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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912962

RESUMO

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and late endosomes/lysosomes (LE/lys) are emerging as critical hubs for diverse cellular events, and changes in their extents are linked to severe neurological diseases. While recent studies show that the synaptotagmin-like mitochondrial-lipid-binding (SMP) domain-containing protein PDZD8 may mediate the formation of ER-LE/lys MCSs, the cellular functions of PDZD8 remain largely elusive. Here, we attempt to investigate the lipid transfer activities of PDZD8 and the extent to which its cellular functions depend on its lipid transfer activities. In accordance with recent studies, we demonstrate that PDZD8 is a protrudin (ZFYVE27)-interacting protein and that PDZD8 acts as a tether at ER-LE/lys MCSs. Furthermore, we discover that the SMP domain of PDZD8 binds glycerophospholipids and ceramides both in vivo and in vitro, and that the SMP domain can transport lipids between membranes in vitro. Functionally, PDZD8 is required for LE/lys positioning and neurite outgrowth, which is dependent on the lipid transfer activity of the SMP domain.

2.
Mol Biol Cell ; : mbcE21030097, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34133214

RESUMO

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and mitochondria are emerging as critical hubs for diverse cellular events, and alterations in the extent of these contacts are linked to neurodegenerative diseases. However, the mechanisms that control ER-mitochondrial interactions are so far elusive. Here, we demonstrate a key role of vacuolar protein sorting-associated protein 13D (VPS13D) in the negative regulation of ER-mitochondrial MCSs. VPS13D suppression results in extensive ER-mitochondrial tethering, a phenotype that can be substantially rescued by suppression of the tethering proteins VAPB and PTPIP51. VPS13D interacts with valosin-containing protein (VCP/p97) to control the level of ER-resident VAPB at contacts. VPS13D is required for the stability of p97. Functionally, VPS13D suppression leads to severe defects in the mitochondrial morphology, mitochondrial cellular distribution and mitochondrial DNA synthesis. Together our results suggest that VPS13D negatively regulates the ER-mitochondrial MCSs partially through its interactions with VCP/p97. [Media: see text].

3.
Nat Commun ; 12(1): 1252, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623047

RESUMO

Upon starvation, cells rewire their metabolism, switching from glucose-based metabolism to mitochondrial oxidation of fatty acids, which require the transfer of FAs from lipid droplets (LDs) to mitochondria at mitochondria-LD membrane contact sites (MCSs). However, factors responsible for FA transfer at these MCSs remain uncharacterized. Here, we demonstrate that vacuolar protein sorting-associated protein 13D (VPS13D), loss-of-function mutations of which cause spastic ataxia, coordinates FA trafficking in conjunction with the endosomal sorting complex required for transport (ESCRT) protein tumor susceptibility 101 (TSG101). The VPS13 adaptor-binding domain of VPS13D and TSG101 directly remodels LD membranes in a cooperative manner. The lipid transfer domain of human VPS13D binds glycerophospholipids and FAs in vitro. Depletion of VPS13D, TSG101, or ESCRT-III proteins inhibits FA trafficking from LDs to mitochondria. Our findings suggest that VPS13D mediates the ESCRT-dependent remodeling of LD membranes to facilitate FA transfer at mitochondria-LD contacts.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Fluorescência , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas/química
4.
J Cell Sci ; 132(18)2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31413070

RESUMO

Recent studies show that mitochondria and actin filaments work together in two contexts: (1) increased cytoplasmic calcium induces cytoplasmic actin polymerization that stimulates mitochondrial fission and (2) mitochondrial depolarization causes actin assembly around mitochondria, with roles in mitophagy. It is unclear whether these two processes utilize similar actin assembly mechanisms. Here, we show that these are distinct actin assembly mechanisms in the acute phase after treatment (<10 min). Calcium-induced actin assembly is INF2 dependent and Arp2/3 complex independent, whereas depolarization-induced actin assembly is Arp2/3 complex dependent and INF2 independent. The two types of actin polymerization are morphologically distinct, with calcium-induced filaments throughout the cytosol and depolarization-induced filaments as 'clouds' around depolarized mitochondria. We have previously shown that calcium-induced actin stimulates increases in both mitochondrial calcium and recruitment of the dynamin GTPase Drp1 (also known as DNM1L). In contrast, depolarization-induced actin is temporally associated with extensive mitochondrial dynamics that do not result in mitochondrial fission, but in circularization of the inner mitochondrial membrane (IMM). These dynamics are dependent on the protease OMA1 and independent of Drp1. Actin cloud inhibition causes increased IMM circularization, suggesting that actin clouds limit these dynamics.This article has an associated First Person interview with the first author of the paper.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Citoplasma/metabolismo , Imunofluorescência , Humanos , Ionomicina/farmacologia , Microscopia Confocal , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/fisiologia , Multimerização Proteica/efeitos dos fármacos
5.
J Cell Biol ; 217(1): 251-268, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29142021

RESUMO

Mitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through ER-bound inverted formin 2 (INF2) stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER-mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.


