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1.
Cell Rep ; 41(7): 111642, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36384111

RESUMO

The primary cilium, a microtubule-based sensory organelle, undergoes cycles of assembly and disassembly that govern the cell cycle progression critical to cell proliferation and differentiation. Although cilia assembly has been studied extensively, the molecular mechanisms underlying cilia disassembly are less well understood. Here, we uncover a γ-tubulin ring complex (γ-TuRC)-dependent pathway that promotes cilia disassembly and thereby prevents cilia formation. We further demonstrate that Kif2A, a kinesin motor that bears microtubule-depolymerizing activity, is recruited to the cilium basal body in a γ-TuRC-dependent manner. Our mechanistic analyses show that γ-TuRC specifically recruits Kif2A via the GCP2 subunit and its binding partner Mzt2. Hence, despite the long-standing view that γ-TuRC acts mainly as a microtubule template, we illustrate that its functional heterogeneity at the basal body facilitates both microtubule nucleation and Kif2A recruitment-mediated regulation of ciliogenesis, ensuring cell cycle progression.


Assuntos
Proteínas Associadas aos Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Cílios/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo
2.
Science ; 378(6621): eabq7361, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-36395215

RESUMO

Meiotic spindle assembly ensures proper chromosome segregation in oocytes. However, the mechanisms behind spindle assembly in human oocytes remain largely unknown. We used three-dimensional high-resolution imaging of more than 2000 human oocytes to identify a structure that we named the human oocyte microtubule organizing center (huoMTOC). The proteins TACC3, CCP110, CKAP5, and DISC1 were found to be essential components of the huoMTOC. The huoMTOC arises beneath the oocyte cortex and migrates adjacent to the nuclear envelope before nuclear envelope breakdown (NEBD). After NEBD, the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Disrupting the huoMTOC led to spindle assembly defects and oocyte maturation arrest. These results reveal a physiological mechanism of huoMTOC-regulated spindle assembly in human oocytes.


Assuntos
Oócitos , Fuso Acromático , Humanos , Fuso Acromático/metabolismo , Oócitos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Cinetocoros/metabolismo , Segregação de Cromossomos , Proteínas Associadas aos Microtúbulos/metabolismo
3.
J Cell Sci ; 135(21)2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36217793

RESUMO

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.


Assuntos
Centrossomo , Centro Organizador dos Microtúbulos , Humanos , Centro Organizador dos Microtúbulos/metabolismo , Centrossomo/metabolismo , Intestinos , Diferenciação Celular , Proteínas/metabolismo , Mucosa Intestinal/metabolismo
4.
J Cell Biol ; 221(12)2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36214847

RESUMO

Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis.


Assuntos
Centrossomo , Células Dendríticas , Pontos de Checagem do Ciclo Celular , Movimento Celular , Centrossomo/metabolismo , Quimiotaxia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Centro Organizador dos Microtúbulos , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/metabolismo
5.
Nat Commun ; 13(1): 5635, 2022 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-36163468

RESUMO

In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is hampered by the lack of structural information on the augmin complex. Here, we elucidate the molecular architecture and conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.


Assuntos
Fuso Acromático , Tubulina (Proteína) , Proteínas Associadas aos Microtúbulos/química , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo
6.
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
7.
Development ; 149(16)2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35980365

RESUMO

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation.


Assuntos
Centríolos , Centro Organizador dos Microtúbulos , Animais , Centríolos/metabolismo , Centrossomo/metabolismo , Masculino , Centro Organizador dos Microtúbulos/metabolismo , Sêmen/metabolismo , Tubulina (Proteína)/metabolismo , Peixe-Zebra/metabolismo
8.
EMBO J ; 41(20): e111631, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-35916262

RESUMO

The orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell-sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture.


