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1.
Nature ; 585(7825): 440-446, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32908304

RESUMEN

Centrosomes catalyse the formation of microtubules needed to assemble the mitotic spindle apparatus1. Centrosomes themselves duplicate once per cell cycle, in a process that is controlled by the serine/threonine protein kinase PLK4 (refs. 2,3). When PLK4 is chemically inhibited, cell division proceeds without centrosome duplication, generating centrosome-less cells that exhibit delayed, acentrosomal spindle assembly4. Whether PLK4 inhibitors can be leveraged as a treatment for cancer is not yet clear. Here we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 levels accelerate acentrosomal spindle assembly and improve proliferation following PLK4 inhibition, whereas high TRIM37 levels inhibit acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer5-8, rendering these cancer types highly sensitive to PLK4 inhibition. We find that inactivating TRIM37 improves acentrosomal mitosis because TRIM37 prevents PLK4 from self-assembling into centrosome-independent condensates that serve as ectopic microtubule-organizing centres. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly through a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is an essential determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes-including 17q gain in neuroblastoma and 17q23 amplification in breast cancer-may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.


Asunto(s)
Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos Par 17/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Centro Organizador de los Microtúbulos/efectos de los fármacos , Centro Organizador de los Microtúbulos/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéutico , Ubiquitina/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Cancer Sci ; 114(4): 1324-1336, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-36441110

RESUMEN

Bile duct cancer (BDC) frequently invades the nerve fibers, making complete surgical resection difficult. A single tumor mass contains cells of variable malignancy and cell-differentiation states, with cancer stem cells (CSCs) considered responsible for poor clinical outcomes. This study aimed to investigate the contribution of autosynthesized dopamine to CSC-related properties in BDC. Sphere formation assays using 13 commercially available BDC cell lines demonstrated that blocking dopamine receptor D1 (DRD1) signaling promoted CSC-related anchorage-independent growth. Additionally, we newly established four new BDC patient-derived organoids (PDOs) and found that blocking DRD1 increased resistance to chemotherapy and enabled xenotransplantation in vivo. Single-cell analysis revealed that the BDC PDO cells varied in their cell-differentiation states and responses to dopamine signaling. Further, DRD1 inhibition increased WNT7B expression in cells with bile duct-like phenotype, and it induced proliferation of other cell types expressing Wnt receptors and stem cell-like signatures. Reagents that inhibited Wnt function canceled the effect of DRD1 inhibition and reduced cell proliferation in BDC PDOs. In summary, in BDCs, DRD1 is a crucial protein involved in autonomous CSC proliferation through the regulation of endogenous WNT7B. As such, inhibition of the DRD1 feedback signaling may be a potential treatment strategy for BDC.


Asunto(s)
Neoplasias de los Conductos Biliares , Vía de Señalización Wnt , Humanos , Neoplasias de los Conductos Biliares/patología , Dopamina , Fenotipo , Receptores Dopaminérgicos/genética
3.
BMC Cancer ; 22(1): 489, 2022 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-35505283

RESUMEN

BACKGROUND: Pancreatic cancer is one of the most lethal tumors. The aim of this study is to provide an effective therapeutic discovery platform for pancreatic cancer by establishing and characterizing patient-derived organoids (PDOs). METHODS: PDOs were established from pancreatic tumor surgical specimens, and the mutations were examined using a panel sequence. Expression of markers was assessed by PCR, immunoblotting, and immunohistochemistry; tumorigenicity was examined using immunodeficient mice, and drug responses were examined in vitro and in vivo. RESULTS: PDOs were established from eight primary and metastatic tumors, and the characteristic mutations and expression of cancer stem cell markers and CA19-9 were confirmed. Tumorigenicity of the PDOs was confirmed in subcutaneous transplantation and in the peritoneal cavity in the case of PDOs derived from disseminated nodules. Gemcitabine-sensitive/resistant PDOs showed consistent responses in vivo. High throughput screening in PDOs identified a compound effective for inhibiting tumor growth of a gemcitabine-resistant PDO xenograft model. CONCLUSIONS: This PDO-based platform captures important aspects of treatment-resistant pancreatic cancer and its metastatic features, suggesting that this study may serve as a tool for the discovery of personalized therapies.


Asunto(s)
Organoides , Neoplasias Pancreáticas , Animales , Descubrimiento de Drogas , Humanos , Ratones , Organoides/patología , Páncreas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas
4.
PLoS Biol ; 16(9): e2004874, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30256801

RESUMEN

Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell-germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.


Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Fertilidad , Células de Sertoli/metabolismo , Espermatogénesis , Actomiosina/metabolismo , Uniones Adherentes/metabolismo , Animales , Adhesión Celular , Células Cultivadas , Forminas , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Polimerizacion , Túbulos Seminíferos/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo
5.
J Cell Sci ; 126(Pt 8): 1773-84, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23444367

RESUMEN

Cytokinesis is initiated by constriction of the cleavage furrow, and completed with separation of the two daughter cells by abscission. Control of transition from constriction to abscission is therefore crucial for cytokinesis. However, the underlying mechanism is largely unknown. Here, we analyze the role of Citron kinase (Citron-K) that localizes at the cleavage furrow and the midbody, and dissect its action mechanisms during this transition. Citron-K forms a stable ring-like structure at the midbody and its depletion affects the maintenance of the intercellular bridge, resulting in fusion of two daughter cells after the cleavage furrow ingression. RNA interference (RNAi) targeting Citron-K reduced accumulation of RhoA, Anillin, and septins at the intercellular bridge in mid telophase, and impaired concentration and maintenance of KIF14 and PRC1 at the midbody in late telophase. RNAi rescue experiments revealed that these functions of Citron-K are mediated by its coiled-coil (CC) domain, and not by its kinase domain. The C-terminal part of CC contains a Rho-binding domain and a cluster-forming region and is important for concentrating Citron-K from the cleavage furrow to the midbody. The N-terminal part of CC directly binds to KIF14, and this interaction is required for timely transfer of Citron-K to the midbody after furrow ingression. We propose that the CC-domain-mediated translocation and actions of Citron-K ensure proper stabilization of the midbody structure during the transition from constriction to abscission.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular , Proteínas Contráctiles/metabolismo , Citocinesis/genética , Citocinesis/fisiología , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Microscopía Fluorescente , Células 3T3 NIH , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Septinas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo
6.
J Cell Sci ; 125(Pt 1): 108-20, 2012 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-22266902

RESUMEN

Regulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-α as an mDia-binding protein. The binding is mediated through the central region of Liprin-α and through the N-terminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-α competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-α fragment containing the mDia-binding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-α by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/metabolismo , Proteínas/metabolismo , Fibras de Estrés/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Forminas , Células HeLa , Humanos , Ratones , Transporte de Proteínas
7.
Elife ; 102021 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34427181

RESUMEN

Microtubules that assemble the mitotic spindle are generated by centrosomal nucleation, chromatin-mediated nucleation, and nucleation from the surface of other microtubules mediated by the augmin complex. Impairment of centrosomal nucleation in apical progenitors of the developing mouse brain induces p53-dependent apoptosis and causes non-lethal microcephaly. Whether disruption of non-centrosomal nucleation has similar effects is unclear. Here, we show, using mouse embryos, that conditional knockout of the augmin subunit Haus6 in apical progenitors led to spindle defects and mitotic delay. This triggered massive apoptosis and abortion of brain development. Co-deletion of Trp53 rescued cell death, but surviving progenitors failed to organize a pseudostratified epithelium, and brain development still failed. This could be explained by exacerbated mitotic errors and resulting chromosomal defects including increased DNA damage. Thus, in contrast to centrosomes, augmin is crucial for apical progenitor mitosis, and, even in the absence of p53, for progression of brain development.


Asunto(s)
Apoptosis/genética , Encéfalo/embriología , Proteínas Asociadas a Microtúbulos/genética , Células-Madre Neurales/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Femenino , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
8.
J Cell Biol ; 219(12)2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33170211

RESUMEN

Centrosomes, composed of centrioles that recruit a pericentriolar material (PCM) matrix assembled from PCNT and CDK5RAP2, catalyze mitotic spindle assembly. Here, we inhibit centriole formation and/or remove PCNT-CDK5RAP2 in RPE1 cells to address their relative contributions to spindle formation. While CDK5RAP2 and PCNT are normally dispensable for spindle formation, they become essential when centrioles are absent. Acentriolar spindle assembly is accompanied by the formation of foci containing PCNT and CDK5RAP2 via a microtubule and Polo-like kinase 1-dependent process. Foci formation and spindle assembly require PCNT-CDK5RAP2-dependent matrix assembly and the ability of CDK5RAP2 to recruit γ-tubulin complexes. Thus, the PCM matrix can self-organize independently of centrioles to generate microtubules for spindle assembly; conversely, an alternative centriole-anchored mechanism supports spindle assembly when the PCM matrix is absent. Extension to three cancer cell lines revealed similar results in HeLa cells, whereas DLD1 and U2OS cells could assemble spindles in the absence of centrioles and PCNT-CDK5RAP2, suggesting cell type variation in spindle assembly mechanisms.


