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2.
Nat Commun ; 12(1): 4578, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321481

RESUMO

Mitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end. However, dynein-mediated TRAK2 transport is minimal unless the dynein-binding protein LIS1 is present at a sufficient level. Using co-immunoprecipitation and co-localization experiments, we demonstrate that TRAK2 forms a complex containing both kinesin-1 and dynein-dynactin. These motors are functionally linked by TRAK2 as knockdown of either kinesin-1 or dynein-dynactin reduces the initiation of TRAK2 transport toward either microtubule end. We propose that TRAK2 coordinates kinesin-1 and dynein-dynactin as an interdependent motor complex, providing integrated control of opposing motors for the proper transport of mitochondria.


Assuntos
Dineínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesina/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Proteínas de Transporte/metabolismo , Complexo Dinactina/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Transporte Proteico/fisiologia , Transcriptoma
3.
Zhongguo Dang Dai Er Ke Za Zhi ; 23(6): 639-644, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34130788

RESUMO

OBJECTIVE: To study the effect of dexamethasone (DEX) on the expression of Dynein heavy chain (DHC) and Dynactin in the cytoplasm of fetal rat cerebral cortical neurons cultured in vitro. METHODS: Primary cerebral cortical neurons of fetal rats were cultured in vitro and were used to establish a cellular model of DEX intervention. According to the final concentration of DEX, the neurons were divided into three groups:control (without DEX), 0.1 µmol/L DEX, and 1.0 µmol/L DEX. On days 1, 3, and 7 after intervention, the quantitative PCR was used to observe the effect of DEX on the mRNA expression of DHC and Dynactin. The Western blot was used to observe the effect of DEX on the protein expression of DHC and Dynactin. RESULTS: There was no significant difference in the mRNA expression levels of DHC and Dynactin among the three groups at all time points (P > 0.05). On day 7 after DEX intervention, the protein expression of DHC in the 1.0 µmol/L DEX group gradually increased and reached the peak over time, which was significantly higher than that in the control and 0.1 µmol/L DEX groups (P < 0.05). The control and 0.1 µmol/L DEX groups had a significant increase in the protein expression of Dynactin from day 1 to days 3 and 7 after DEX intervention (P < 0.05). The control group had a significant increase in the protein expression of Dynactin from day 3 to day 7 after intervention (P < 0.05), while the 0.1 µmol/L DEX group had a significant reduction in the protein expression of Dynactin from day 3 to day 7 after intervention (P < 0.05). On days 3 and 7 after DEX intervention, the 0.1 µmol/L DEX and 1.0 µmol/L DEX groups had a significantly lower protein expression level of Dynactin in the cerebral cortical neurons than the control group (P < 0.05). On day 7 after DEX intervention, the 1.0 µmol/L DEX group had a significantly lower protein expression level of Dynactin than the 0.1 µmol/L DEX group (P < 0.05). CONCLUSIONS: DEX affects the protein expression of DHC and Dynactin in the fetal rat cerebral cortical neurons cultured in vitro, possibly in a concentration- and time-dependent manner.


Assuntos
Dineínas , Neurônios , Animais , Citoplasma , Dexametasona/farmacologia , Complexo Dinactina/genética , Ratos
4.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34014261

