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1.
Zhonghua Bing Li Xue Za Zhi ; 49(7): 675-680, 2020 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-32610377

RESUMO

Objective: To investigate the clinical and pathological features of pediatric NTRK-rearranged tumors. Methods: Four NTRK-rearranged soft tissue tumors and one renal tumor at Shanghai Children's Medical Center, Shanghai Jiaotong University and Singapore KK Women's and Children's Hospital from January 2017 to September 2019 were identified. Pan-TRK immunohistochemistry, and the ALK and ETV6 gene break-apart fluorescence in situ hybridizations (FISH) were performed. NTRK gene rearrangement was detected using sequencing-based methods. Results: There were 3 males and 2 females in this study. The patients were between 3 months and 13 years of age. Histologically, the tumors were infiltrative spindle cell tumors with variable accompanying inflammatory cells. Immunohistochemistry showed positive reactivity for pan-TRK in all tumors, with nuclear staining for NTRK3 fusion, and cytoplasmic staining for NTRK1 fusion. The molecular testing revealed NTRK gene fusions (one each of TPM3-NTRK1, ETV6-NTRK3 and DCTN1-NTRK1, and two cases of LMNA-NTRK1). Two patients were receiving larotrectinib. The others were are well without disease, with follow-up durations of 9 to 29 months. Conclusions: NTRK-rearranged mesenchymal tumors from soft tissue sites and kidney are identified. A novel DCTN1-NTRK1 fusion is described. Pan-TRK immunohistochemistry is useful for diagnosis. NTRK-targeted therapy may be an option for unresectable, recurrent or metastatic cases.


Assuntos
Neoplasias de Tecido Conjuntivo e de Tecidos Moles , Adolescente , Criança , Pré-Escolar , China , Complexo Dinactina , Feminino , Rearranjo Gênico , Humanos , Imuno-Histoquímica , Lactente , Masculino , Receptor trkA
2.
N Z Med J ; 133(1513): 116-118, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32325477

RESUMO

Perry syndrome is a rare neurological condition characterised clinically by depression, sleep disturbance, central hypoventilation and parkinsonism. Perry syndrome is a TAR DNA-binding protein 43 (TDP-43) proteinopathy associated with mutated dynactin-1 protein, inherited in an autosomal dominant manner. Several pathogenic mutations in exon 2 in the dynactin 1 gene have been identified; p. F521, p. G67d, p. G71R, p. G71E, p. G71A, p. T72p, p. Q74p and p. Y78C. We present the second known case Perry syndrome with confirmed DCTN1 mutation (p. Y78C) in New Zealand, who initially was thought to have a depressive illness. Perry syndrome should be considered in the differential diagnosis of young parkinsonism, especially if there is family history of sleep disorders, weight loss and/or marked depression.


Assuntos
Complexo Dinactina/genética , Hipoventilação , Mutação/genética , Transtornos Parkinsonianos , Depressão/diagnóstico , Depressão/genética , Depressão/fisiopatologia , Feminino , Humanos , Hipoventilação/diagnóstico , Hipoventilação/genética , Hipoventilação/fisiopatologia , Pessoa de Meia-Idade , Transtornos Parkinsonianos/diagnóstico , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/fisiopatologia , Linhagem
3.
Nat Cell Biol ; 22(5): 570-578, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341547

RESUMO

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favours the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells.