Asunto(s)
Antígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Antígenos/genética , Proteínas de Ciclo Celular/genética , Centriolos/genética , Células HeLa , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Quinasa Tipo Polo 1
9.
FEBS Lett ; 580(14): 3375-80, 2006 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-16716304

RESUMEN

Here we used RNA interference and examined possible redundancy amongst Rho GTPases in their mitotic role. Chromosome misalignment is induced significantly in HeLa cells by Cdc42 depletion and not by depletion of either one or all of the other four Cdc42-like GTPases (TC10, TCL, Wrch1 or Wrch2), four Rac-like GTPases or three Rho-like GTPases. Notably, combined depletion of Cdc42 and all of the other four Cdc42-like GTPases significantly enhances chromosomal misalignment. These observations suggest that Cdc42 is the primary GTPase functioning during mitosis but that the other four Cdc42-like GTPases can also assume the mitotic role in its absence.


Asunto(s)
Mitosis/fisiología , Proteína de Unión al GTP cdc42/fisiología , Secuencia de Bases , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Interferencia de ARN
10.
Cell Rep ; 15(1): 54-60, 2016 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-27052165

RESUMEN

Errors during cell division in oocytes and early embryos are linked to birth defects in mammals. Bipolar spindle assembly in early mouse embryos is unique in that three or more acentriolar microtubule-organizing centers (MTOCs) are initially formed and are then clustered into two spindle poles. Using a knockout mouse and live imaging of spindles in embryos, we demonstrate that MTOC clustering during the blastocyst stage requires augmin, a critical complex for MT-dependent MT nucleation within the spindle. Functional analyses in cultured cells with artificially increased numbers of centrosomes indicate that the lack of intra-spindle MT nucleation, but not loss of augmin per se or overall reduction of spindle MTs, is the cause of clustering failure. These data suggest that onset of mitosis with three or more MTOCs is turned into a typical bipolar division through augmin-dependent intra-spindle MT assembly.


Asunto(s)
Blastocisto/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Blastocisto/ultraestructura , Células Cultivadas , Ratones , Proteínas Asociadas a Microtúbulos/genética , Centro Organizador de los Microtúbulos/ultraestructura , Mitosis
11.
Dev Cell ; 29(1): 5-6, 2014 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-24735876

RESUMEN

Microtubule networks generate various forces, and the forces are applied to microtubule-associated proteins (MAPs). Forth et al. (2014) show in a recent issue of Cell that asymmetric frictional force between MAPs and microtubules leads to directional movement of MAPs along microtubules, providing insight into the mechanism of microtubule network self-organization.


Asunto(s)
Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo
12.
Eur J Cell Biol ; 92(10-11): 303-15, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24183240

RESUMEN

Rho GTPase is a master regulator controlling cytoskeleton in multiple contexts such as cell migration, adhesion and cytokinesis. Of several Rho GTPases in mammals, the best characterized is the Rho subfamily including ubiquitously expressed RhoA and its homologs RhoB and RhoC. Upon binding GTP, Rho exerts its functions through downstream Rho effectors, such as ROCK, mDia, Citron, PKN, Rhophilin and Rhotekin. Until recently, our knowledge about functions of Rho and Rho effectors came mostly from in vitro studies utilizing cultured cells, and their physiological roles in vivo were largely unknown. However, gene-targeting studies of Rho and its effectors have now unraveled their tissue- and cell-specific roles and provide deeper insight into the physiological function of Rho signaling in vivo. In this article, we briefly describe previous studies of the function of Rho and its effectors in vitro and then review and discuss recent studies on knockout mice of Rho and its effectors.