RESUMO

Autophagy is a degradative pathway required to maintain homeostasis. Neuronal autophagosomes form constitutively at the axon terminal and mature via lysosomal fusion during dynein-mediated transport to the soma. How the dynein-autophagosome interaction is regulated is unknown. Here, we identify multiple dynein effectors on autophagosomes as they transit along the axons of primary neurons. In the distal axon, JIP1 initiates autophagosomal transport. Autophagosomes in the mid-axon require HAP1 and Huntingtin. We find that HAP1 is a dynein activator, binding the dynein-dynactin complex via canonical and noncanonical interactions. JIP3 is on most axonal autophagosomes, but specifically regulates the transport of mature autolysosomes. Inhibiting autophagosomal transport disrupts maturation, and inhibiting autophagosomal maturation perturbs the association and function of dynein effectors; thus, maturation and transport are tightly linked. These results reveal a novel maturation-based dynein effector handoff on neuronal autophagosomes that is key to motility, cargo degradation, and the maintenance of axonal health.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagossomos/genética , Axônios/metabolismo , Proteína Huntingtina/genética , Proteínas do Tecido Nervoso/genética , Autofagia/genética , Transporte Axonal/genética , Complexo Dinactina/genética , Dineínas/genética , Homeostase , Humanos , Lisossomos/genética , Proteínas Associadas aos Microtúbulos/genética , Neurônios/metabolismo , Neurônios/patologia , Fagossomos/genética
5.
Childs Nerv Syst ; 37(7): 2147-2151, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34014367

RESUMO

PURPOSE: Inflammatory myofibroblastic tumor (IMT) is a rare neoplastic tumor type of intermediate biological potential, only recently distinguished from the non-neoplastic category of inflammatory pseudotumor (IP). The literature describes very few cases of IMTs arising in the central nervous system (CNS), and the distinguishing clinical, pathological, and molecular features of IMT-CNS are not well understood. Our purpose is to publish a case of an IMT-CNS with a novel DCTN1-ALK gene fusion, furthering in the literature's characterization of a rare tumor type. METHODS: Review of the literature included a PubMed Database search of articles found by the following searches: "Inflammatory myofibroblastic tumor;" "Inflammatory myofibroblastic tumor central nervous system;" "ALK gene fusion;" and "DCTN1-ALK gene fusion." Inclusion of articles discovered by these search terms was determined through critical appraisal of article relevance, number of citations, cross-citation within articles of interest, and rare findings with conflicting conclusions in an effort to reduce publication bias. RESULTS: We present a case of IMT-CNS with several distinctive molecular features including a DCTN1-ALK gene fusion, the first of its kind described in an intracranial IMT. CONCLUSION: IMT is an infrequent tumor type and its presentation within the CNS is exceedingly rare. The paucity of cases, along with the ambiguity of terminology in the literature, has stunted accurate clinical, pathological, and molecular characterization of IMT-CNS. Our case report improves the characterization of the recently appreciated category of IMT-CNS so that connections between phenotype and prognosis, and between genotype and treatment, can eventually be made.


Assuntos
Granuloma de Células Plasmáticas , Quinase do Linfoma Anaplásico , Sistema Nervoso Central , Complexo Dinactina , Fusão Gênica , Granuloma de Células Plasmáticas/diagnóstico por imagem , Granuloma de Células Plasmáticas/genética , Granuloma de Células Plasmáticas/cirurgia , Humanos , Receptores Proteína Tirosina Quinases/genética
6.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34014309

RESUMO

In animal cells, a single cytoplasmic dynein motor mediates microtubule minus-end-directed transport, counterbalancing dozens of plus-end-directed kinesins. The remarkable ability of dynein to interact with a diverse cargo spectrum stems from its tightly regulated recruitment of cargo-specific adaptor proteins, which engage the dynactin complex to make a tripartite processive motor. Adaptor binding is governed by the homologous dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which exist in mutually exclusive dynein complexes that can perform both unique and overlapping functions. The intrinsically disordered and variable C-terminal domains of the LICs are indispensable for engaging a variety of structurally divergent adaptors. Here, we hypothesize that numerous spatiotemporally regulated permutations of posttranslational modifications of the LICs, as well as of the adaptors and cargoes, exponentially expand the spectrum of dynein-adaptor-cargo complexes. We thematically illustrate the possibilities that could generate a vast set of biochemical variations required to support the wide range of dynein functions.


