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1.
EMBO J ; 37(3): 321-336, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29311115

RESUMO

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho-GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double-knockout mouse embryos and single- and double-knockout embryonic fibroblasts, we demonstrate the essential and non-redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double-knockout cells. In contrast, we show that TRAK2-mediated retrograde mitochondrial transport is Miro1-dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin-dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double-knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine-tune actin- and tubulin-dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.


Assuntos
Dineínas/metabolismo , Cinesinas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Miosinas/metabolismo , Proteínas rho de Ligação ao GTP/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Transporte Biológico , Proteínas de Transporte/metabolismo , Linhagem Celular Transformada , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
2.
J Biol Chem ; 291(2): 613-29, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26553875

RESUMO

The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder, and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but its effects in dendrites have yet to be studied. Further, the mechanisms of this regulation and its role in neuronal development and brain function are poorly understood. Here we have demonstrated that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane GTPase proteins Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins (mitofusins). Using live cell imaging, we show that disruption of the DISC1-Miro-TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localizes to the mitochondria, where it similarly disrupts mitochondrial dynamics. We also show by super resolution microscopy that DISC1 is localized to endoplasmic reticulum contact sites and that the DISC1-Boymaw fusion protein decreases the endoplasmic reticulum-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro-TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate the transport, fusion, and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function leading to abnormal neuronal development.


Assuntos
Dendritos/metabolismo , Dinâmica Mitocondrial , Morfogênese , Proteínas do Tecido Nervoso/metabolismo , Animais , Axônios/metabolismo , Transporte Biológico , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Tecido Nervoso/química , Ligação Proteica , RNA Longo não Codificante , Proteínas Recombinantes de Fusão/metabolismo , Esquizofrenia/metabolismo , Relação Estrutura-Atividade
3.
Glia ; 64(7): 1252-64, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27189737

RESUMO

The astrocytic GLT-1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live-cell imaging to study the mechanisms regulating GLT-1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP-time lapse imaging, we show that GLT-1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity-dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT-1 is more stable than diffuse GLT-1 and that glutamate increases GLT-1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT-1 isoforms expressed in the brain, GLT-1a and GLT-1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT-1b more so. GLT-1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT-1 isoforms. Altogether, these data reveal that astrocytic GLT-1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252-1264.


Assuntos
Astrócitos/fisiologia , Transporte Biológico/fisiologia , Córtex Cerebral/citologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Neurônios/fisiologia , 4-Aminopiridina/farmacologia , Anestésicos Locais/farmacologia , Animais , Animais Recém-Nascidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacologia , Astrócitos/efeitos dos fármacos , Transporte Biológico/genética , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Transgênicos , Tetrodotoxina/farmacologia
4.
J Biol Chem ; 289(21): 14569-82, 2014 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-24671417

RESUMO

Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2-3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.


Assuntos
Proteínas Mitocondriais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Feminino , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/genética , Lisina/metabolismo , Masculino , Microscopia Confocal , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Serina/genética , Serina/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas rho de Ligação ao GTP/genética
5.
Biochem Soc Trans ; 41(6): 1525-31, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24256248

RESUMO

Correct mitochondrial dynamics are essential to neuronal function. These dynamics include mitochondrial trafficking and quality-control systems that maintain a precisely distributed and healthy mitochondrial network, so that local energy demands or Ca2+-buffering requirements within the intricate architecture of the neuron can be met. Mitochondria make use of molecular machinery that couples these organelles to microtubule-based transport via kinesin and dynein motors, facilitating the required long-range movements. These motors in turn are associated with a variety of adaptor proteins allowing additional regulation of the complex dynamics demonstrated by these organelles. Over recent years, a number of new motor and adaptor proteins have been added to a growing list of components implicated in mitochondrial trafficking and distribution. Yet, there are major questions that remain to be addressed about the regulation of mitochondrial transport complexes. One of the core components of this machinery, the mitochondrial Rho GTPases Miro1 (mitochondrial Rho 1) and Miro2 have received special attention due to their Ca2+-sensing and GTPase abilities, marking Miro an exceptional candidate for co-ordinating mitochondrial dynamics and intracellular signalling pathways. In the present paper, we discuss the wealth of literature regarding Miro-mediated mitochondrial transport in neurons and recently highlighted involvement of Miro proteins in mitochondrial turnover, emerging as a key process affected in neurodegeneration.


