RESUMEN
Expansion of a hexanucleotide repeat in a noncoding region of the C9ORF72 gene is responsible for a significant fraction of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) cases, but identifying specific toxic gene products and mechanisms has been difficult. Pathogenesis was proposed to involve the production of toxic RNA species and/or accumulation of toxic dipeptide repeats (DPRs), but distinguishing between these mechanisms has been challenging. In this study, we first use complementary model systems for analyzing pathogenesis in adult-onset neurodegenerative diseases to characterize the pathogenicity of DPRs produced by Repeat Associated Non-ATG (RAN) translation of C9ORF72 in specific cellular compartments: isolated axoplasm and giant synapse from the squid. Results showed selective axonal and presynaptic toxicity of GP-DPRs, independent of associated RNA. These effects involved downstream ASK1 signaling pathways that affect fast axonal transport and synaptic function, a pathogenic mechanism shared with other mutant proteins associated with familial ALS, like SOD1 and FUS. These pathways are sufficient to produce the "dying-back" axonopathy seen in ALS. However, other mutant genes (e.g., SOD1) that activate this mechanism rarely produce FTD. When parallel studies in primary motor neurons from rats were conducted, an additional pathogenic mechanism was revealed. The GR- and PR-DPRs, which had no effect on axonal transport or synaptic transmission, were found to disrupt the nuclei of transfected neurons, leading to "dying-forward" neuropathy. All C9-DRP-mediated toxic effects observed here are independent of whether the corresponding mRNAs contained hexanucleotide repeats or alternative codons. These studies establish the divergent toxicity of C9-DPRs that cause neurodegeneration in ALS and FTD, suggesting that these two independent pathogenic mechanisms may contribute to disease heterogeneity and/or synergize on disease progression in C9ORF72 patients with both ALS and FTD symptoms.
RESUMEN
Huntington's disease (HD) results from expansion of a polyglutamine tract (polyQ) in mutant huntingtin (mHTT) protein, but mechanisms underlying polyQ expansion-mediated toxic gain-of-mHTT function remain elusive. Here, deletion and antibody-based experiments revealed that a proline-rich domain (PRD) adjacent to the polyQ tract is necessary for mutant huntingtin (mHTT) to inhibit fast axonal transport and promote axonal pathology in cultured mammalian neurons. Further, polypeptides corresponding to subregions of the PRD sufficed to elicit the toxic effect on fast axonal transport, which was mediated by JNK kinases and involved PRD binding to one or more SH3-domain containing proteins. Collectively, these data suggested a mechanism whereby polyQ tract expansion in mHTT promotes aberrant PRD exposure and interactions of this domain with SH3 domain-containing proteins including some involved in activation of JNK kinases. In support, biochemical and immunohistochemical experiments linked aberrant PRD exposure to increased JNK activation in striatal tissues of the zQ175 mouse model and from post-mortem HD patients. Collectively, these findings support a critical role of PRD on mHTT toxicity, suggesting a novel framework for the potential development of therapies aimed to halt or reduce axonal pathology in HD.
RESUMEN
A benzimidazole-containing diamine monomer was prepared via a simple one-step synthesis process. A two-step procedure involving polycondensation in the presence of aromatic dianhydrides (4,4'-oxydiphthalic anhydride, ODPA) followed by thermal imidization was then performed to prepare a benzimidazole-based polyimide (BI-PI). BI-PI membranes were fabricated using an electrospinning technique and were hot pressed for 30 min at 200 °C under a pressure of 50 kgf /cm2. Finally, the hot-pressed membranes were assembled into supercapacitors, utilizing high-porosity-activated water chestnut shell biochar as the active material. The TGA results showed that the BI-PI polymer produced in the two-step synthesis process had a high thermal stability (Td5% = 527 °C). Moreover, the hot-press process reduced the pore size in the BI-PI membrane and improved the pore-size uniformity. The hot-press procedure additionally improved the mechanical properties of the BI-PI membrane, resulting in a high tensile modulus of 783 MPa and a tensile strength of 34.8 MPa. The cyclic voltammetry test results showed that the membrane had a specific capacitance of 121 F/g and a capacitance retention of 77%. By contrast, a commercial cellulose separator showed a specific capacitance value of 107 F/g and a capacitance retention of 49% under the same scanning conditions. Finally, the membrane showed both a small equivalent series resistance (Rs) and a small interfacial resistance (Rct). Overall, the results showed that the BI-PI membrane has significant potential as a separator for high-performance supercapacitor applications.
