RESUMEN
Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
Asunto(s)
Núcleo Celular , Dineínas , Cinesinas , Proteínas Asociadas a Microtúbulos , Microtúbulos , Proteínas del Tejido Nervioso , Neuronas , Animales , Microtúbulos/metabolismo , Neuronas/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Dineínas/metabolismo , Núcleo Celular/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transporte Activo de Núcleo Celular , Complejo Dinactina/metabolismo , Complejo Dinactina/genética , Movimiento Celular , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Cerebelo/metabolismo , Cerebelo/citología , Sitios de Unión , HumanosRESUMEN
Respiratory syncytial virus (RSV) hijacks cholesterol or autophagy pathways to facilitate optimal replication. However, our understanding of the associated molecular mechanisms remains limited. Here, we show that RSV infection blocks cholesterol transport from lysosomes to the endoplasmic reticulum by downregulating the activity of lysosomal acid lipase, activates the SREBP2-LDLR axis, and promotes uptake and accumulation of exogenous cholesterol in lysosomes. High cholesterol levels impair the VAP-A-binding activity of ORP1L and promote the recruitment of dynein-dynactin, PLEKHM1, or HOPS VPS39 to Rab7-RILP, thereby facilitating minus-end transport of autophagosomes and autolysosome formation. Acidification inhibition and dysfunction of cholesterol-rich lysosomes impair autophagy flux by inhibiting autolysosome degradation, which promotes the accumulation of RSV fusion protein. RSV-F storage is nearly abolished after cholesterol depletion or knockdown of LDLR. Most importantly, the knockout of LDLR effectively inhibits RSV infection in vivo. These findings elucidate the molecular mechanism of how RSV co-regulates lysosomal cholesterol reprogramming and autophagy and reveal LDLR as a novel target for anti-RSV drug development.
Asunto(s)
Autofagia , Colesterol , Lisosomas , Receptores de LDL , Infecciones por Virus Sincitial Respiratorio , Proteínas de Transporte Vesicular , Replicación Viral , Proteínas de Unión al GTP rab , Proteínas de Unión a GTP rab7 , Lisosomas/metabolismo , Colesterol/metabolismo , Humanos , Animales , Receptores de LDL/metabolismo , Receptores de LDL/genética , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Ratones , Complejo Dinactina/metabolismo , Retículo Endoplásmico/metabolismo , Dineínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Virus Sincitial Respiratorio Humano/fisiología , Autofagosomas/metabolismo , Proteínas Virales de Fusión/metabolismo , Proteínas Virales de Fusión/genética , Células HeLa , Células A549RESUMEN
Dynactin subunit 2 (DCTN2) has been reported to play a role in progression of several tumours; however, the involvement of DCTN2 in potential mechanism or the tumour immune microenvironment among various cancers still remains largely unknown. Therefore, the objective of this study was to comprehensively investigate the expression status and potential function of DCTN2 in various malignancies through different database, such as The Cancer Genome Atlas, the Genotype-Tissue Expression and Gene Expression Omnimus databases. We discovered that DCTN2 expression was high in many type of tumours tissues compared to adjacent non-tumour ones. High DCTN2 signified poor prognosis for patients with tumours. Additionally, Gene Set Enrichment Analysis (GSEA) analysis revealed that DCTN2 was positively correlated with oncogenic pathways, including cell cycle, tumour metastasis-related pathway, while it was negatively with anti-tumour immune signalling pathway, such as INF-γ response. More importantly, we elucidated the functional impact of DCTN2 on hepatocellular carcinoma (HCC) progression and its underlying mechanisms. DCTN2 expression was much higher in HCC tissues than in adjacent non-tumour tissues. Silencing DCTN2 dramatically suppressed the proliferative and metastasis capacities of tumour cell in vitro. Mechanistically, DCTN2 exerted tumour-promoting effects by modulating the AKT signalling pathway. DCTN2 knockdown in HCC cells inhibited AKT phosphorylation and its downstream targets as well. Rescue experiments revealed that the anti-tumour effects of DCTN2 knockdown were partially reversed upon AKT pathway activation. Overall, DCTN2 may be a potent biomarker signifying tumour prognosis and a promising therapeutic target for tumour treatment, particularly in HCC.
Asunto(s)
Carcinoma Hepatocelular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Complejo Dinactina/metabolismo , Complejo Dinactina/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Microambiente Tumoral/genéticaRESUMEN
Chaperonin Containing Tailless complex polypeptide 1 (CCT) is a molecular chaperone composed of eight distinct subunits that can exist as individual monomers or as components of a double oligomeric ring, which is essential for the folding of actin and tubulin and other substrates. Here we assess the role of CCT subunits in the context of cell cycle progression by individual subunit depletions upon siRNA treatment in mammalian cells. The depletion of individual CCT subunits leads to variation in the distribution of cell cycle phases and changes in mitotic index. Mitotic defects, such as unaligned chromosomes occur when CCTδ is depleted, concurrent with a reduction in spindle pole-localised p150Glued, a component of the dynactin complex and a binding partner of monomeric CCTδ. In CCTδ-depleted cells, changes in the elution profile of p150Glued are observed consistent with altered conformations and or assembly states with the dynactin complex. Addition of monomeric CCTδ, in the form of GFP-CCTδ, restores correct p150Glued localisation to the spindle poles and rescues the mitotic segregation defects that occur when CCTδ is depleted. This study demonstrates a requirement for CCTδ in its monomeric form for correct chromosome segregation via a mechanism that promotes the correct localisation of p150Glued, thus revealing further complexities to the interplay between CCT, tubulin folding and microtubule dynamics.