Asunto(s)
Proteínas de Unión al GTP rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , Forminas , Proteínas de Unión al GTP , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/metabolismo
13.
Cell Rep ; 5(4): 926-32, 2013 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-24239357

RESUMEN

The small GTPase Rho and mDia2, a Rho-regulated actin nucleator, function as critical regulators of cytokinesis in cultured cells. However, their involvement in cytokinesis during mammalian development remains unknown. Here, we generated mice deficient in mDia2 and examined the role of Rho signaling in cytokinesis during development. mDia2-deficient mice survive until embryonic day 11.5 (E11.5), exhibit severe anemia with multinucleate erythroblasts, and die in utero by E12.5. mDia2-deficient erythroid cells differentiate normally, though in a delayed manner, but exhibit cytokinesis failure with decreased accumulation of F-actin in the cleavage furrow during late differentiation from proerythroblasts. On the other hand, inactivation of Rho induces cytokinesis failure from the earlier progenitor stage. mDia2-deficient erythroblasts, however, are able to enucleate their nuclei. Our findings have thus revealed that mDia2 functions critically in cytokinesis in vivo during erythropoiesis and further suggest that the cytokinesis mechanism in development diverges downstream of Rho. They also demonstrate that cytokinesis and enucleation utilize different mechanisms.


Asunto(s)
Citocinesis/genética , Embrión de Mamíferos/fisiología , Eritropoyesis/genética , Proteínas Asociadas a Microtúbulos/genética , NADPH Deshidrogenasa/genética , Proteínas de Unión al GTP rho/genética , Actinas , Animales , Núcleo Celular , Células Cultivadas , Citocinesis/fisiología , Eritroblastos/citología , Humanos , Ratones , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño
14.
Mol Biol Cell ; 21(18): 3193-204, 2010 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-20660154

RESUMEN

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.


Asunto(s)
Proteínas Contráctiles/metabolismo , Citocinesis/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , NADPH Deshidrogenasa/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Proteínas Contráctiles/genética , Células HeLa , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , NADPH Deshidrogenasa/genética , Células 3T3 NIH , Isoformas de Proteínas/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rho/genética
15.
J Biol Chem ; 284(9): 5753-62, 2009 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-19117945

RESUMEN

Mammalian homolog of Drosophila diaphanous (mDia) consisting of three isoforms, mDia1, mDia2, and mDia3, is an effector of Rho GTPases that catalyzes actin nucleation and polymerization. Although the mDia actions on actin dynamics in the cytoplasm have been well studied, whether mDia accumulates and functions in the nucleus remains largely unknown. Given the presence of actin and actin-associated proteins in the nucleus, we have examined nuclear localization of mDia isoforms. We expressed each of mDia isoforms as a green fluorescent protein fusion protein and examined their localization. Although all the mDia isoforms were localized predominantly in the cytoplasm under the steady-state conditions, mDia2 and not mDia1 or mDia3 accumulated extensively in the nucleus upon treatment with leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The LMB-induced nuclear accumulation was confirmed for endogenous mDia2 by using an antibody specific to mDia2. Studies using green fluorescent protein fusions of various truncation mDia2 mutants and point mutants of some of these proteins identified a functional nuclear localization signal in the N terminus of mDia2 and at least one functional nuclear export signal in the C terminus. The nuclear localization signal of mDia2 bound to importin-alpha and was imported into the nucleus by importin-alpha/beta complex in an in vitro transport assay. Consistently, depletion of importin-beta with RNA interference suppressed the LMB-induced nuclear localization of endogenous mDia2. These results suggest that mDia2 continuously shuttles between the nucleus and the cytoplasm using specific nuclear transport machinery composing of importin-alpha/beta and CRM1.


Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antifúngicos/farmacología , Proteínas Portadoras/genética , Núcleo Celular/genética , Citoplasma/genética , Ácidos Grasos Insaturados/farmacología , Técnica del Anticuerpo Fluorescente , Forminas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Carioferinas/genética , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Isoformas de Proteínas , ARN Interferente Pequeño/farmacología , Receptores Citoplasmáticos y Nucleares/genética , alfa Carioferinas/antagonistas & inhibidores , alfa Carioferinas/genética , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , Proteína Exportina 1
16.
Mol Biol Cell ; 19(5): 2328-38, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18287523

RESUMEN

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.


Asunto(s)
Actinas/metabolismo , Citocinesis , Fibroblastos/citología , Proteínas Asociadas a Microtúbulos/metabolismo , NADPH Deshidrogenasa/metabolismo , Anafase/efectos de los fármacos , Animales , Citocinesis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Ratones , Proteínas Asociadas a Microtúbulos/deficiencia , NADPH Deshidrogenasa/deficiencia , Células 3T3 NIH , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN/efectos de los fármacos , Telofase/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo
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