Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
J Cell Sci ; 135(3)2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-35006275

RESUMEN

Insulin secretion in pancreatic ß-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5ß (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5ß or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.


Asunto(s)
Células Secretoras de Insulina , Proteínas del Citoesqueleto/metabolismo , Exocitosis , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo
2.
J Biol Chem ; 291(39): 20617-29, 2016 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-27528609

RESUMEN

During cytokinesis, the antiparallel array of microtubules forming the central spindle organizes the midbody, a structure that anchors the ingressed cleavage furrow and guides the assembly of abscission machinery. Here, we identified a role for the flavoprotein monooxygenase MICAL3, an actin disassembly factor, in organizing midbody-associated protein complexes. By combining cell biological assays with cross-linking mass spectrometry, we show that MICAL3 is recruited to the central spindle and the midbody through a direct interaction with the centralspindlin component MKLP1. Knock-out of MICAL3 leads to an increased frequency of cytokinetic failure and a delayed abscission. In a mechanism independent of its enzymatic activity, MICAL3 targets the adaptor protein ELKS and Rab8A-positive vesicles to the midbody, and the depletion of ELKS and Rab8A also leads to cytokinesis defects. We propose that MICAL3 acts as a midbody-associated scaffold for vesicle targeting, which promotes maturation of the intercellular bridge and abscission.


Asunto(s)
Citocinesis/fisiología , Oxigenasas de Función Mixta/metabolismo , Huso Acromático/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Oxigenasas de Función Mixta/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Huso Acromático/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo
3.
Proc Natl Acad Sci U S A ; 113(11): E1498-505, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26903625

RESUMEN

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.


Asunto(s)
Proteína Axina/metabolismo , Folículo Piloso/citología , Células Madre/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Comunicación Autocrina , Proteína Axina/genética , Regulación de la Expresión Génica , Folículo Piloso/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Mutantes , Ratones Transgénicos , Células Madre/citología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
4.
Nat Commun ; 5: 4317, 2014 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-25007825

RESUMEN

Mical is a reduction-oxidation (redox) enzyme that functions as an unusual F-actin disassembly factor during Drosophila development. Although three Molecule interacting with CasL (MICAL) proteins exist in vertebrate species, their mechanism of action remains poorly defined and their role in vivo unknown. Here, we report that vertebrate MICAL-1 regulates the targeting of secretory vesicles containing immunoglobulin superfamily cell adhesion molecules (IgCAMs) to the neuronal growth cone membrane through its ability to control the actin cytoskeleton using redox chemistry, thereby maintaining appropriate IgCAM cell surface levels. This precise regulation of IgCAMs by MICAL-1 is essential for the lamina-specific targeting of mossy fibre axons onto CA3 pyramidal neurons in the developing mouse hippocampus in vivo. These findings reveal the first in vivo role for a vertebrate MICAL protein, expand the repertoire of cellular functions controlled through MICAL-mediated effects on the cytoskeleton, and provide insights into the poorly characterized mechanisms underlying neuronal protein cell surface expression and lamina-specific axonal targeting.


Asunto(s)
Citoesqueleto/fisiología , Hipocampo/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Oxigenasas de Función Mixta/fisiología , Fibras Musgosas del Hipocampo/fisiología , Vesículas Secretoras/fisiología , Actinas/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/fisiología , Femenino , Hipocampo/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Oxigenasas de Función Mixta/deficiencia , Oxigenasas de Función Mixta/genética , Modelos Animales , Oxidación-Reducción , Sinapsis/fisiología , Técnicas de Cultivo de Tejidos
5.
Dev Cell ; 27(2): 145-160, 2013 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-24120883

RESUMEN

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/ß1 and the components of the LL5ß-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.


Asunto(s)
Enfermedades Hereditarias del Ojo/metabolismo , Fibrosis/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Trastornos de la Motilidad Ocular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Línea Celular , Chlorocebus aethiops , Proteínas del Citoesqueleto , Enfermedades Hereditarias del Ojo/genética , Inhibidores de Crecimiento , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Morfogénesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Oftalmoplejía , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Supresoras de Tumor/metabolismo
6.
EMBO J ; 32(14): 2056-72, 2013 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-23736259