Assuntos
Dineínas do Citoplasma , Dineínas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Dineínas/genética , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo
7.
Technol Cancer Res Treat ; 20: 15330338211010143, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33896271

RESUMO

OBJECTIVE: This present study aims to investigate the potential prognostic values of dynactin genes (DCTN) for predicting the overall survival (OS) in low-grade glioma (LGG) patients. METHODS: The DCTN mRNA expression data were downloaded from The Cancer Genome Atlas database containing 518 patients with LGG. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses for DCTN genes were performed by using Database for Annotation, Visualization, and Integrated Discovery platform, and their enrichment results were verified by using the Biological Networks Gene Ontology tool. Next, the correlations between DCTN genes and LGG were identified by Pearson correlation coefficient analysis. The OS was estimated by Kaplan-Meier survival analysis. The cBio Cancer Genomics Portal was used to analyze the mutations of DCTN genes and their effects on the prognosis of LGG. The correlation between the abundance of immune infiltration and tumor purity of DCTN genes were predicted by The Tumor Immune Estimation Resource. RESULTS: Our research showed that the mRNA expression of DCTN4 in tumor tissues was much higher (P < 0.01) than that in normal tissues. Meanwhile, there was a certain correlation between the DCTN genes. Survival analysis showed that the high expression of DCTN1, DCTN3, DCTN4, DCTN6, and their co-expression were significantly correlated with favorable OS in LGG patients (P < 0.05). In DCTN2, a high mutation rate was observed. Further research showed that the genetic alteration in DCTN genes was related to a poor OS and progression-free survival of LGG patients. The expression of DCTN genes had a certain correlation with immune infiltrating cells. CONCLUSION: Our study showed that the high expressions of DCTN1, DCTN3, DCTN4, and DCTN6 were associated with a favorable OS of LGG patients, indicating that these DCTN genes are potential biomarkers for evaluating the prognosis of LGG patients.


Assuntos
Neoplasias Encefálicas/genética , Complexo Dinactina/genética , Glioma/genética , Biomarcadores Tumorais/genética , Encéfalo/metabolismo , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Bases de Dados Genéticas , Células Dendríticas/imunologia , Ontologia Genética , Glioma/imunologia , Glioma/patologia , Humanos , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral , Macrófagos/imunologia , Taxa de Mutação , Gradação de Tumores , Neutrófilos/imunologia , Intervalo Livre de Progressão , RNA Mensageiro/metabolismo , Taxa de Sobrevida , Regulação para Cima
8.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924373

RESUMO

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexo Dinactina/metabolismo , Agregados Proteicos , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Complexo Dinactina/química , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Sinais de Localização Nuclear/metabolismo , Mutação Puntual/genética , Ligação Proteica , Frações Subcelulares/metabolismo
9.
EMBO J ; 40(8): e106164, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33734450

RESUMO

Dynactin is a 1.1 MDa complex that activates the molecular motor dynein for ultra-processive transport along microtubules. In order to do this, it forms a tripartite complex with dynein and a coiled-coil adaptor. Dynactin consists of an actin-related filament whose length is defined by its flexible shoulder domain. Despite previous cryo-EM structures, the molecular architecture of the shoulder and pointed end of the filament is still poorly understood due to the lack of high-resolution information in these regions. Here we combine multiple cryo-EM datasets and define precise masking strategies for particle signal subtraction and 3D classification. This overcomes domain flexibility and results in high-resolution maps into which we can build the shoulder and pointed end. The unique architecture of the shoulder securely houses the p150 subunit and positions the four identical p50 subunits in different conformations to bind dynactin's filament. The pointed end map allows us to build the first structure of p62 and reveals the molecular basis for cargo adaptor binding to different sites at the pointed end.


Assuntos
Complexo Dinactina/química , Microscopia Crioeletrônica , Complexo Dinactina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
10.
BMJ Case Rep ; 14(2)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33541941

RESUMO

A 72-year-old man initially presented to the ENT outpatient department after 20 years with increasing intermittent episodes of dyspnoea and stridor. Flexible nasendoscopy revealed bilateral vocal cord paralysis with the cords in a medial position. He subsequently underwent urgent tracheostomy. He has six similarly affected family members across three generations all requiring tracheostomy to maintain an adequate airway. Follow-up and genetic testing have revealed mutation of the dynactin 1 gene leading to distal hereditary motor neuropathy type 7b. This is a rare occurrence causing this condition to be reported in only three families previously throughout the world.