Assuntos
Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Humanos , Mitocôndrias/metabolismo , Neurônios/enzimologia
6.
iScience ; 25(1): 103536, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-34977501

RESUMO

Ataxin-2 (Atx2) is a highly conserved RNA binding protein. Atx2 undergoes polyglutamine expansion leading to amyotrophic lateral sclerosis (ALS) or spinocerebellar ataxia type 2 (SCA2). However, the physiological functions of Atx2 in neurons remain unknown. Here, using the powerful genetics of Drosophila, we show that Atx2 is essential for normal neuronal cytoskeletal dynamics and organelle trafficking. Upon neuron-specific Atx2 loss, the microtubule and actin networks were abnormally stabilized and cargo transport was drastically inhibited. Depletion of Atx2 caused multiple morphological defects in the nervous system of third instar larvae. These include reduced brain size, impaired axon development, and decreased dendrite outgrowth. Defects in the nervous system caused loss of the ability to crawl and lethality at the pupal stage. Taken together, these data mark Atx2 as a major regulator of cytoskeletal dynamics and denote Atx2 as an essential gene in neurodevelopment, as well as a neurodegenerative factor.

7.
Front Cell Dev Biol ; 8: 449, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32637409

RESUMO

The disrupted in schizophrenia 1 (DISC1) protein is implicated in major mental illnesses including schizophrenia and bipolar disorder. A key feature of psychiatric disease is aberrant synaptic communication. Correct synaptic transmission is dependent on spatiotemporally regulated energy provision and calcium buffering. This can be achieved by precise distribution of mitochondria throughout the elaborate architecture of the neuron. Central to this process is the calcium sensor and GTPase Miro1, which allows mitochondrial trafficking by molecular motors. While the role of Miro1-calcium binding in mitochondrial transport is well described, far less is known regarding the functions of the two GTPase domains. Here, we investigate the effects of a psychiatric disease-associated mutation in DISC1 on mitochondrial trafficking. We show that this DISC1 mutation impairs Miro1's ability to transport mitochondria. We also demonstrate the necessity of the first Miro1 GTPase domain in determining direction of mitochondrial transport and the involvement of DISC1 in this process. Finally, we describe the effects of mutant DISC1 on positioning of mitochondria at synapses.

8.
Elife ; 92020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32022690

RESUMO

Correct neuronal development requires tailored neurite outgrowth. Neurite outgrowth is driven in part by microtubule-sliding - the transport of microtubules along each other. We have recently demonstrated that a 'mitotic' kinesin-6 (Pavarotti in Drosophila) effectively inhibits microtubule-sliding and neurite outgrowth. However, mechanisms regulating Pavarotti itself in interphase cells and specifically in neurite outgrowth are unknown. Here, we use a combination of live imaging and biochemical methods to show that the inhibition of microtubule-sliding by Pavarotti is controlled by phosphorylation. We identify the Ser/Thr NDR kinase Tricornered (Trc) as a Pavarotti-dependent regulator of microtubule sliding in neurons. Further, we show that Trc-mediated phosphorylation of Pavarotti promotes its interaction with 14-3-3 proteins. Loss of 14-3-3 prevents Pavarotti from associating with microtubules. Thus, we propose a pathway by which microtubule-sliding can be up- or downregulated in neurons to control neurite outgrowth, and establish parallels between microtubule-sliding in mitosis and post-mitotic neurons.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Crescimento Neuronal/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Drosophila , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Larva/citologia , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/genética , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética
9.
Dev Cell ; 48(6): 746-748, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30913405

RESUMO

Kinetochore-microtubule attachments are essential to direct proper chromosome segregation during cell division. In this issue of Developmental Cell, Cheerambathur et al. (2019) and Zhao et al. (2019) uncover an unexpected role in neuronal development, unrelated to cell division, for components of the highly conserved kinetochore-microtubule attachment complex.


Assuntos
Segregação de Cromossomos , Cinetocoros , Microtúbulos , Morfogênese , Sistema Nervoso
10.
Trends Cell Biol ; 29(11): 901-911, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31597609

RESUMO

At first look, cell division and neurite formation seem to be two different, essential biological processes. However, both processes require extensive reorganization of the cytoskeleton, and especially microtubules. Remarkably, in recent years, independent work from several groups has shown that multiple cytoskeletal components previously considered specific for the mitotic machinery play important roles in neurite initiation and extension. In this review article, we describe how several cytoplasmic and mitotic microtubule motors, components of mitotic kinetochores, and cortical actin participate in reorganization of the microtubule network required to form and maintain axons and dendrites. The emerging similarities between these two biological processes will certainly generate new insights into the mechanisms generating the unique morphology of neurons.


Assuntos
Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Neuritos/metabolismo , Neurogênese/fisiologia , Animais , Drosophila , Cinetocoros/metabolismo , Mitose/fisiologia , Neurônios/citologia , Fuso Acromático/fisiologia
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