RESUMEN
Tau protein is subject to phosphorylation by multiple kinases at more than 80 different sites. Some of these sites are associated with tau pathology and neurodegeneration, but other sites are modified in normal tau as well as in pathological tau. Although phosphorylation of tau at residues in the microtubule-binding repeats is thought to reduce tau association with microtubules, the functional consequences of other sites are poorly understood. The AT8 antibody recognizes a complex phosphoepitope site on tau that is detectable in a healthy brain but significantly increased in Alzheimer's disease (AD) and other tauopathies. Previous studies showed that phosphorylation of tau at the AT8 site leads to exposure of an N-terminal sequence that promotes activation of a protein phosphatase 1 (PP1)/glycogen synthase 3 (GSK3) signaling pathway, which inhibits kinesin-1-based anterograde fast axonal transport (FAT). This finding suggests that phosphorylation may control tau conformation and function. However, the AT8 includes three distinct phosphorylated amino acids that may be differentially phosphorylated in normal and disease conditions. To evaluate the effects of specific phosphorylation sites in the AT8 epitope, recombinant, pseudophosphorylated tau proteins were perfused into the isolated squid axoplasm preparation to determine their effects on axonal signaling pathways and FAT. Results from these studies suggest a mechanism where specific phosphorylation events differentially impact tau conformation, promoting activation of independent signaling pathways that differentially affect FAT. Implications of findings here to our understanding of tau function in health and disease conditions are discussed.
RESUMEN
Overexpression of Aurora kinases is largely observed in many cancers, including hematologic malignancies. In this study, we investigated the effects and molecular mechanisms of Aurora kinase inhibitors in acute lymphoblastic leukemia (ALL). Western blot analysis showed that both Aurora-A and Aurora-B are overexpressed in ALL cell lines and primary ALL cells. Both VE-465 and VX-680 effectively inhibited Aurora kinase activities in nine ALL cell lines, which exhibited different susceptibilities to the inhibitors. Cells sensitive to Aurora kinase inhibitors underwent apoptosis at an IC50 of â¼10-30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 µM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility of ALL cell lines was not correlated with the expression level or activation status of Aurora kinases. Interestingly, RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora kinase-A inhibitors treatment. Complementary DNA (cDNA) microarray analysis suggested that CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Most importantly, primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to Aurora kinase inhibitors. Our study suggests CDKN1A could be a potential biomarker in determining the drug responsiveness of Aurora kinase inhibitors in ALL, particularly in MLL-AF4-positive patients.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.
RESUMEN
The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser(83) by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser(539), probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.
Asunto(s)
Cromosomas Humanos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitosis/fisiología , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Aurora Quinasas , Centrosoma/metabolismo , Cromosomas Humanos/genética , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosfoproteínas/genética , Fosforilación , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Aurora B kinase forms the enzymatic core of the Chromosomal Passenger Complex (CPC) and is a master regulator of mitosis. Understanding the regulation of Aurora B is critical to illuminate its role in mitosis. INCENP, Survivin and Borealin have all been known to promote Aurora B activation. In this study, we have identified the Aurora A activator protein TPX2 as a novel scaffold and co-activator protein of the CPC. Studies utilizing M-phase Xenopus egg extracts (XEE) revealed that the immunodepletion of endogenous TPX2 from XEE decreases Aurora B-Survivin and Aurora B-INCENP interactions, leading to a consequent reduction in Aurora B activity. Further, residues 138 to 328 of Xenopus TPX2 (TPX2 B) are sufficient to enhance Aurora B-Survivin association and Aurora B kinase activity in vitro. Importantly, experiments with pancreatic cancer cell lines suggest that this mechanism of Aurora B activation by TPX2 is likely to be conserved in human cells. Strikingly, the overexpression of human TPX2 B in HeLa cells causes defects in metaphase chromosome alignment and INCENP localization. Thus, in addition to its already established role as an Aurora A activator, our data support the role of TPX2 as a novel co-activator of Aurora kinase B.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Animales , Aurora Quinasa B/aislamiento & purificación , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/aislamiento & purificación , Células Cultivadas , Proteínas Cromosómicas no Histona/aislamiento & purificación , Células HEK293 , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Mitosis , Proteínas Nucleares/aislamiento & purificación , XenopusRESUMEN
