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1.
Mol Ther ; 31(7): 2089-2104, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-36945773

RESUMO

CAR T cells recognizing CD19 effectively treat relapsed and refractory B-ALL and DLBCL. However, CD19 loss is a frequent cause of relapse. Simultaneously targeting a second antigen, CD22, may decrease antigen escape, but is challenging: its density is approximately 10-fold less than CD19, and its large structure may hamper immune synapse formation. The characteristics of the optimal CD22 CAR are underexplored. We generated 12 distinct CD22 antibodies and tested CARs derived from them to identify a CAR based on the novel 9A8 antibody, which was sensitive to low CD22 density and lacked tonic signaling. We found no correlation between affinity or membrane proximity of recognition epitope within Ig domains 3-6 of CD22 with CART function. The optimal strategy for CD19/CD22 CART co-targeting is undetermined. Co-administration of CD19 and CD22 CARs is costly; single CARs targeting CD19 and CD22 are challenging to construct. The co-expression of two CARs has previously been achieved using bicistronic vectors. Here, we generated a dual CART product by co-transduction with 9A8-41BBζ and CAT-41BBζ (obe-cel), the previously described CD19 CAR. CAT/9A8 CART eliminated single- and double-positive target cells in vitro and eliminated CD19- tumors in vivo. CAT/9A8 CART is being tested in a phase I clinical study (NCT02443831).


Assuntos
Linfoma de Burkitt , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T , Recidiva Local de Neoplasia , Imunoterapia Adotiva , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD19 , Anticorpos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico
2.
J Virol ; 95(19): e0068521, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287040

RESUMO

The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here, we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7, B.1.351, and P.1 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralizing antibody approaches. Furthermore, we report a preclinical characterization package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralization capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralized four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections. IMPORTANCE Mutational drift of SARS-CoV-2 risks rendering both therapeutics and vaccines less effective. Receptor decoy strategies utilizing soluble human ACE2 may overcome the risk of viral mutational escape since mutations disrupting viral interaction with the ACE2 decoy will by necessity decrease virulence, thereby preventing meaningful escape. The solution described here of a soluble ACE2 receptor decoy is significant for the following reasons: while previous ACE2-based therapeutics have been described, ours has novel features, including (i) mutations within ACE2 to remove catalytical activity and systemic interference with the renin/angiotensin system, (ii) abrogated FcγR engagement, reduced risk of antibody-dependent enhancement of infection, and reduced risk of hyperinflammation, and (iii) streamlined antibody-like purification process and scale-up manufacturability indicating that this receptor decoy could be produced quickly and easily at scale. Finally, we demonstrate that ACE2-based therapeutics confer a broad-spectrum neutralization potency for ACE2-tropic viruses, including SARS-CoV-2 variants of concern in contrast to therapeutic MAb.


Assuntos
Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Antivirais/imunologia , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Facilitadores , COVID-19/imunologia , Células HEK293 , Humanos , Cinética , Mutação , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
ACS Chem Biol ; 19(2): 308-324, 2024 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-38243811

RESUMO

A versatile, safe, and effective small-molecule control system is highly desirable for clinical cell therapy applications. Therefore, we developed a two-component small-molecule control system based on the disruption of protein-protein interactions using minocycline, an FDA-approved antibiotic with wide availability, excellent biodistribution, and low toxicity. The system comprises an anti-minocycline single-domain antibody (sdAb) and a minocycline-displaceable cyclic peptide. Here, we show how this versatile system can be applied to OFF-switch split CAR systems (MinoCAR) and universal CAR adaptors (MinoUniCAR) with reversible, transient, and dose-dependent suppression; to a tunable T cell activation module based on MyD88/CD40 signaling; to a controllable cellular payload secretion system based on IL12 KDEL retention; and as a cell/cell inducible junction. This work represents an important step forward in the development of a remote-controlled system to precisely control the timing, intensity, and safety of therapeutic interventions.