RESUMEN

The vesicle-associated membrane protein (VAMP) associated protein B (VAPB) is an integral membrane protein localized to the endoplasmic reticulum (ER). The P56S mutation in VAPB has been linked to motor neuron degeneration in amyotrophic lateral sclerosis type 8 (ALS8) and forms ER-like inclusions in various model systems. However, the role of wild-type and mutant VAPB in neurons is poorly understood. Here, we identified Yip1-interacting factor homologue A (YIF1A) as a new VAPB binding partner and important component in the early secretory pathway. YIF1A interacts with VAPB via its transmembrane regions, recycles between the ER and Golgi and is mainly localized to the ER-Golgi intermediate compartments (ERGICs) in rat hippocampal neurons. VAPB strongly affects the distribution of YIF1A and is required for intracellular membrane trafficking into dendrites and normal dendritic morphology. When VAPB-P56S is present, YIF1A is recruited to the VAPB-P56S clusters and loses its ERGIC localization. These data suggest that both VAPB and YIF1A are important for ER-to-Golgi transport and that missorting of YIF1A may contribute to VAPB-associated motor neuron disease.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Células Cultivadas , Dendritas/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Aparato de Golgi/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Enfermedad de la Neurona Motora/etiología , Enfermedad de la Neurona Motora/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Ratas , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
7.
Methods Mol Biol ; 777: 293-316, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21773937

RESUMEN

Microtubule organization and dynamics are controlled by a large set of cellular factors. An important group of microtubule regulators is microtubule plus-end-tracking proteins (+TIPs), which accumulate specifically at the growing microtubule ends, affect different phases of dynamic instability, and link microtubules to various cellular structures. +TIPs include a very diverse set of proteins with widely different structural properties. One of the most conserved and ubiquitous +TIP families are end-binding (EB) proteins, which can track growing microtubule ends autonomously in the absence of any other factors. In contrast, the majority of other known +TIPs cannot recognize the growing microtubule plus ends on their own; instead, they "hitchhike" to the plus ends by interacting with one of the members of the EB family. Therefore, the association with EBs and the ability to track growing microtubule ends are tightly linked, and binding to the EBs can be used to identify new +TIPs. In this chapter, we describe two affinity purification techniques, glutathione S-transferase and biotinylation tag-based pull-down assays that proved to be very useful for the identification of new EB-interacting +TIPs and their binding partners by mass spectrometry. We also discuss cytological techniques that can be applied to confirm plus-end localization of newly identified proteins.


Asunto(s)
Microtúbulos/metabolismo , Biotina/química , Cromatografía de Afinidad , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Células HEK293 , Humanos , Microtúbulos/química , Unión Proteica
8.
Curr Biol ; 21(11): 967-74, 2011 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-21596566

RESUMEN

Rab6 is a conserved small GTPase that localizes to the Golgi apparatus and cytoplasmic vesicles and controls transport and fusion of secretory carriers [1]. Another Rab implicated in trafficking from the trans-Golgi to the plasma membrane is Rab8 [2-5]. Here we show that Rab8A stably associates with exocytotic vesicles in a Rab6-dependent manner. Rab8A function is not needed for budding or motility of exocytotic carriers but is required for their docking and fusion. These processes also depend on the Rab6-interacting cortical factor ELKS [1], suggesting that Rab8A and ELKS act in the same pathway. We show that Rab8A and ELKS can be linked by MICAL3, a member of the MICAL family of flavoprotein monooxygenases [6]. Expression of a MICAL3 mutant with an inactive monooxygenase domain resulted in a strong accumulation of secretory vesicles that were docked at the cell cortex but failed to fuse with the plasma membrane, an effect that correlated with the strongly reduced mobility of MICAL3. We propose that the monooxygenase activity of MICAL3 is required to regulate its own turnover and the concomitant remodeling of vesicle-docking protein complexes in which it is engaged. Taken together, the results of our study illustrate cooperation of two Rab proteins in constitutive exocytosis and implicates a redox enzyme in this process.


Asunto(s)
Exocitosis/fisiología , Oxigenasas de Función Mixta/fisiología , Proteínas de Unión al GTP rab/fisiología , Transporte Biológico , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Células HeLa , Humanos , Fusión de Membrana , Oxigenasas de Función Mixta/análisis , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/metabolismo
9.
PLoS Biol ; 8(4): e1000350, 2010 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-20386726

RESUMEN

BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.


Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Mitosis/fisiología , Poro Nuclear/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Núcleo Celular/ultraestructura , Complejo Dinactina , Humanos , Cinesinas/genética , Proteínas de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Huso Acromático/metabolismo , Técnicas del Sistema de Dos Híbridos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...