Assuntos
Actinas/metabolismo , Divisão Celular/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Dinaminas , Forminas , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Transporte de Íons/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
6.
J Cell Biol ; 216(12): 4123-4139, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29158231

RESUMO

Drp1 is a dynamin guanosine triphosphatase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51, and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is associated with ER in mammalian cells and is distinct from mitochondrial or peroxisomal Drp1 populations. Subpopulations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, and ER bound) and mitochondrial division, whereas Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division.


Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/genética , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Linhagem Celular Tumoral , Dinaminas , Retículo Endoplasmático/ultraestrutura , Forminas , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Multimerização Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
7.
Mol Biol Cell ; 27(20): 3109-3121, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27559132

RESUMO

Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds actin filaments, and the off-rate of actin-bound Drp1 is significantly increased by unbound Drp1. GDP and GTP accelerate and decelerate Drp1/actin binding dynamics, respectively. Actin has a biphasic effect on Drp1 GTP hydrolysis, increasing at low actin:Drp1 ratio but returning to baseline at high ratio. Drp1 also bundles filaments. Bundles have reduced dynamics but follow the same trends as single filaments. Drp1 preferentially incorporates into bundles at higher ionic strength. We measure Drp1 concentration to be ∼0.5 µM in U2OS cell cytosol, suggesting the actin-binding affinity measured here (Kd = 0.6 µM) is in the physiologically relevant range. The ability of Drp1 to bind actin filaments in a highly dynamic manner provides potential for actin filaments to serve as reservoirs of oligomerization-competent Drp1 that can be accessed for mitochondrial fission.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Técnicas de Cultura de Células , Citosol/metabolismo , Dinaminas , GTP Fosfo-Hidrolases/farmacocinética , GTP Fosfo-Hidrolases/fisiologia , Humanos , Hidrólise , Proteínas Associadas aos Microtúbulos/farmacocinética , Proteínas Associadas aos Microtúbulos/fisiologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/farmacocinética , Proteínas Mitocondriais/fisiologia , Peroxissomos/metabolismo , Ligação Proteica , Multimerização Proteica
8.
Elife ; 4: e11553, 2015 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-26609810

RESUMO

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.


Assuntos
Citoesqueleto de Actina/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Linhagem Celular , Dinaminas , Humanos , Ligação Proteica , Transporte Proteico
9.
Proc Natl Acad Sci U S A ; 111(15): 5574-9, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24706897

RESUMO

The mammalian small ubiquitin-like modifiers (SUMOs) are actively involved in regulating differentiation of different cell types. However, the functional differences between SUMO isoforms and their mechanisms of action remain largely unknown. Using the ocular lens as a model system, we demonstrate that different SUMOs display distinct functions in regulating differentiation of epithelial cells into fiber cells. During lens differentiation, SUMO1 and SUMO2/3 displayed different expression, localization, and targets, suggesting differential functions. Indeed, overexpression of SUMO2/3, but not SUMO1, inhibited basic (b) FGF-induced cell differentiation. In contrast, knockdown of SUMO1, but not SUMO2/3, also inhibited bFGF action. Mechanistically, specificity protein 1 (Sp1), a major transcription factor that controls expression of lens-specific genes such as ß-crystallins, was positively regulated by SUMO1 but negatively regulated by SUMO2. SUMO2 was found to inhibit Sp1 functions through several mechanisms: sumoylating it at K683 to attenuate DNA binding, and at K16 to increase its turnover. SUMO2 also interfered with the interaction between Sp1 and the coactivator, p300, and recruited a repressor, Sp3 to ß-crystallin gene promoters, to negatively regulate their expression. Thus, stable SUMO1, but diminishing SUMO2/3, during lens development is necessary for normal lens differentiation. In support of this conclusion, SUMO1 and Sp1 formed complexes during early and later stages of lens development. In contrast, an interaction between SUMO2/3 and Sp1 was detected only during the initial lens vesicle stage. Together, our results establish distinct roles of different SUMO isoforms and demonstrate for the first time, to our knowledge, that Sp1 acts as a major transcription factor target for SUMO control of cell differentiation.


Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Cristalino/crescimento & desenvolvimento , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fator de Transcrição Sp1/metabolismo , Sumoilação/fisiologia , Animais , Western Blotting , Imunoprecipitação da Cromatina , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Crescimento de Fibroblastos/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Cristalino/citologia , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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