Assuntos
Comunicação Celular , Minociclina , Minociclina/farmacologia , Distribuição Tecidual , Antibacterianos/farmacologia , Transdução de Sinais
5.
Nat Commun ; 15(1): 1583, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38383515

RESUMO

Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor ß-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2, with preclinical evidence to support their efficacy in T cell malignancies.


Assuntos
Neoplasias , Linfócitos T , Humanos , Imunoterapia , Receptores de Antígenos de Linfócitos T
6.
Mol Ther Nucleic Acids ; 32: 603-621, 2023 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-37200859

RESUMO

The hostile tumor microenvironment limits the efficacy of adoptive cell therapies. Activation of the Fas death receptor initiates apoptosis and disrupting these receptors could be key to increasing CAR T cell efficacy. We screened a library of Fas-TNFR proteins identifying several novel chimeras that not only prevented Fas ligand-mediated kill, but also enhanced CAR T cell efficacy by signaling synergistically with the CAR. Upon binding Fas ligand, Fas-CD40 activated the NF-κB pathway, inducing greatest proliferation and IFN-γ release out of all Fas-TNFRs tested. Fas-CD40 induced profound transcriptional modifications, particularly genes relating to the cell cycle, metabolism, and chemokine signaling. Co-expression of Fas-CD40 with either 4-1BB- or CD28-containing CARs increased in vitro efficacy by augmenting CAR T cell proliferation and cancer target cytotoxicity, and enhanced tumor killing and overall mouse survival in vivo. Functional activity of the Fas-TNFRs were dependent on the co-stimulatory domain within the CAR, highlighting crosstalk between signaling pathways. Furthermore, we show that a major source for Fas-TNFR activation derives from CAR T cells themselves via activation-induced Fas ligand upregulation, highlighting a universal role of Fas-TNFRs in augmenting CAR T cell responses. We have identified Fas-CD40 as the optimal chimera for overcoming Fas ligand-mediated kill and enhancing CAR T cell efficacy.

7.
Cancer Immunol Res ; 11(9): 1203-1221, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37352396

RESUMO

Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing nonnative dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs coexpressed with a clinically relevant solid tumor-specific GD2-specific chimeric antigen receptor (CAR). Based on this characterization, we suggest that the coexpression of either the common ß-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.


Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Citocinas , Neoplasias/patologia , Citocinas
8.
Mol Ther Methods Clin Dev ; 31: 101123, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-37886606

RESUMO

Base editing is a revolutionary gene-editing technique enabling the introduction of point mutations into the genome without generating detrimental DNA double-stranded breaks. Base-editing enzymes are commonly delivered in the form of modified linear messenger RNA (mRNA) that is costly to produce. Here, we address this problem by developing a simple protocol for manufacturing base-edited cells using circular RNA (circRNA), which is less expensive to synthesize. Compared with linear mRNA, higher editing efficiencies were achieved with circRNA, enabling an 8-fold reduction in the amount of RNA required. We used this protocol to manufacture a clinical dose (1 × 108 cells) of base-edited chimeric antigen receptor (CAR) T cells lacking expression of the inhibitory receptor, PD-1. Editing efficiencies of up to 86% were obtained using 0.25 µg circRNA/1 × 106 cells. Increased editing efficiencies with circRNA were attributed to more efficient translation. These results suggest that circRNA, which is less expensive to produce than linear mRNA, is a viable option for reducing the cost of manufacturing base-edited cells at scale.

