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/genéticaRESUMO
Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Dineínas do Citoplasma/genética , Dineínas/metabolismo , Vírus da Leucemia Murina/crescimento & desenvolvimento , Replicação Viral/genética , Células 3T3 , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular , Núcleo Celular/virologia , Dineínas/genética , Produtos do Gene gag/genética , Células HEK293 , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Camundongos , Microtúbulos/metabolismoRESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG nucleotide expansion, which encodes the amino acid glutamine, in the huntingtin gene. HD is characterized by motor, cognitive, and psychiatric dysfunctions. In a previous study, we showed by qPCR that some genes altered in an HD mouse model were also altered in blood of HD patients. These alterations were mainly with respect to the dynein family. Therefore, this study aimed to investigate whether dynein light chain Tctex type 1 (DYNLT1) is altered in HD patients and if there is a correlation between DYNLT1 gene expression changes and disease progression. We assessed the DYNLT1 gene expression in the blood of 19 HD patients and 20 healthy age-matched controls. Also, in 6 of these patients, we analyzed the DYNLT1 expression at two time points, 3 years apart. The DYNLT1 gene expression in the whole blood of HD patients was significantly downregulated and this difference was widened in later stages. These data suggest that DYNLT1 could emerge as a peripheral prognostic indicator in HD and, also, might be a target for potential intervention in the future.
Assuntos
Dineínas/genética , Doença de Huntington , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Dineínas/sangue , Expressão Gênica , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , CamundongosRESUMO
Acute lymphoblastic leukemia is the most common type of childhood cancer worldwide. Mexico City has one of the highest incidences and mortality rates of this cancer. It has previously been recognized that chromosomal translocations are important in cancer etiology. Specific fusion genes have been considered as important treatment targets in childhood acute lymphoblastic leukemia (ALL). The present research aimed at the identification and characterization of novel fusion genes with potential clinical implications in Mexican children with acute lymphoblastic leukemia. The RNA-sequencing approach was used. Four fusion genes not previously reported were identified: CREBBP-SRGAP2B, DNAH14-IKZF1, ETV6-SNUPN, ETV6-NUFIP1. Although a fusion gene is not sufficient to cause leukemia, it could be involved in the pathogenesis of the disease. Notably, these new translocations were found in genes encoding for hematopoietic transcription factors which are known to play an important role in leukemogenesis and disease prognosis such as IKZF1, CREBBP, and ETV6. In addition, they may have an impact on the prognosis of Mexican pediatric patients with ALL, with the potential to be included in the current risk stratification schemes or used as therapeutic targets.
Assuntos
Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Adolescente , Adulto , Proteína de Ligação a CREB/genética , Criança , Pré-Escolar , Dineínas/genética , Feminino , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Humanos , Fator de Transcrição Ikaros/genética , Lactente , Masculino , México , Proteínas Nucleares/genética , Prognóstico , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Adulto Jovem , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Molecular motors play relevant roles on the regulation of mitochondria size and shape, essential properties for the cell homeostasis. In this work, we tracked single rod-shaped mitochondria with nanometer precision to explore the performance of microtubule motor teams during processive anterograde and retrograde transport. We analyzed simultaneously the organelle size and verified that mitochondria retracted during retrograde transport with their leading tip moving slower in comparison with the rear tip. In contrast, mitochondria preserved their size during anterograde runs indicating a different performance of plus-end directed teams. These results were interpreted considering the different performance of dynein and kinesin teams and provide valuable information on the collective action of motors during mitochondria transport.
Assuntos
Homeostase/genética , Microtúbulos/genética , Mitocôndrias/genética , Forma das Organelas/genética , Animais , Dineínas/genética , Cinesinas/genética , Microtúbulos/metabolismo , Análise de Célula Única , Xenopus laevis/genéticaRESUMO
The dynamic formation of stress granules (SGs), processing bodies (PBs), and related RNA organelles regulates diverse cellular processes, including the coordination of functionally connected messengers, the translational regulation at the synapse, and the control of viruses and retrotransposons. Recent studies have shown that pyruvate kinase and other enzymes localize in SGs and PBs, where they become protected from stress insults. These observations may have implications for enzyme regulation and metabolic control exerted by RNA-based organelles. The formation of these cellular bodies is governed by liquid-liquid phase separation (LLPS) processes, and it needs to be strictly controlled to prevent pathogenic aggregation. The intracellular concentration of key metabolites, such as ATP and sterol derivatives, may influence protein solubility, thus affecting the dynamics of liquid organelles. LLPS in vitro depends on the thermal diffusion of macromolecules, which is limited inside cells, where the condensation and dissolution of membrane-less organelles are helped by energy-driven processes. The active transport by the retrograde motor dynein helps SG assembly, whereas the anterograde motor kinesin mediates SG dissolution; a tug of war between these two molecular motors allows transient SG formation. There is evidence that the efficiency of dynein-mediated transport increases with the number of motor molecules associated with the cargo. The dynein-dependent transport may be influenced by cargo size as larger cargos can load a larger number of motors. We propose a model based on this emergent property of dynein motors, which would be collectively stronger during SG condensation and weaker during SG breakdown, thus allowing kinesin-mediated dispersion.