Selective small-molecule kinase inhibitors have encouraging clinical efficacy in several malignancies. These agents are still limited to a subset of patients, indicating the need to develop therapeutic biomarkers that influence clinical benefit. In this study, we demonstrate that treatment with MK-8745, a novel Aurora-A specific inhibitor, leads to cell cycle arrest at the G2/M phase with accumulation of tetraploid nuclei followed by cell death in non-Hodgkin lymphoma (NHL) cell lines. The sensitivity of the cell lines to MK-8745 is correlated with the expression level of Aurora-A activator. The siRNA knockdown of Aurora-A activator TPX2 (targeting protein for Xenopus kinase-like protein 2) increased MK-8745 sensitivity in less-MK-8745-sensitive NHL cell lines, whereas overexpression of TPX2 in high-MK-8745-sensitive NHL cell lines increased drug resistance. Our results indicate that TPX2 may serve as a biomarker for identifying subpopulations of patients sensitive to Aurora-A inhibitor treatment.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/análisis , Linfoma no Hodgkin/patología , Proteínas Asociadas a Microtúbulos/análisis , Proteínas de Neoplasias/análisis , Proteínas Nucleares/análisis , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Tiazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Aurora Quinasa A , Aurora Quinasas , Biomarcadores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral/química , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Ciclina B/biosíntesis , Ciclina B/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser(539) as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser(539) phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser(539) plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitosis/fisiología , Fosfoproteínas/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Aurora Quinasas , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosfoproteínas/genética , Fosforilación/fisiología , Proteína Fosfatasa 1/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Unperturbed mitosis is a prerequisite for the generation of two genetically identical daughter cells. Nucleolar-spindle associated protein (NuSAP) is an important mitotic regulator. The activity of NuSAP is essential for a variety of cellular events that occur during mitosis starting from spindle assembly to cytokinesis. In addition to playing crucial roles during mitosis, NuSAP has been in the spotlight recently due to different studies exhibiting its importance in embryogenesis and cancer. In this review, we have extensively mined the current literature and made connections between different studies involving NuSAP. Importantly, we have assembled data pertaining to NuSAP from several proteomic studies and analyzed it thoroughly. Our review focuses on the role of NuSAP in mitosis and cancer, and brings to light several unanswered questions regarding the regulation of NuSAP in mitosis and its role in carcinogenesis.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Neoplasias/genética , Neoplasias/terapia , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , Transcripción GenéticaRESUMEN
Here we provide evidence in support of an inherent role for Arpc1b, a component of the Arp2/3 complex, in regulation of mitosis and demonstrate that its depletion inhibits Aurora A activation at the centrosome and impairs the ability of mammalian cells to enter mitosis. We discovered that Arpc1b colocalizes with gamma-tubulin at centrosomes and stimulates Aurora A activity. Aurora A phosphorylates Arpc1b on threonine 21, and expression of Arpc1b but not a nonphosphorylatable Arpc1b mutant in mammalian cells leads to Aurora A kinase activation and abnormal centrosome amplification in a Pak1-independent manner. Together, these findings reveal a new function for Arpc1b in centrosomal homeostasis. Arpc1b is both a physiological activator and substrate of Aurora A kinase and these interactions help to maintain mitotic integrity in mammalian cells.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Centrosoma/metabolismo , Activadores de Enzimas/metabolismo , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Aurora Quinasas , Línea Celular Tumoral , Humanos , Mutación , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Oxythiamine (OT), a transketolase inhibitor, is known to inhibit pancreatic cancer cell proliferation. In this study, we investigated the effect of inhibition of the transketolase pathway on signaling pathways in MIA PaCa cancer cells using in-house proteomic techniques. We hypothesized that OT alter protein phosphorylation thus affecting cell cycle arrest and cell proliferation. MIA PaCa-2 cells were cultured in media containing an algal (15)N amino acid mixture at 50% enrichment, with and without OT, to determine protein expression and synthesis. Analysis of cell lysates using two-dimensional gel electrophoresis matrix assisted laser desorption and ionization time-of-flight and time-of-flight mass spectrometry (2-DE-MALDI-TOF/TOF MS) identified 12 phosphor proteins that were significantly suppressed by OT treatment. Many of these proteins are involved in regulation of cycle activities and apoptosis. Among the proteins identified, expression of the phosphor heat shock protein 27 (Hsp27) was dramatically inhibited by OT treatment while the level of its total protein remained unchanged. Hsp27 expression and phosphorylation is known to be associated with drug resistance and cancer cell survival. The changes in phosphorylation of key proteins of cancer proliferation and survival suggest that protein phosphorylation is the confluence of the effects of OT on metabolic and signaling pathways.