9.
Front Immunol ; 14: 1119350, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37334382

RESUMO

SHP1 and SHP2 are SH2 domain-containing proteins which have inhibitory phosphatase activity when recruited to phosphorylated ITIMs and ITSMs on inhibitory immune receptors. Consequently, SHP1 and SHP2 are key proteins in the transmission of inhibitory signals within T cells, constituting an important point of convergence for diverse inhibitory receptors. Therefore, SHP1 and SHP2 inhibition may represent a strategy for preventing immunosuppression of T cells mediated by cancers hence improving immunotherapies directed against these malignancies. Both SHP1 and SHP2 contain dual SH2 domains responsible for localization to the endodomain of inhibitory receptors and a protein tyrosine phosphatase domain which dephosphorylates and thus inhibits key mediators of T cell activation. We explored the interaction of the isolated SH2 domains of SHP1 and SHP2 to inhibitory motifs from PD1 and identified strong binding of both SH2 domains from SHP2 and more moderate binding in the case of SHP1. We next explored whether a truncated form of SHP1/2 comprising only of SH2 domains (dSHP1/2) could act in a dominant negative fashion by preventing docking of the wild type proteins. When co-expressed with CARs we found that dSHP2 but not dSHP1 could alleviate immunosuppression mediated by PD1. We next explored the capacity of dSHP2 to bind with other inhibitory receptors and observed several potential interactions. In vivo we observed that the expression of PDL1 on tumor cells impaired the ability of CAR T cells to mediate tumor rejection and this effect was partially reversed by the co-expression of dSHP2 albeit at the cost of reduced CAR T cell proliferation. Modulation of SHP1 and SHP2 activity in engineered T cells through the expression of these truncated variants may enhance T cell activity and hence efficacy in the context of cancer immunotherapy.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Linfócitos T , Proteínas de Transporte , Imunidade , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas/metabolismo , Linfócitos T/metabolismo
10.
Mol Ther Methods Clin Dev ; 28: 116-128, 2023 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-36620071

RESUMO

γ-Retroviral vectors (γ-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for γ-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced γ-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for γ-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 107-108 TU/mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in product- and process-related impurities. Furthermore, product characterization of clinical-grade γ-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade γ-RV produced by transient transfection and purified by high-speed centrifugation was similar to γ-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using γ-RV to bridge vector supply between early- and late-stage clinical trials.

11.
Biotechniques ; 72(4): 143-154, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35234525

RESUMO

The development of multicistronic vectors enabling differential transgene expression is a goal of gene therapy and poses a significant engineering challenge. Current approaches rely on the insertion of long regulatory sequences that occupy valuable space in vectors, which have a finite and limited packaging capacity. Here we describe a simple method of achieving differential transgene expression by inserting stop codons and translational readthrough motifs (TRMs) to suppress stop codon termination. TRMs reduced downstream transgene expression ∼sixfold to ∼140-fold, depending on the combination of stop codon and TRM used. We show that a TRM can facilitate the controlled secretion of the highly potent cytokine IL-12 at therapeutically beneficial levels in an aggressive immunocompetent mouse melanoma model to prevent tumor growth. Given their compact size (6 bp) and ease of introduction, we envisage that TRMs will be widely adopted in recombinant DNA engineering to facilitate differential transgene expression.


Assuntos
Biossíntese de Proteínas , Animais , Códon de Terminação , Camundongos , Biossíntese de Proteínas/genética , Transgenes
12.
MAbs ; 13(1): 1864084, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33382949

RESUMO

Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics.


Assuntos
Anticorpos Monoclonais/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/isolamento & purificação , Antígenos CD/imunologia , Teorema de Bayes , Proteínas Ligadas por GPI/imunologia , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Ratos Wistar , Receptores Imunológicos/imunologia
13.
Bio Protoc ; 11(16): e4194, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34541054

RESUMO

The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike (CoV-2 S) proteins and green fluorescent protein (GFP) reporters. The virus titer is determined by the GFP reporter (fluorescent) expression with a flow cytometer. High titers (>1.00 E+06 infectious units/ml) are produced using codon-optimized CoV-2 S, harbouring the prevalent D614G mutation and lacking its ER retention signal. Enhanced and consistent cell entry is achieved by using permissive HEK293T/17 cells that were genetically engineered to stably express the SARS-CoV-2 human receptor ACE2 along with the cell surface protease TMPRSS2 required for efficient fusion. For the widespread use of this protocol, its reagents have been made publicly available. Graphic abstract: Production and quantification of lentiviral vectors pseudotyped with the SARS-CoV-2 Spike glycoprotein.