Assuntos
Actinas , Centrossomo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Centrossomo/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo
9.
Curr Opin Cell Biol ; 77: 102114, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35841745

RESUMO

Mammalian preimplantation embryogenesis depends on the spatio-temporal dynamics of the microtubule cytoskeleton to enable exceptionally fast changes in cell number, function, architecture, and fate. Microtubule organising centres (MTOCs), which coordinate the remodelling of microtubules, are therefore of fundamental significance during the first days of a new life. Despite its indispensable role during early mammalian embryogenesis, the origin of microtubule growth remains poorly understood. In this review, we summarise the most recent discoveries on microtubule organisation and function during early human embryogenesis and compare these to innovative studies conducted in alternative mammalian models. We emphasise the differences and analogies of centriole inheritance and their role during the first cleavage. Furthermore, we highlight the significance of non-centrosomal MTOCs for embryo viability and discuss the potential of novel in vitro models and light-inducible approaches towards unravelling microtubule formation in research and assisted reproductive technologies.


Assuntos
Centrossomo , Centro Organizador dos Microtúbulos , Animais , Blastocisto , Centrossomo/fisiologia , Citoesqueleto , Humanos , Mamíferos , Microtúbulos/fisiologia
10.
J Hazard Mater ; 436: 129248, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35739767

RESUMO

Diesel exhaust particles (DEPs) are major components of ambient particulate matter and are associated with various adverse health effects. Typically, DEPs contain a vast number of organic compounds, among which 9,10-phenanthrenequinone (9,10-PQ), the quinone derivative of the polycyclic aromatic hydrocarbon phenanthrene, is one of the most abundant and toxic. 9,10-PQ can produce excessive reactive oxygen species (ROS) via redox cycling and exhibit cytotoxicity in various cells. However, the underlying mechanisms involved in cytotoxicity of 9,10-PQ remain elusive. In this study, we investigated the effects of exposure to 9,10-PQ using mouse oocytes as a model system. We found that 9,10-PQ compromised meiotic maturation by impairing acentriolar microtubule organizing center (MTOC) assembly and subsequent spindle formation during meiotic maturation. Moreover, 9,10-PQ exposure prevented cell cycle progression by inhibiting Cdk1 activation via disturbance of cyclin B1 accumulation. Importantly, meiotic defects induced by 9,10-PQ exposure were not rescued by decreasing ROS levels, revealing that 9,10-PQ has ROS-independent activity that regulates cell cycle progression and spindle assembly. Therefore, our findings reveal that 9,10-PQ has novel activity that regulates cell-cycle progression and spindle formation in an ROS-independent manner during meiotic maturation in mouse oocytes.


Assuntos
Meiose , Oócitos , Animais , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Fenantrenos , Espécies Reativas de Oxigênio/metabolismo
11.
Annu Rev Cell Dev Biol ; 38: 1-23, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-35759800

RESUMO

The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.


Assuntos
Centro Organizador dos Microtúbulos , Fuso Acromático , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo
12.
Elife ; 112022 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-35758262

RESUMO

Centrosomes act as the main microtubule organizing center (MTOC) in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, the most upstream facilitator of centriole duplication. Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms. Recent work has shown that the E3 ligase TRIM37 is required for cell cycle arrest in acentrosomal cells. To gain additional insights into this process, we undertook a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest triggered by treatment with centrinone B, a selective PLK4 inhibitor. We found that TRIM37 is a key mediator of growth arrest after partial or full PLK4 inhibition. Interestingly, PLK4 cellular mobility decreased in a dose-dependent manner after centrinone B treatment. In contrast to recent work, we found that growth arrest after PLK4 inhibition correlated better with PLK4 activity than with mitotic length or centrosome number. These data provide insights into the global response to changes in centrosome number and PLK4 activity and extend the role for TRIM37 in regulating the abundance, localization, and function of centrosome proteins.


