RESUMEN
For more than 15 years, TPX2 has been studied as a factor critical for mitosis and spindle assembly. These functions of TPX2 are attributed to its Ran-regulated microtubule-associated protein properties and to its control of the Aurora A kinase. Overexpressed in cancers, TPX2 is being established as marker for the diagnosis and prognosis of malignancies. During interphase, TPX2 resides preferentially in the nucleus where its function had remained elusive until recently. The latest finding that TPX2 plays a role in amplification of the DNA damage response, combined with the characterization of TPX2 knockout mice, open new perspectives to understand the biology of this protein. This review provides an historic overview of the discovery of TPX2 and summarizes its cytoskeletal and signaling roles with relevance to cancer therapies. Finally, the review aims to reconcile discrepancies between the experimental and pathological effects of TPX2 overexpression and advances new roles for compartmentalized TPX2.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Huso Acromático/metabolismo , Animales , Ciclo Celular , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/genética , Mitosis , Neoplasias/genética , Neoplasias/patología , Neoplasias/terapia , Proteínas Nucleares/análisis , Proteínas Nucleares/genéticaRESUMEN
In acute and chronic neurodegeneration, Ca(2+) mishandling and disruption of the cytoskeleton compromise neuronal integrity, yet abnormalities in the signaling roles of cytoskeletal proteins remain largely unexplored. We now report that the microtubule-associated protein p600 (also known as UBR4) promotes neuronal survival. Following depletion of p600, glutamate-induced Ca(2+) influx through NMDA receptors, but not AMPA receptors, initiates a degenerative process characterized by endoplasmic reticulum fragmentation and endoplasmic reticulum Ca(2+) release via inositol 1,4,5-trisphosphate receptors. Downstream of NMDA receptors, p600 associates with the calmodulin·calmodulin-dependent protein kinase IIα complex. A direct and atypical p600/calmodulin interaction is required for neuronal survival. Thus, p600 counteracts specific Ca(2+)-induced death pathways through regulation of Ca(2+) homeostasis and signaling.
Asunto(s)
Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas de Unión a Calmodulina/genética , Supervivencia Celular/fisiología , Células Cultivadas , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Ratas , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The large microtubule-associated/Ca(2+)-signalling protein p600 (also known as UBR4) is required for hippocampal neuronal survival upon Ca(2+) dyshomeostasis induced by glutamate treatment. During this process, p600 prevents aggregation of the Ca(2+)/calmodulin-dependent kinase IIα (CaMKIIα), a proxy of neuronal death, via direct binding to calmodulin in a microtubuleindependent manner. Using photoconductive stimulation coupled with live imaging of single neurons, we identified a distinct mechanism of prevention of CaMKIIα aggregation by p600. Upon direct depolarization, CaMKIIα translocates to microtubules. In the absence of p600, this translocation is interrupted in favour of a sustained self-aggregation that is prevented by the microtubule-stabilizing drug paclitaxel. Thus, during photoconductive stimulation, p600 prevents the aggregation of CaMKIIα by stabilizing microtubules. The effectiveness of this stabilization for preventing CaMKIIα aggregation during direct depolarization but not during glutamate treatment suggests a model wherein p600 has two modes of action depending on the source of cytosolic Ca(2+).
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Células Cultivadas , Dendritas/metabolismo , Hipocampo/citología , Luz , Proteínas Asociadas a Microtúbulos/genética , Neuronas/citología , Neuronas/efectos de la radiación , Interferencia de ARN , Ratas , Análisis de la Célula Individual/métodos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Rayos gamma/efectos adversos , Histonas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/efectos de la radiación , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/genética , Apoptosis/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Línea Celular , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fase G1/genética , Fase G1/efectos de la radiación , Histonas/genética , Humanos , Proteínas Asociadas a Microtúbulos/genética , Mitosis/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/efectos de la radiación , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Apical neural progenitors (aNPs) drive neurogenesis by means of a program consisting of self-proliferative and neurogenic divisions. The balance between these two manners of division sustains the pool of apical progenitors into late neurogenesis, thereby ensuring their availability to populate the brain with terminal cell types. Using knockout and in utero electroporation mouse models, we report a key role for the microtubule-associated protein 600 (p600) in the regulation of spindle orientation in aNPs, a cellular event that has been associated with cell fate and neurogenesis. We find that p600 interacts directly with the neurogenic protein Ndel1 and that aNPs knockout for p600, depleted of p600 by shRNA or expressing a Ndel1-binding p600 fragment all display randomized spindle orientation. Depletion of p600 by shRNA or expression of the Ndel1-binding p600 fragment also results in a decreased number of Pax6-positive aNPs and an increased number of Tbr2-positive basal progenitors destined to become neurons. These Pax6-positive aNPs display a tilted mitotic spindle. In mice wherein p600 is ablated in progenitors, the production of neurons is significantly impaired and this defect is associated with microcephaly. We propose a working model in which p600 controls spindle orientation in aNPs and discuss its implication for neurogenesis.