Assuntos
Dineínas/genética , Cinesinas/genética , Organelas/genética , RNA/genética , Trifosfato de Adenosina/química , Transporte Biológico/genética , Citoplasma/química , Citoplasma/genética , Dineínas/química , Humanos , Cinesinas/química , Membranas/química , Microtúbulos/química , Organelas/química , Piruvato Quinase/química , RNA/química , SolubilidadeRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor dysfunction, cognitive deficits, and psychiatric symptoms. The primary genetic cause is an expansion of cytosine adenine guanine (CAG) nucleotides of the huntingtin gene, which codes an important protein involved with neuronal signaling. The severity of HD correlates with the number of CAG repeats and individuals with longer expansions have an earlier onset and more severe symptoms. A microarray study conducted by our research group showed alteration in DNAH6 gene (encoding dynein axonemal heavy chain 6). DNAH6 belongs to dynein family, whose members are constituents of the microtubule-associated motor proteins and is downregulated in the striatum of a HD mouse model (knockin HdhQ111/Q111). In this manner, our goal was to confirm these downregulations in the mouse model and verify if the same alteration in the axonemal DNAH6 gene expression is observed in blood samples of HD patients. Blood samples were collected from 17 patients with clinical diagnosis of HD and 12 healthy individuals and RNA extracted for qPCR analysis. Microarray data were confirmed by qPCR in knockin HdhQ111/Q111, and DNAH6 was severely decreased in those mice, as compared to control mice (HdhQ20/Q20). Notably, decreased expression of DNAH6 gene was also observed in HD patients when compared to control group and negatively correlates with the CAG expansion. Although further studies are necessary to underlie the molecular mechanisms of dynein-htt interaction, this data highlights DNAH6 as a potential new blood marker for HD.
Assuntos
Dineínas/sangue , Doença de Huntington/sangue , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Regulação para Baixo , Dineínas/genética , Dineínas/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Pesquisa Translacional BiomédicaRESUMO
How dynein motors accurately move cargoes is an important question. In budding yeast, dynein moves the mitotic spindle to the predetermined site of cytokinesis by pulling on astral microtubules. In this study, using high-resolution imaging in living cells, we discover that spindle movement is regulated by changes in microtubule plus-end dynamics that occur when dynein generates force. Mutants that increase plus-end stability increase the frequency and duration of spindle movements, causing positioning errors. We find that dynein plays a primary role in regulating microtubule dynamics by destabilizing microtubules. In contrast, the dynactin complex counteracts dynein and stabilizes microtubules through a mechanism involving the shoulder subcomplex and the cytoskeletal-associated protein glycine-rich domain of Nip100/p150glued Our results support a model in which dynein destabilizes its microtubule substrate by using its motility to deplete dynactin from the plus end. We propose that interplay among dynein, dynactin, and the stability of the microtubule substrate creates a mechanism that regulates accurate spindle positioning.
Assuntos
Ciclo Celular , Dineínas/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Dineínas/genética , Mutação , Estabilidade Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
La disquinesia ciliar primaria (DCP) corresponde a una enfermedad genética heterogénea, que se produce por una alteración estructural o funcional de los cilios. Es de difícil diagnóstico tanto por su variada sintomatología como por la existencia de métodos de screening y diagnóstico complejos. El método que hasta ahora ha sido considerado como gold standard es el análisis de la estructura ciliar por medio de la microscopía electrónica de transmisión (MET). Esta técnica tiene limitaciones porque permite analizar un número limitado de axonemas ciliares y puede excluir del diagnóstico a pacientes con axonema normal pero con alteración funcional y clínica clásicas. En los últimos años se han desarrollado métodos diagnósticos sobre la base de un mejor conocimiento de la estructura proteica de los cilios, de los genes que codifican estas proteínas y de las mutaciones asociadas a DCP. Estos nuevos métodos consisten en un análisis genético y un estudio de la expresión de proteínas ciliares en los pacientes afectados. Esta publicación tiene como objetivo realizar una revisión de la fisiopatología de la DCP, los métodos diagnósticos actuales y resumir el desarrollo del diagnóstico genético en la literatura internacional y su posible aplicación en nuestro medio.
Primary cilliary dyskinesia (PCD) is an heterogeneous genetic disease caused by a structural and/or functional alteration of the ciliary skeleton. It is a diagnostic challenge due to its protean clinical presentation and to the complexity of screening and diagnostic methods. The method hitherto regarded as the gold standard is the analysis of ciliary structure by transmission electron microscopy (TEM). This presents limitations because analyzes a limited number of ciliary axonemes, and may exclude cases with typical functional and clinical presentation. In recent years new diagnostic methods have been developed based on novel knowledge of the structural ciliary proteins, the genes encoding these proteins and mutations associated to DCP. These new methods include genetic analysis and the study of protein expression in cilia of the affected patients. This paper reviews DCP pathophysiology, the current diagnostic methods applied, and summarizes the international literature regarding the diagnosis of DCP based on genetic screening.
Assuntos
Humanos , Dineínas/genética , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/fisiopatologia , Síndrome de Kartagener/genética , Mutação , Testes Genéticos , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/fisiopatologia , Transtornos da Motilidade Ciliar/genéticaRESUMO
The unusual life cycle of Dictyostelium discoideum, in which an extra-cellular stressor such as starvation induces the development of a multicellular fruiting body consisting of stalk cells and spores from a culture of identical amoebae, provides an excellent model for investigating the molecular control of differentiation and the transition from single- to multi-cellular life, a key transition in development. We utilized serial analysis of gene expression (SAGE), a molecular method that is unbiased by dependence on previously identified genes, to obtain a transcriptome from a high-density culture of amoebae, in order to examine the transition to multi-cellular development. The SAGE method provides relative expression levels, which allows us to rank order the expressed genes. We found that a large number of ribosomal proteins were expressed at high levels, while various components of the proteosome were expressed at low levels. The only identifiable transmembrane signaling system components expressed in amoebae are related to quorum sensing, and their expression levels were relatively low. The most highly expressed gene in the amoeba transcriptome, dutA untranslated RNA, is a molecule with unknown function that may serve as an inhibitor of translation. These results suggest that high-density amoebae have not initiated development, and they also suggest a mechanism by which the transition into the development program is controlled.