Assuntos
Obstrução das Vias Respiratórias/cirurgia , Complexo Dinactina/genética , Traqueostomia , Paralisia das Pregas Vocais/diagnóstico , Paralisia das Pregas Vocais/genética , Idoso , Obstrução das Vias Respiratórias/fisiopatologia , Dispneia/etiologia , Humanos , Masculino , Sons Respiratórios/etiologia
11.
Development ; 148(3)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33462114

RESUMO

The microtubule motor cytoplasmic dynein 1 (dynein) and its essential activator dynactin have conserved roles in spindle assembly and positioning during female meiosis and mitosis, but their contribution to male meiosis remains poorly understood. Here, we characterize the G33S mutation in the C. elegans dynactin subunit DNC-1, which corresponds to G59S in human p150Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents the segregation of homologous chromosomes. By contrast, chromosomes segregate without errors in the early dnc-1(G33S) embryo. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to the inability of DNC-1 to interact with microtubules. Instead, our results suggest that the DNC-1(G33S) protein, which is aggregation prone in vitro, is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.


Assuntos
Segregação de Cromossomos , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Espermatócitos/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Cromossomos , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/genética , Feminino , Humanos , Masculino , Meiose , Mitose , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Mutação , Fuso Acromático/metabolismo
12.
Neurol Sci ; 42(9): 3695-3705, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33443672

RESUMO

The Dynactin 1 (DCTN1) encodes the p150 subunit of dynactin, which engages retrograde axonal transport. Missense mutations in DCTN1 have been linked to a series of neurodegenerative diseases, including distal hereditary motor neuropathies (dHMN) and Perry syndrome. A few pathogenic DCTN1 mutations related with Perry syndrome have been described within, or adjacent to, the highly conserved N-terminal cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain. But to our best knowledge, only the pathogenic G59S mutation in DCTN1 has been reported in dHMN7B families. Herein, we provided a novel heterozygous mutation in DCTN1 which caused both dHMN7B and Perry syndrome from a Chinese family. Whole exome sequencing (WES) was performed to identify the disease-associated genes. Single nucleotide variants (SNVs) and small insertions/deletions (INDELs) were further predicted with Mutation Taster, Polymorphism Phenotyping v2 (PolyPhen-2), and Sorting Intolerant From Tolerant (SIFT) and compared to the Single Nucleotide Polymorphism Database(dbSNP), Exome Aggregation Consortium (ExAC), and the 1000 Genomes Project. Furthermore, a novel missense mutation c.279G>C (Q93H) in DCTN1 was identified as the candidate loci. The mutation was confirmed with Sanger sequencing in the family members and cosegregated with various phenotypes. In silico analysis and molecular structural modeling, the mutation not only caused the loss of a hydrogen bond within the p150 protein but also affected the formation of hydrogen bonds between p150 and EB. Therefore, the new Q93H mutation in DCTN1 caused both familial dHMN7B and Perry syndrome. Our findings could expand the clinical and pathogenic spectrum and strengthen the clinical diagnostic role of the DCTN1 gene.


Assuntos
Mutação de Sentido Incorreto , Transtornos Parkinsonianos , China , Depressão , Complexo Dinactina/genética , Humanos , Hipoventilação , Mutação , Mutação de Sentido Incorreto/genética
13.
J Cell Biol ; 220(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416861

RESUMO

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase-based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein-dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.