14.
Mol Biol Cell ; 18(9): 3667-80, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17626165

RESUMO

Pericentrin is an integral centrosomal component that anchors regulatory and structural molecules to centrosomes. In a yeast two-hybrid screen with pericentrin we identified chromodomain helicase DNA-binding protein 4 (CHD4/Mi2beta). CHD4 is part of the multiprotein nucleosome remodeling deacetylase (NuRD) complex. We show that many NuRD components interacted with pericentrin by coimmunoprecipitation and that they localized to centrosomes and midbodies. Overexpression of the pericentrin-binding domain of CHD4 or another family member (CHD3) dissociated pericentrin from centrosomes. Depletion of CHD3, but not CHD4, by RNA interference dissociated pericentrin and gamma-tubulin from centrosomes. Microtubule nucleation/organization, cell morphology, and nuclear centration were disrupted in CHD3-depleted cells. Spindles were disorganized, the majority showing a prometaphase-like configuration. Time-lapse imaging revealed mitotic failure before chromosome segregation and cytokinesis failure. We conclude that pericentrin forms complexes with CHD3 and CHD4, but a distinct CHD3-pericentrin complex is required for centrosomal anchoring of pericentrin/gamma-tubulin and for centrosome integrity.


Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos/metabolismo , Autoantígenos/metabolismo , Centrossomo/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/deficiência , Animais , Autoantígenos/química , Células COS , Chlorocebus aethiops , Citocinese , DNA Helicases/química , DNA Helicases/deficiência , Histona Desacetilases/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Camundongos , Microtúbulos/metabolismo , Mitose , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismo
15.
J Cell Biol ; 161(3): 535-45, 2003 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-12732615

RESUMO

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.


Assuntos
Proteínas de Ciclo Celular/isolamento & purificação , Divisão Celular/genética , Centríolos/metabolismo , Células Eucarióticas/metabolismo , Fase S/genética , Sequência de Aminoácidos/genética , Animais , Anticorpos/farmacologia , Sequência de Bases/genética , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centríolos/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA Complementar/análise , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Células Eucarióticas/ultraestrutura , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , tRNA Metiltransferases
16.
Mol Biol Cell ; 15(8): 3642-57, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15146056

RESUMO

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.


Assuntos
Antígenos/metabolismo , Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismo , Animais , Antígenos/genética , Antígenos/imunologia , Apoptose/genética , Linhagem Celular , Humanos , Imunoprecipitação , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Fuso Acromático/genética , Fuso Acromático/fisiologia
17.
J Cell Biol ; 216(11): 3713-3728, 2017 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-28993469

RESUMO

Primary cilia are sensory organelles located at the cell surface. Their assembly is primed by centrosome migration to the apical surface, yet surprisingly little is known about this initiating step. To gain insight into the mechanisms driving centrosome migration, we exploited the reproducibility of cell architecture on adhesive micropatterns to investigate the cytoskeletal remodeling supporting it. Microtubule network densification and bundling, with the transient formation of an array of cold-stable microtubules, and actin cytoskeleton asymmetrical contraction participate in concert to drive apical centrosome migration. The distal appendage protein Cep164 appears to be a key actor involved in the cytoskeleton remodeling and centrosome migration, whereas intraflagellar transport 88's role seems to be restricted to axoneme elongation. Together, our data elucidate the hitherto unexplored mechanism of centrosome migration and show that it is driven by the increase and clustering of mechanical forces to push the centrosome toward the cell apical pole.