Assuntos
Complexo Dinactina/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Linhagem Celular , Humanos , Cinesina/metabolismo , Microtúbulos/metabolismo , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Células Sf9 , Suínos
4.
Nat Cell Biol ; 22(5): 518-525, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341549

RESUMO

Cytoplasmic dynein-1 is a molecular motor that drives nearly all minus-end-directed microtubule-based transport in human cells, performing functions that range from retrograde axonal transport to mitotic spindle assembly1,2. Activated dynein complexes consist of one or two dynein dimers, the dynactin complex and an 'activating adaptor', and they show faster velocity when two dynein dimers are present3-6. Little is known about the assembly process of this massive ~4 MDa complex. Here, using purified recombinant human proteins, we uncover a role for the dynein-binding protein LIS1 in promoting the formation of activated dynein-dynactin complexes that contain two dynein dimers. Complexes activated by proteins representing three families of activating adaptors-BicD2, Hook3 and Ninl-all show enhanced motile properties in the presence of LIS1. Activated dynein complexes do not require sustained LIS1 binding for fast velocity. Using cryo-electron microscopy, we show that human LIS1 binds to dynein at two sites on the motor domain of dynein. Our research suggests that LIS1 binding at these sites functions in multiple stages of assembling the motile dynein-dynactin-activating adaptor complex.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Camundongos , Microtúbulos/metabolismo , Ligação Proteica/fisiologia , Proteínas Recombinantes/metabolismo
6.
Drug Discov Ther ; 14(1): 35-41, 2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32023558

RESUMO

Lysosomes are involved in many cellular functions, and in turn lysosomal dysfunction underlies a variety of diseases, including cancer and neurodegenerative diseases. Lysosomes are distributed broadly in the cytoplasm and can move throughout the cell in kinesin- and dynein-dependent manners. Although many mechanisms of lysosomal transport have been reported, how lysosomal transport is regulated has yet to be fully elucidated. In this study we analyzed c-Jun NH2-terminal kinase-associated leucine zipper protein (JLP), an adaptor of kinesin and dynein motor proteins, and found that lysosomes were localized toward the cell periphery in JLP knockdown cells, leading to the impairment of autophagosome-lysosome fusion. Furthermore, we performed rescue experiments using wild-type JLP and its various deletion mutants. The results indicated that JLP may regulate lysosome localization and autophagy through interaction of JLP with kinesin-1 heavy chain, but not with dynactin p150Glued or lysosomal transmembrane protein 55b. Our findings provide new insights into the mechanisms of lysosomal trafficking regulation. This study contributes to the understanding of how lysosomes exert their multiple functions, potentially leading to the identification of molecular targets for diseases caused by lysosomal dysfunction.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Lisossomos/metabolismo , Complexo Dinactina/metabolismo , Humanos , Cinesina/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo
7.
Nat Chem Biol ; 15(11): 1093-1101, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31501589

RESUMO

Cytoplasmic dynein is an ATP-driven motor that transports intracellular cargos along microtubules. Dynein adopts an inactive conformation when not attached to a cargo, and motility is activated when dynein assembles with dynactin and a cargo adaptor. It was unclear how active dynein-dynactin complexes step along microtubules and transport cargos under tension. Using single-molecule imaging, we showed that dynein-dynactin advances by taking 8 to 32-nm steps toward the microtubule minus end with frequent sideways and backward steps. Multiple dyneins collectively bear a large amount of tension because the backward stepping rate of dynein is insensitive to load. Recruitment of two dyneins to dynactin increases the force generation and the likelihood of winning against kinesin in a tug-of-war but does not directly affect velocity. Instead, velocity is determined by cargo adaptors and tail-tail interactions between two closely packed dyneins. Our results show that cargo adaptors modulate dynein motility and force generation for a wide range of cellular functions.


Assuntos
Complexo Dinactina/metabolismo , Animais , Complexo Dinactina/química , Dineínas/química , Dineínas/metabolismo , Humanos , Ligação Proteica
8.
Am J Physiol Gastrointest Liver Physiol ; 317(4): G373-G386, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31373507

