOCs and packed into lysosome-localizing areas. Conversely, the wild-type F. novicida exhibited intracellular growth distant from MTOCs. In addition, IglE expressed in 293T cells colocalized with dynein. These results suggest that IglE helps to prevent dynein- and MTOC-mediated intracellular trafficking in host cells to inhibit the transport of F. novicida toward lysosomes.
Assuntos
Proteínas de Bactérias/metabolismo , Francisella tularensis/patogenicidade , Ilhas Genômicas , Centro Organizador dos Microtúbulos/microbiologia , Tularemia/microbiologia , Proteínas de Bactérias/genética , Linhagem Celular , Dineínas/genética , Dineínas/metabolismo , Francisella tularensis/genética , Francisella tularensis/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , Transporte Proteico , Tularemia/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Polaridade Celular , Forma Celular , Cães , Células Epiteliais/citologia , Técnicas de Inativação de Genes , Humanos , Células Madin Darby de Rim Canino , CamundongosRESUMO
The execution of female meiosis and the establishment of the zygote is arguably the most critical stage of mammalian development. The egg can be arrested in the prophase of meiosis I for decades, and when it is activated, the spindle is assembled de novo. This spindle must function with the highest of fidelity and yet its assembly is unusually achieved in the absence of conventional centrosomes and with minimal influence of chromatin. Moreover, its dramatic asymmetric positioning is achieved through remarkable properties of the actin cytoskeleton to ensure elimination of the polar bodies. The second meiotic arrest marks a uniquely prolonged metaphase eventually interrupted by egg activation at fertilization to complete meiosis and mark a period of preparation of the male and female pronuclear genomes not only for their entry into the mitotic cleavage divisions but also for the imminent prospect of their zygotic expression.
Assuntos
Desenvolvimento Embrionário , Mamíferos/embriologia , Meiose , Mitose , Animais , Cromossomos de Mamíferos/metabolismo , Humanos , Fuso Acromático/metabolismoRESUMO
The cellular functions of the trans-Golgi network protein TGN38 remain unknown. In this research, we studied the expression, localization and functions of TGN38 in the meiotic maturation of mouse oocytes. TGN38 was expressed at every stage of oocyte meiotic maturation and colocalized with γ-tubulin at metaphase I and metaphase II. The spindle microtubule disturbing agents nocodazole and taxol did not affect the colocalization of TGN38 and γ-tubulin. Depletion of TGN38 with specific siRNAs resulted in increased metaphase I arrest, accompanied with spindle assembly checkpoint activation and decreased first polar extrusion (PB1). In the oocytes that had extruded the PB1 after the depletion of TGN38, symmetric division occurred, leading to the production of 2 similarly sized cells. Moreover, the peripheral migration of metaphase I spindle and actin cap formation were impaired in TGN38-depleted oocytes. Our data suggest that TGN38 may regulate the metaphase I/anaphase I transition and asymmetric cell division in mouse oocytes.