Assuntos
Centríolos , Centrossomo , Proteínas Serina-Treonina Quinases , Pirimidinas , Sulfonas , Animais , Proteínas de Ciclo Celular/metabolismo , Centríolos/efeitos dos fármacos , Centríolos/metabolismo , Centrossomo/metabolismo , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Fuso Acromático/metabolismo , Sulfonas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo
13.
Elife ; 112022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35762204

RESUMO

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.


Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.


Assuntos
Espectrina , Tubulina (Proteína) , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Espectrina/metabolismo , Tubulina (Proteína)/metabolismo
14.
Elife ; 112022 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-35758650

RESUMO

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically.


Assuntos
Centro Organizador dos Microtúbulos , Estresse Fisiológico , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Fosforilação , Proteínas/metabolismo
15.
Mol Biol Cell ; 33(8): ar68, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35511803

RESUMO

Osteoclasts are highly specialized, multinucleated cells responsible for the selective resorption of the dense, calcified bone matrix. Microtubules (MTs) contribute to the polarization and trafficking events involved in bone resorption by osteoclasts; however, the origin of these elaborate arrays is less clear. Osteoclasts arise through cell fusion of precursor cells. Previous studies have suggested that centrosome MT nucleation is lost during this process, with the nuclear membrane and its surrounding Golgi serving as the major MT organizing centers (MTOCs) in these cells. Here we reveal that precursor cell centrosomes are maintained and functional in the multinucleated osteoclast and interestingly form large MTOC clusters, with the clusters organizing significantly more MTs compared with individual centrosomes. MTOC cluster formation requires dynamic MTs and minus-end directed MT motor activity. Inhibition of these centrosome clustering elements had a marked impact on both F-actin ring formation and bone resorption. Together these findings show that multinucleated osteoclasts employ unique centrosomal clusters to organize the extensive MTs during bone attachment and resorption.


Assuntos
Reabsorção Óssea , Osteoclastos , Reabsorção Óssea/metabolismo , Centrossomo/metabolismo , Humanos , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo
16.
Life Sci Alliance ; 5(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35550346

RESUMO

The centriole/basal body (CBB) is an evolutionarily conserved organelle acting as a microtubule organising centre (MTOC) to nucleate cilia, flagella, and the centrosome. SAS4/CPAP is a conserved component associated with BB biogenesis in many model flagellated cells. Plasmodium, a divergent unicellular eukaryote and causative agent of malaria, displays an atypical, closed mitosis with an MTOC (or centriolar plaque), reminiscent of an acentriolar MTOC, embedded in the nuclear membrane. Mitosis during male gamete formation is accompanied by flagella formation. There are two MTOCs in male gametocytes: the acentriolar nuclear envelope MTOC for the mitotic spindle and an outer centriolar MTOC (the basal body) that organises flagella assembly in the cytoplasm. We show the coordinated location, association and assembly of SAS4 with the BB component, kinesin-8B, but no association with the kinetochore protein, NDC80, indicating that SAS4 is part of the BB and outer centriolar MTOC in the cytoplasm. Deletion of the SAS4 gene produced no phenotype, indicating that it is not essential for either male gamete formation or parasite transmission.


Assuntos
Parasitos , Plasmodium , Animais , Corpos Basais/metabolismo , Centríolos/metabolismo , Masculino , Centro Organizador dos Microtúbulos/metabolismo
17.
J Cell Biol ; 221(7)2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35604367

RESUMO

Branching microtubule (MT) nucleation is mediated by the augmin complex and γ-tubulin ring complex (γ-TuRC). However, how these two complexes work together to promote this process remains elusive. Here, using purified components from native and recombinant sources, we demonstrate that human augmin and γ-TuRC are sufficient to reconstitute the minimal MT branching machinery, in which NEDD1 bridges between augmin holo complex and GCP3/MZT1 subcomplex of γ-TuRC. The single-molecule experiment suggests that oligomerization of augmin may activate the branching machinery. We provide direct biochemical evidence that CDK1- and PLK1-dependent phosphorylation are crucial for NEDD1 binding to augmin, for their synergistic MT-binding activities, and hence for branching MT nucleation. In addition, we unveil that NEDD1 possesses an unanticipated intrinsic affinity for MTs via its WD40 domain, which also plays a pivotal role in the branching process. In summary, our study provides a comprehensive understanding of the underlying mechanisms of branching MT nucleation in human cells.