RESUMO

Although steatosis (fatty liver) is a clinically well-described early stage of alcoholic liver disease, surprisingly little is known about how it promotes hepatotoxicity. We have shown that ethanol consumption leads to microtubule hyperacetylation that can explain ethanol-induced defects in protein trafficking. Because almost all steps of the lipid droplet life cycle are microtubule dependent and because microtubule acetylation promotes adipogenesis, we examined droplet dynamics in ethanol-treated cells. In WIF-B cells treated with ethanol and/or oleic acid (a fatty acid associated with the "Western" diet), we found that ethanol dramatically increased lipid droplet numbers and led to the formation of large, peripherally located droplets. Enhanced droplet formation required alcohol dehydrogenase-mediated ethanol metabolism, and peripheral droplet distributions required intact microtubules. We also determined that ethanol-induced microtubule acetylation led to impaired droplet degradation. Live-cell imaging revealed that droplet motility was microtubule dependent and that droplets were virtually stationary in ethanol-treated cells. To determine more directly whether microtubule hyperacetylation could explain impaired droplet motility, we overexpressed the tubulin-specific acetyltransferase αTAT1 to promote microtubule acetylation in the absence of alcohol. Droplet motility was impaired in αTAT1-expressing cells but to a lesser extent than in ethanol-treated cells. However, in both cases, the large immotile droplets (but not small motile ones) colocalized with dynein and dynactin (but not kinesin), implying that altered droplet-motor microtubule interactions may explain altered dynamics. These studies further suggest that modulating cellular acetylation is a potential strategy for treating alcoholic liver disease.NEW & NOTEWORTHY Chronic alcohol consumption with the "Western diet" enhances the development of fatty liver and leads to impaired droplet motility, which may have serious deletrious effects on hepatocyte function.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Acetilação , Acetiltransferases/metabolismo , Álcool Desidrogenase/metabolismo , Linhagem Celular , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Humanos , Proteínas dos Microtúbulos/metabolismo , Ácidos Oleicos/farmacologia
9.
Reprod Toxicol ; 88: 48-55, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31260804

RESUMO

The storage of surplus oocytes by cryopreservation (OC) is a widely used tool in assisted reproductive technology, but there is a great debate about the effects of cryopreservation on oocyte competence. It is known that OC may affect meiotic spindles but remains unclear if OC may increase the risk of aneuploidy. The aim of this study was to test the effects of OC and women aging on the expression of cytokinesis-related genes playing an important role in the segregation of chromosomes (DCTN1, DCTN2, DCTN3, DCTN6 and PLK1). Results highlighted that OC do not modify the expression of the selected genes, whereas women aging modulate the expression of all transcripts, confirming that aging is the crucial factor affecting meiosis and aneuploidy risk. A new role for Dynactin and PLK1 was shed in light, providing information on the ageing process in the oocyte which may be associated to reduced fertility.


Assuntos
Envelhecimento/metabolismo , Criopreservação , Complexo Dinactina/fisiologia , Regulação da Expressão Gênica , Oócitos/metabolismo , Vitrificação , Adulto , Fatores Etários , Aneuploidia , Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Humanos , Recuperação de Oócitos , Oócitos/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Técnicas de Reprodução Assistida
10.
Nat Commun ; 10(1): 2208, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101817

RESUMO

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.


Assuntos
Antígenos Nucleares/metabolismo , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/isolamento & purificação , Células CACO-2 , Cristalografia por Raios X , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
11.
Folia Neuropathol ; 57(1): 51-62, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038188

RESUMO

Traumatic brain injury (TBI) is characterized by various micro- and macrostructural neuropathological changes which can be identified in the light microscope examination. The most common pathophenotype of TBI visualized in postmortem neuropathological assessment includes neuron injury with involvement of all of its structural regions followed by its progressive degeneration defined as traumatic axonal injury (TAI). This is directly related with disruption of the axolemmal cytoskeletal network architecture resulting in breakdown, dissolution and accumulation of a number of neuronal proteins. Regarding the availability and progress in the development of specific antibodies against neuronal proteins, their usage is restricted due to low specificity for injured axons in the pathomechanism of TBI followed by TAI. Taking this into account with relation to expanding the role of axonal cytoskeleton and its based biomarkers we have presented a study documenting neuropathological features concerning the expression of dynein (DNAH9), dynactin (DCTN1) and kinesin (KIF5B) in the brain specimens obtained during forensic autopsies from TBI victims. The study was carried out using cases (n = 21) of severe head injury suspected to be the cause of death and control cases (n = 17) of sudden death in the mechanism of cardiopulmonary failure along with a positive control case which died after suicidal gunshot injury. In our study, we documented that DNAH9, DCTN1, and KIF5B staining should be considered as a supplemental diagnostic tool for TBI in postmortem neuropathological examination and forensic autopsy. This additional motor protein immunohistochemical staining procedure could be useful in the evaluation of lesions that may remain undiagnosed during a routine examination and aid in more accurate identification of TBI followed by TAI.