Assuntos
Complexo Dinactina/metabolismo , Dineínas/metabolismo , Septinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Endossomos/metabolismo , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Ratos , Septinas/química , Proteínas rab de Ligação ao GTP/metabolismo
14.
Mol Brain ; 14(1): 14, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33461576

RESUMO

Mitochondrial movement in neurons is finely regulated to meet the local demand for energy and calcium buffering. Elaborate transport machinery including motor complexes is required to deliver and localize mitochondria to appropriate positions. Defects in mitochondrial transport are associated with various neurological disorders without a detailed mechanistic information. In this study, we present evidence that dystrobrevin-binding protein 1 (dysbindin), a schizophrenia-associated factor, plays a critical role in axonal mitochondrial movement. We observed that mitochondrial movement was impaired in dysbindin knockout mouse neurons. Reduced mitochondrial motility caused by dysbindin deficiency decreased the density of mitochondria in the distal part of axons. Moreover, the transport and distribution of mitochondria were regulated by the association between dysbindin and p150glued. Furthermore, altered mitochondrial distribution in axons led to disrupted calcium dynamics, showing abnormal calcium influx in presynaptic terminals. These data collectively suggest that dysbindin forms a functional complex with p150glued that regulates axonal mitochondrial transport, thereby affecting presynaptic calcium homeostasis.


Assuntos
Axônios/metabolismo , Complexo Dinactina/metabolismo , Disbindina/metabolismo , Mitocôndrias/metabolismo , Esquizofrenia/metabolismo , Animais , Cálcio/metabolismo , Células HEK293 , Homeostase , Humanos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Modelos Biológicos , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica
15.
J Oncol Pharm Pract ; 27(2): 485-489, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32693686

RESUMO

INTRODUCTION: Neurotrophic receptor tyrosine kinase fusions cause overexpression or activation of kinase and are believed to confer oncogenic potential in some non-rhabdomyosarcoma soft tissue sarcomas. TRK inhibitors have recently been shown to induce responses in these tumours though current experience with these agents is still limited. CASE REPORT: We report a case of an adolescent with treatment-refractory non-rhabdomyosarcoma soft tissue sarcomas, carrying a novel DCTN1-NTRK1 gene fusion whose progressive disease was treated with multi-kinase and TRK inhibitors.Management and outcome: Our patient was started on pan-TRK inhibitor larotrectinib, as his disease progressed after chemotherapy, radiation therapy and surgery, based on next-generation sequencing test showing DCTN1-NTRK1 gene fusion. He responded quickly to larotrectinib with the improvement of symptoms and reduction of masses. However, this response was short-lived due to the development of acquired solvent front resistance mutation. This patient did not respond to next-generation TRK inhibitor selitrectinib and eventually succumbed to his disease. DISCUSSION: The initial rapid and drastic response of our patient to larotrectinib was not sustained due to the development of acquired resistance. This case emphasizes the need for upfront and periodic next-generation sequencing testing to guide treatment of patients with refractory non-rhabdomyosarcoma soft tissue sarcomas.


Assuntos
Antineoplásicos/uso terapêutico , Compostos Aza/uso terapêutico , Complexo Dinactina/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Receptor trkA/genética , Sarcoma/tratamento farmacológico , Neoplasias de Tecidos Moles/tratamento farmacológico , Adolescente , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos/genética , Evolução Fatal , Humanos , Imageamento por Ressonância Magnética , Masculino , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Resultado do Tratamento
16.
J Cell Physiol ; 236(4): 2706-2724, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32869310