Assuntos
Proteínas Associadas aos Microtúbulos , Centro Organizador dos Microtúbulos , Microtúbulos , Tubulina (Proteína) , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tubulina (Proteína)/metabolismo , Repetições WD40
18.
Front Immunol ; 13: 871106, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35558071

RESUMO

NK cell-mediated cytotoxicity is a critical element of our immune system required for protection from microbial infections and cancer. NK cells bind to and eliminate infected or cancerous cells via direct secretion of cytotoxic molecules toward the bound target cells. In this review, we summarize the current understanding of the molecular regulations of NK cell cytotoxicity, focusing on lytic granule development and degranulation processes. NK cells synthesize apoptosis-inducing proteins and package them into specialized organelles known as lytic granules (LGs). Upon activation of NK cells, LGs converge with the microtubule organizing center through dynein-dependent movement along microtubules, ultimately polarizing to the cytotoxic synapse where they subsequently fuse with the NK plasma membrane. From LGs biogenesis to degranulation, NK cells utilize several strategies to protect themselves from their own cytotoxic molecules. Additionally, molecular pathways that enable NK cells to perform serial killing are beginning to be elucidated. These advances in the understanding of the molecular pathways behind NK cell cytotoxicity will be important to not only improve current NK cell-based anti-cancer therapies but also to support the discovery of additional therapeutic opportunities.


Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais , Grânulos Citoplasmáticos , Imunoterapia Adotiva , Centro Organizador dos Microtúbulos
19.
Methods Mol Biol ; 2430: 315-336, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35476342

RESUMO

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. How γTuRC nucleates microtubules, and how nucleation is regulated is not understood. To gain an understanding of γTuRC activity and regulation at the molecular level, it is important to measure quantitatively how γTuRC interacts with tubulin and potential regulators in space and time. Here, we describe a total internal reflection fluorescence microscopy-based assay on chemically functionalized glass slides for the in vitro study of surface immobilized purified γTuRC. The assay allows to measure microtubule nucleation by γTuRC in real time and at a single molecule level over a wide variety of assay conditions, in the absence and presence of potential regulators. This setup provides a previously unavailable opportunity for quantitative studies of the kinetics of microtubule nucleation by γTuRC.


Assuntos
Centrossomo , Proteínas Associadas aos Microtúbulos , Microscopia , Proteínas Associadas aos Microtúbulos/química , Centro Organizador dos Microtúbulos , Microtúbulos/química
20.
Mol Biol Cell ; 33(5)2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35420887

RESUMO

Protein localization is intrinsic to cellular function and specialized activities, such as migration or proliferation. Many localized proteins enrich at defined organelles, forming subdomains of functional activity further specified by interacting protein assemblies. One well-studied organelle showing dynamic, functional changes in protein composition is the centrosome. Centrosomes are microtubule-organizing centers with diverse cellular functions largely defined by the composition of the pericentriolar material, an ordered matrix of proteins organized around a central pair of centrioles. Also localizing to the pericentriolar material are mRNAs. Although RNA was identified at centrosomes decades ago, the characterization of specific RNA transcripts and their functional contributions to centrosome biology remained largely unstudied. While the identification of RNA localized to centrosomes accelerated with the development of high-throughput screening methods, this discovery still outpaces functional characterization. Recent work indicates RNA localized to centrosomes is biologically significant and further implicates centrosomes as sites for local protein synthesis. Distinct RNA localization and translational activities likely contribute to the diversity of centrosome functions within cells.


Assuntos
Centríolos , Centrossomo , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Centro Organizador dos Microtúbulos , Proteínas/metabolismo , RNA/metabolismo
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