Assuntos
Citoesqueleto de Actina/fisiologia , Centrossomo/fisiologia , Células Epiteliais/fisiologia , Microtúbulos/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Citoesqueleto de Actina/metabolismo , Linhagem Celular , Centrossomo/metabolismo , Cílios/metabolismo , Cílios/fisiologia , Células Epiteliais/metabolismo , Humanos , Mecanotransdução Celular , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Estabilidade Proteica , Interferência de RNA , Epitélio Pigmentado da Retina/metabolismo , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
18.
Nat Cell Biol ; 18(1): 65-75, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26655833

RESUMO

Microtubules and actin filaments are the two main cytoskeleton networks supporting intracellular architecture and cell polarity. The centrosome nucleates and anchors microtubules and is therefore considered to be the main microtubule-organizing centre. However, recurring, yet unexplained, observations have pointed towards a connection between the centrosome and actin filaments. Here we have used isolated centrosomes to demonstrate that the centrosome can directly promote actin-filament assembly. A cloud of centrosome-associated actin filaments could be identified in living cells as well. Actin-filament nucleation at the centrosome was mediated by the nucleation-promoting factor WASH in combination with the Arp2/3 complex. Pericentriolar material 1 (PCM1) seemed to modulate the centrosomal actin network by regulating Arp2/3 complex and WASH recruitment to the centrosome. Hence, our results reveal an additional facet of the centrosome as an intracellular organizer and provide mechanistic insights into how the centrosome can function as an actin-filament-organizing centre.


Assuntos
Actinas/metabolismo , Polaridade Celular/fisiologia , Centrossomo/fisiologia , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Células Cultivadas , Humanos
19.
J Mol Biol ; 322(4): 785-97, 2002 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-12270714

RESUMO

Protein kinase CK1 (formerly termed casein kinase I) is ubiquitous in eukaryotic cells and comprises a family of as many as 14 isoforms (including splice variants) in mammalian cells. Mammalian CK1delta and CK1epsilon, which are highly related to each other, are enriched at the centrosomes in interphase cells and at the spindle during mitosis. In the present study we have isolated, using the yeast two-hybrid system, a 182 amino acid residue fragment of the centrosomal and golgi N-kinase anchoring protein (CG-NAP, also known as AKAP450), which specifically interacts with CK1delta and CK1epsilon, but not with other CK1 isoforms. The 182 amino acid residue CG-NAP fragment, or full length CG-NAP, co-immunoprecipitates with CK1delta and CK1epsilon from mammalian cells. Consistent with this association, endogenous CG-NAP/AKAP450 and CK1delta co-localize in cells. Moreover, when expressed in the presence of CK1delta the 182 amino acid residue CG-NAP fragment adopts the same sub-cellular localization as CK1delta. Strikingly, attachment of the CG-NAP fragment to the plasma membrane is sufficient to re-localize a significant level of CK1delta to the membrane. These findings support a model in which sub-cellular localization of CK1delta/epsilon molecules at the centrosome is mediated, at least in part, through the action of CG-NAP/AKAP450 and provide a potential mechanism by which the contribution to cell cycle progression by CK1delta/epsilon may be regulated.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Centrossomo/metabolismo , Proteínas do Citoesqueleto , Proteínas Quinases/metabolismo , Proteínas de Ancoragem à Quinase A , Sequência de Aminoácidos , Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Caseína Quinases , Catálise , Chlorocebus aethiops , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Testes de Precipitina , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Especificidade por Substrato
20.
PLoS One ; 8(4): e62963, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23638170

RESUMO

Arfaptin2 contains a Bin/Amphiphysin/Rvs (BAR) domain and directly interacts with proteins of the Arf/Arl family in their active GTP-bound state. It has been proposed that BAR domains are able to sense membrane curvature and to induce membrane tubulation. We report here that active Arf1 is required for the recruitment of Arfaptin2 to artificial liposomes mimicking the Golgi apparatus lipid composition. The Arf1-dependent recruitment of Arfaptin2 increases with membrane curvature, while the recruitment of Arf1 itself is not sensitive to curvature. At high protein concentrations, the binding of Arfaptin2 induces membrane tubulation. Finally, membrane-bound Arfaptin2 is released from the liposome when ArfGAP1 catalyzes the hydrolysis of GTP to GDP in Arf1. These results show that both Arf1 activation and high membrane curvature are required for efficient recruitment of Arfaptin2 to membranes.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Lipossomos/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Transporte Proteico , Especificidade por Substrato , Lipossomas Unilamelares/metabolismo
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