Assuntos
Autopsia/métodos , Dineínas do Axonema/análise , Lesões Encefálicas Traumáticas/diagnóstico , Complexo Dinactina/análise , Cinesina/análise , Biomarcadores/análise , Encéfalo/metabolismo , Encéfalo/patologia , Patologia Legal/métodos , Humanos
12.
J Cell Biol ; 218(5): 1429-1431, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31010854

RESUMO

Many different adaptor proteins activate the processivity of dynein-dynactin complexes and determine the specific cargo for retrograde transport by binding cargo receptors such as Rab GTP-binding (G) proteins. In this issue, Wang et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201806097) identify two GTPases that can function directly as dynein adaptors during endocytosis and are regulated by calcium.


Assuntos
Cálcio , Dineínas , Sinalização do Cálcio , Complexo Dinactina , Proteínas Associadas aos Microtúbulos
13.
Biochemistry ; 58(8): 1026-1031, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30702276

RESUMO

Cytoplasmic dynein is a retrograde microtubule-based motor transporting cellular cargo, including organelles, vesicular intermediates, RNA granules, and proteins, thus regulating their subcellular distribution and function. Mammalian dynein associates with dynactin, a multisubunit protein complex that is necessary for the processive motility of dynein along the microtubule tracks. Recent studies have shown that the interaction between dynein and dynactin is enhanced in the presence of a coiled-coil activating adaptor protein, which performs dual functions of recruiting dynein and dynactin to their cargoes and inducing the superprocessive motility of the motor complex. One such family of coiled-coil activating adaptor proteins is the Hook family of proteins that are conserved across evolution with three paralogs in the case of mammals, namely, HOOK1-HOOK3. This Perspective aims to provide an overview of the Hook protein structure and the cellular functions of Hook proteins, with an emphasis on the recent developments in understanding their role as activating dynein adaptors.


Assuntos
Movimento Celular , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Transporte Biológico , Humanos , Microtúbulos/metabolismo
14.
PLoS One ; 14(1): e0209820, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30640917

RESUMO

Microtubules (Mts) are dynamic cytoskeleton structures that play a key role in vesicular transport. The Mts-mediated transport depends on motor proteins named kinesins and the dynein/dynactin motor complex. The Rab7 adapter protein FYCO1 controls the anterograde transport of the endocytic compartments through the interaction with the kinesin KIF5. Rab7 and its partner RILP induce the recruitment of dynein/dynactin to late endosomes regulating its retrograde transport to the perinuclear area to fuse with lysosomes. The late endosomal-lysosomal fusion is regulated by the HOPS complex through its interaction with RILP and the GTPase Arl8. Coxiella burnetii (Cb), the causative agent of Q fever, is an obligate intracellular pathogen, which generates a large compartment with autophagolysosomal characteristics named Cb-containing vacuole (CCV). The CCV forms through homotypic fusion between small non-replicative CCVs (nrCCV) and through heterotypic fusion with other compartments, such as endosomes and lysosomes. In this work, we characterise the role of Mts, motor proteins, RILP/Rab7 and Arl8 on the CCV biogenesis. The formation of the CCV was affected when either the dynamics and/or the acetylation state of Mts were modified. Similarly, the overexpression of the dynactin subunit non-functional mutants p150Glued and RILP led to the formation of small nrCCVs. This phenomenon is not observed in cells overexpressing WT proteins, the motor KIF5 or its interacting protein FYCO1. The formation of the CCV was normal in infected cells that overexpressed Arl8 alone or together with hVps41 (a HOPS subunit) or in cells co-overexpressing hVps41 and RILP. The dominant negative mutant of Arl8 and the non-functional hVps41 inhibited the formation of the CCV. When the formation of CCV was affected, the bacterial multiplication diminished. Our results suggest that nrCCVs recruit the molecular machinery that regulate the Mts-dependent retrograde transport, Rab7/RILP and the dynein/dynactin system, as well as the tethering processes such as HOPS complex and Arl8 to finally originate the CCV where C. burnetii multiplies.