Asunto(s)
Chaperonina con TCP-1 , Complejo Dinactina , Mitosis , Polos del Huso , Complejo Dinactina/metabolismo , Chaperonina con TCP-1/metabolismo , Chaperonina con TCP-1/genética , Humanos , Polos del Huso/metabolismo , Células HeLa , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Segregación CromosómicaRESUMEN
Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection.IMPORTANCEMany viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.
Asunto(s)
Membrana Celular , Complejo Dinactina , Dineínas , Herpesvirus Humano 1 , Proteínas Asociadas a Microtúbulos , Neuronas , Proteínas del Envoltorio Viral , Ensamble de Virus , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/metabolismo , Dineínas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virología , Humanos , Neuronas/virología , Neuronas/metabolismo , Complejo Dinactina/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Línea Celular , Animales , Compartimentos de Replicación Viral/metabolismo , Microtúbulos/metabolismoRESUMEN
The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.
Asunto(s)
Autofagia , Complejo Dinactina , Lisosomas , Animales , Humanos , Ratones , Complejo 2 de Proteína Adaptadora/metabolismo , Autofagosomas/metabolismo , Complejo Dinactina/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neuronas/metabolismo , Unión Proteica , Sirolimus/farmacologíaRESUMEN
Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa , Proteínas Adaptadoras Transductoras de Señales , Complejo Dinactina , Dineínas , Proteínas Asociadas a Microtúbulos , Proteínas del Tejido Nervioso , Microscopía por Crioelectrón , Complejo Dinactina/química , Complejo Dinactina/genética , Complejo Dinactina/metabolismo , Dineínas/química , Dineínas/genética , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Unión Proteica , Humanos , Células HeLa , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Repeticiones WD40 , Mapeo de Interacción de ProteínasRESUMEN
Experimental studies in flies, mice, and humans suggest a significant role of impaired axonal transport in the pathogenesis of Alzheimer's disease (AD). The mechanisms underlying these impairments in axonal transport, however, remain poorly understood. Here we report that the Swedish familial AD mutation causes a standstill of the amyloid precursor protein (APP) in the axons at the expense of its reduced anterograde transport. The standstill reflects the perturbed directionality of the axonal transport of APP, which spends significantly more time traveling in the retrograde direction. This ineffective movement is accompanied by an enhanced association of dynactin-1 with APP, which suggests that reduced anterograde transport of APP is the result of enhanced activation of the retrograde molecular motor dynein by dynactin-1. The impact of the Swedish mutation on axonal transport is not limited to the APP vesicles since it also reverses the directionality of a subset of early endosomes, which become enlarged and aberrantly accumulate in distal locations. In addition, it also reduces the trafficking of lysosomes due to their less effective retrograde movement. Altogether, our experiments suggest a pivotal involvement of retrograde molecular motors and transport in the mechanisms underlying impaired axonal transport in AD and reveal significantly more widespread derangement of axonal transport pathways in the pathogenesis of AD.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Transporte Axonal , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal/genética , Axones/metabolismo , Axones/patología , Complejo Dinactina/metabolismo , Complejo Dinactina/genética , Dineínas/metabolismo , Endosomas/metabolismo , Endosomas/genética , Lisosomas/metabolismo , Mutación , Variación GenéticaRESUMEN
In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.
Asunto(s)
Autofagosomas , Dineínas , Proteínas Activadoras de GTPasa , Nexinas de Clasificación , Proteínas de Unión al GTP rab , Humanos , Citoesqueleto de Actina/metabolismo , Autofagosomas/metabolismo , Complejo Dinactina/metabolismo , Dineínas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Lisosomas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Cell polarization requires asymmetric localization of numerous mRNAs, proteins and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors. In the dynein-dynactin-Bicaudal-D transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here, we focus on the role of Drosophila BicD-related (BicDR, CG32137) in the development of the long bristles. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft. BicD and BicDR also support the stable expression and distribution of Rab6 and Spn-F in the bristle shaft, including the distal tip localization of Spn-F, pointing to the role of microtubule-dependent vesicle trafficking for bristle construction. BicDR supports the function of BicD, and we discuss the hypothesis whereby BicDR might transport cargo more locally, with BicD transporting cargo over long distances, such as to the distal tip. We also identified embryonic proteins that interact with BicDR and appear to be BicDR cargo. For one of them, EF1γ (also known as eEF1γ), we show that the encoding gene EF1γ interacts with BicD and BicDR in the construction of the bristles.
Asunto(s)
Proteínas de Drosophila , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Dineínas/genética , Dineínas/metabolismo , Drosophila/metabolismo , Microtúbulos/metabolismo , Complejo Dinactina/genética , Complejo Dinactina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only â¼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of â¼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.