RESUMO

Septins play important roles in regulating development and differentiation. Septin 7 (SEPT7) is a crucial component in orchestrating the septin core complex into highly ordered filamentous structures. Here, we showed that genetic depletion of SEPT7 or treatment with forchlorfenuron (FCF; a compound known to affect septin filament assembly) led to reduced the S phase entry in cell models and zebrafish embryos. In addition to colocalizing with actin filaments, SEPT7 resided in the centrosome, and SEPT7 depletion led to aberrant mitotic spindle pole formation. This mitotic defect was rescued in SEPT7-deficient cells by wild-type SEPT7, suggesting that SEPT7 maintained mitotic spindle poles. In addition, we observed disorganized microtubule nucleation and reduced cell migration with SEPT7 depletion. Furthermore, SEPT7 formed a complex with and maintained the abundance of p150glued , the component of centriole subdistal appendages. Depletion of p150glued resulted in a phenotype reminiscent of SEPT7-deficient cells, and overexpression of p150glued reversed the defective phenotypes. Thus, SEPT7 is a centrosomal protein that maintains proper cell proliferation and microtubule array formation via maintaining the abundance of p150glued .


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Complexo Dinactina/metabolismo , Microtúbulos/metabolismo , Fase S , Septinas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Centrossomo/efeitos dos fármacos , Complexo Dinactina/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Fase S/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular , Septinas/genética , Transdução de Sinais , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
18.
Nat Commun ; 11(1): 5695, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33173051

RESUMO

Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called "activating adaptors" that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 µM. We propose that a tunable LIC1-adaptor interaction modulates dynein's motility in a cargo-specific manner.


Assuntos
Transporte Biológico/fisiologia , Dineínas do Citoplasma/metabolismo , Proteínas Motores Moleculares/metabolismo , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , Cristalografia por Raios X/métodos , Complexo Dinactina/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica
19.
EMBO J ; 39(24): e103661, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33215754

RESUMO

Although subcellular positioning of endosomes significantly impacts on their functions, the molecular mechanisms governing the different steady-state distribution of early endosomes (EEs) and late endosomes (LEs)/lysosomes (LYs) in peripheral and perinuclear eukaryotic cell areas, respectively, are still unsolved. We unveil that such differences arise because, while LE retrograde transport depends on the dynein microtubule (MT) motor only, the one of EEs requires the cooperative antagonism of dynein and kinesin-14 KIFC1, a MT minus end-directed motor involved in cancer progression. Mechanistically, the Ser-x-Ile-Pro (SxIP) motif-mediated interaction of the endoplasmic reticulum transmembrane protein stromal interaction molecule 1 (STIM1) with the MT plus end-binding protein 1 (EB1) promotes its association with the p150Glued subunit of the dynein activator complex dynactin and the distinct location of EEs and LEs/LYs. The peripheral distribution of EEs requires their p150Glued-mediated simultaneous engagement with dynein and SxIP motif-containing KIFC1, via HOOK1 and HOOK3 adaptors, respectively. In sum, we provide evidence that distinct minus end-directed MT motor systems drive the differential transport and subcellular distribution of EEs and LEs in mammalian cells.


Assuntos
Transporte Biológico/fisiologia , Endossomos/metabolismo , Microtúbulos/metabolismo , Adesão Celular , Linhagem Celular , Citoesqueleto , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Retículo Endoplasmático/metabolismo , Inativação Gênica , Humanos , Cinesina/genética , Cinesina/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
20.
Biochimie ; 177: 127-131, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32841682

RESUMO

A-kinase anchoring protein 350 (AKAP350) is a centrosomal/Golgi scaffold protein, critical for the regulation of microtubule dynamics. AKAP350 recruits end-binding protein 1 (EB1) to the centrosome in mitotic cells, ensuring proper spindle orientation in epithelial cells. AKAP350 also interacts with p150glued, the main component of the dynactin complex. In the present work, we found that AKAP350 localized p150glued to the spindle poles, facilitating p150glued/EB1 interaction at these structures. Our results further showed that the decrease in AKAP350 expression reduced p150glued localization at astral microtubules and impaired the elongation of astral microtubules during anaphase. Overall, this study provides mechanistic data on how microtubule regulatory proteins gather to define microtubule dynamics in mitotic cells.


Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Complexo Dinactina/fisiologia , Polos do Fuso/metabolismo , Animais , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cães , Células Madin Darby de Rim Canino , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Polos do Fuso/ultraestrutura
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