Assuntos
Coxiella burnetii/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Transporte Biológico , Chlorocebus aethiops , Coxiella burnetii/patogenicidade , Citoesqueleto/metabolismo , Complexo Dinactina/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Microtúbulos/fisiologia , Transporte Proteico/fisiologia , Febre Q/metabolismo , Vacúolos/metabolismo , Células Vero , Proteínas rab de Ligação ao GTP/metabolismo
15.
PLoS Biol ; 17(1): e3000100, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615611

RESUMO

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.


Assuntos
Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Dineínas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Sequência Conservada , Complexo Dinactina , Dineínas/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Fuso Acromático
16.
Neurosci Lett ; 690: 181-187, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30366015

RESUMO

Dynein-dynactin has an indispensable role in autophagy and p150glued is the largest component of the dynactin complex. Here, we characterized the effects of knockdown (KD) of endogenous p150glued and of the pathogenic mutation of p150glued found in autosomal dominant p150glued-associated disorders [hereditary motor neuronopathy with vocal paresis (HMN7B) and Perry syndrome] on autophagy. Overexpression of the p150glued pathogenic mutant or siRNA KD of p150glued promoted the localization of lysosomes at the cell periphery and increased the number of autophagosomes, suggesting partial blockage of autophagic flux. Surprisingly, although autophagosomes and lysosomes were redistributed predominantly to the cell periphery in p150glued-KD cells, the autolysosome formation ratio was preserved. However, under autophagy activation conditions induced by starvation, the ratio of autophagosome-lysosome fusion in p150glued-KD cells was decreased in the early phase. Our data demonstrate that functional loss of p150glued may cause autophagic insufficiency, which may be associated with the pathogenesis of p150glued-associated disorders.


Assuntos
Autofagossomos/metabolismo , Complexo Dinactina/metabolismo , Lisossomos/metabolismo , Linhagem Celular Tumoral , Complexo Dinactina/genética , Técnicas de Silenciamento de Genes , Humanos , Mutação/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/farmacologia , Regulação para Cima
17.
J Cell Physiol ; 234(7): 10445-10457, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30417365

RESUMO

Chloroquine (CQ) is an antimalaria drug that has been used in clinical practice for several decades. One serious complication of CQ treatment is the macular retinopathy caused by the disruption of the retinal pigmented epithelium, leading to vision loss. Little is known about how CQ affects retinal pigmented epithelium. In this study, we found that cell proliferation was reduced by CQ treatment in time and dose-dependent manners. No obvious cell death was detected; however, what was observed instead was G0/G1 arrest during which primary cilium started to grow in the presence of CQ. Pharmacological inhibition of primary cilium formation led to a reduction of cell viability suggesting that CQ-induced primary cilium protected cells from death. In addition to cell growth, with the CQ treatment the retina pigmented epithelium (RPE) cells less flattened with the spindle-like protrusion. When checking the microtubule networks, the microtubule nucleation activity was disrupted in the presence of CQ. The level of p150 glued , the largest subunit of dynactin, was reduced in CQ-treated RPE1 cells, and depletion of p150 glued resulted in a phenotype reminiscent of CQ-treated cells. Thus, CQ treatment reduced the expression of p150 glued , leading to reduced S phase entry and defective microtubule nucleation.


Assuntos
Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Proteínas Quinases/metabolismo , Retina/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Complexo Dinactina/metabolismo , Células Epiteliais/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Retina/metabolismo
18.
Elife ; 72018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30547880

RESUMO

Accurate chromosome segregation relies on bioriented amphitelic attachments of chromosomes to microtubules of the mitotic spindle, in which sister chromatids are connected to opposite spindle poles. BUB-1 is a protein of the Spindle Assembly Checkpoint (SAC) that coordinates chromosome attachment with anaphase onset. BUB-1 is also required for accurate sister chromatid segregation independently of its SAC function, but the underlying mechanism remains unclear. Here we show that, in Caenorhabditis elegans embryos, BUB-1 accelerates the establishment of non-merotelic end-on kinetochore-microtubule attachments by recruiting the RZZ complex and its downstream partner dynein-dynactin at the kinetochore. In parallel, BUB-1 limits attachment maturation by the SKA complex. This activity opposes kinetochore-microtubule attachment stabilisation promoted by CLS-2CLASP-dependent kinetochore-microtubule assembly. BUB-1 is therefore a SAC component that coordinates the function of multiple downstream kinetochore-associated proteins to ensure accurate chromosome segregation.


Assuntos
Anáfase , Proteínas de Caenorhabditis elegans/genética , Segregação de Cromossomos , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Dineínas/genética , Dineínas/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica , Cinetocoros/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fuso Acromático/ultraestrutura
19.
Curr Biol ; 28(22): R1308-R1310, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30458151

RESUMO

In metazoans, positioning of the mitotic spindle is controlled by the microtubule-dependent motor protein dynein, which associates with the cell cortex. Using optogenetic tools, two new studies examine how the levels and activity of dynein are regulated at the cortex to ensure proper positioning of the mitotic spindle.


Assuntos
Dineínas , Fuso Acromático , Complexo Dinactina , Cinesina , Microtúbulos
20.
Neuropathology ; 38(6): 583-590, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30215870

RESUMO

Dynactin forms a protein complex with dynein that retrogradely transports cargo along microtubules. Dysfunction of this dynein-dynactin complex causes several neurodegenerative diseases such as Perry syndrome, motor neuron diseases and progressive supranuclear palsy. Recently, we reported colocalization of phosphorylated α-synuclein (p-SNCA) and the largest subunit of dynactin (DCTN1) in Lewy body (LB)-like structures in Perry syndrome. Previous reports have not focused on the relationship between dynactin and synucleinopathies. Thus, we examined autopsied human brains from patients with Parkinson's disease, dementia with LBs, and multiple system atrophy using immunohistochemistry for p-SNCA, DCTN1, dynactin 2 (DCTN2, dynamitin) and dynein cytoplasmic 1 intermediate chain 1 (DYNC1I1). We also examined microtubule affinity-regulating kinases (MARKs), which phosphorylate microtubule-associated proteins and trigger microtubule disruption. Both brainstem-type and cortical LBs were immunopositive for DCTN1, DCTN2, DYNC1I1 and p-MARK and their staining often overlapped with p-SNCA. Lewy neurites were also immunopositive for DCTN1, DCTN2 and DYNC1I1. However, p-SNCA-positive inclusions of multiple system atrophy, which included both glial and neuronal cytoplasmic inclusions, were immunonegative for DCTN1, DCTN2, DYNC1I1 and p-MARK. Thus, immunohistochemistry for dynein-dynactin complex molecules, especially DCTN1, can clearly distinguish LBs from neuronal cytoplasmic inclusions. Our results suggest that dynactin is closely associated with LB pathology.


Assuntos
Encéfalo/patologia , Complexo Dinactina/metabolismo , Corpos de Lewy/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Doença por Corpos de Lewy/patologia , Masculino , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/patologia , Doença de Parkinson/patologia
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