Asunto(s)
Neoplasias Pancreáticas/metabolismo , Proteínas/metabolismo , Transducción de Señal , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas , Oxitiamina/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The small guanosine triphosphatase Ran loaded with GTP (RanGTP) can stimulate assembly of the type V intermediate filament protein lamin B into a membranous lamin B spindle matrix, which is required for proper microtubule organization during spindle assembly. Microtubules in turn enhance assembly of the matrix. Here we report that the isolated matrix contains known spindle assembly factors such as dynein and Nudel. Using spindle assembly assays in Xenopus egg extracts, we show that Nudel regulates microtubule organization during spindle assembly independently of its function at kinetochores. Importantly, Nudel interacts directly with lamin B to facilitate the accumulation and assembly of lamin-B-containing matrix on microtubules in a dynein-dependent manner. Perturbing either Nudel or dynein inhibited the assembly of lamin B matrix. However, depleting lamin B still allowed the formation of matrices containing dynein and Nudel. Therefore, dynein and Nudel regulate assembly of the lamin B matrix. Interestingly, we found that whereas depleting lamin B resulted in disorganized spindle and spindle poles, disrupting the function of Nudel or dynein caused a complete lack of spindle pole focusing. We suggest that Nudel regulates microtubule organization in part by facilitating assembly of the lamin B spindle matrix in a dynein-dependent manner.
Asunto(s)
Proteínas Portadoras/metabolismo , Dineínas/metabolismo , Matriz Extracelular/metabolismo , Lamina Tipo B/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Huso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Aurora Quinasas , Bioensayo , Cromatina/metabolismo , Humanos , Cinetocoros/metabolismo , Laminina/metabolismo , Masculino , Microtúbulos/metabolismo , Mitosis , Morfogénesis , Óvulo/citología , Óvulo/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , XenopusRESUMEN
The mitotic spindle apparatus has attracted the attention of cell biologists for decades. Whereas the main function of this microtubule-based system is to segregate chromosomes, spindle morphogenesis and chromosome segregation must also coordinate with the segregation of the whole cell. The finding that RanGTPase stimulates the assembly of a lamin B-containing membranous matrix in mitosis(1) may provide a connection between the segregation of mitotic chromosomes and the partitioning of membrane systems during cell division.
Asunto(s)
Laminina/metabolismo , Huso Acromático/metabolismo , Segregación Cromosómica , Humanos , Interfase , Mitosis , Huso Acromático/fisiología , Proteína de Unión al GTP ran/fisiologíaRESUMEN
Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.
Asunto(s)
Lamina Tipo B/fisiología , Mitosis , Huso Acromático/fisiología , Proteína de Unión al GTP ran/fisiología , Animales , Guanosina Trifosfato/metabolismo , Humanos , Lamina Tipo B/análisis , Lamina Tipo B/genética , Microtúbulos/metabolismo , Interferencia de ARN , Huso Acromático/química , Huso Acromático/ultraestructura , Xenopus , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismoRESUMEN
The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts . Recently, we have shown that AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis . However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear . Here, we report the development of functional assays that allow us to show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads , AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs.
Asunto(s)
Centro Organizador de los Microtúbulos/metabolismo , Óvulo/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Huso Acromático/fisiología , Animales , Aurora Quinasas , Proteínas de Ciclo Celular/metabolismo , Microesferas , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Huso Acromático/metabolismo , Xenopus , Proteína de Unión al GTP ran/metabolismoRESUMEN
The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.