RESUMEN
p600 is a multifunctional protein implicated in cytoskeletal organization, integrin-mediated survival signaling, calcium-calmodulin signaling and the N-end rule pathway of ubiquitin-proteasome-mediated proteolysis. While push, the Drosophila counterpart of p600, is dispensable for development up to adult stage, the role of p600 has not been studied during mouse development. Here we generated p600 knockout mice to investigate the in vivo functions of p600. Interestingly, we found that homozygous deletion of p600 results in lethality between embryonic days 11.5 and 13.5 with severe defects in both embryo and placenta. Since p600 is required for placental development, we performed conditional disruption of p600, which deletes selectively p600 in the embryo but not in the placenta. The conditional mutant embryos survive longer than knockout embryos but ultimately die before embryonic day 14.5. The mutant embryos display severe cardiac problems characterized by ventricular septal defects and thin ventricular walls. These anomalies are associated with reduced activation of FAK and decreased expression of MEF2, which is regulated by FAK and plays a crucial role in cardiac development. Moreover, we observed pleiotropic defects in the liver and brain. In sum, our study sheds light on the essential roles of p600 in fetal development.
Asunto(s)
Potenciales Evocados , Desarrollo Fetal/fisiología , Animales , Potenciales Evocados/genética , Eliminación de Gen , Ratones , Ratones NoqueadosRESUMEN
Cytoskeleton dynamics, membranes trafficking and positioning are essential for the proper functioning of any mammalian cell. The identification of the molecules and mechanisms that allow these cellular processes to interface is vital for understanding cell behaviors. Ndel1, the mammalian homolog of the Aspergillus nidulans NudE, organizes the cytoskeleton and regulates molecular motors, thereby impacting on the positioning of membranes. Hypothetically, Ndel1 can act in concert with enzymes controlling membrane trafficking (vesicle-mediated transport) per se, but this idea has never been investigated. We now report that a pool of Ndel1 associates directly with Dynamin 2 (Dyn2), a large cytosolic GTPase involved in the trafficking of the AMPA receptor subunit GluR1. In vitro, Ndel1 enhances Dyn2 GTPase activity in its unassembled and assembled forms, without promoting oligomerization of the enzyme. In cells, gain and loss of function of Ndel1 recapitulate the effects of overexpression of Dyn2 and Dyn2 dominant negative with reduced GTPase activity on the intracellular localization of GluR1, respectively, without affecting the stability of microtubules. Together, these results indicate that Ndel1 regulates Dyn2 GTPase activity and impacts GluR1-containing membranes distribution in a manner reminiscent of Dyn2.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas del Citoesqueleto/metabolismo , Dinamina II/metabolismo , Receptores AMPA/metabolismo , Transporte Biológico , Línea Celular , Membrana Celular , GTP Fosfohidrolasas/metabolismo , HumanosRESUMEN
Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.
Asunto(s)
Axones/fisiología , Proteínas Portadoras/metabolismo , Filamentos Intermedios/metabolismo , Regeneración Nerviosa , Animales , Axones/patología , Proteínas Portadoras/genética , Ganglios Espinales/patología , Ratones , Compresión Nerviosa , Neuritas/patología , Proteínas de Neurofilamentos/metabolismo , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Nervio Ciático/patología , Regulación hacia Arriba , Vimentina/genéticaRESUMEN
Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1-6). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein alphaCOP. Ndel1 promotes the interaction between Lis1, alphaCOP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/alphaCOP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration.