RESUMO
Axonal transport is required for neuronal development and survival. Transport from the axon to the soma is driven by the molecular motor cytoplasmic dynein, yet it remains unclear how dynein is spatially and temporally regulated. We find that the dynein effector Hook1 mediates transport of TrkB-BDNF-signaling endosomes in primary hippocampal neurons. Hook1 comigrates with a subpopulation of Rab5 endosomes positive for TrkB and BDNF, which exhibit processive retrograde motility with faster velocities than the overall Rab5 population. Knockdown of Hook1 significantly reduced the motility of BDNF-signaling endosomes without affecting the motility of other organelles. In microfluidic chambers, Hook1 depletion resulted in a significant decrease in the flux and processivity of BDNF-Qdots along the mid-axon, an effect specific for Hook1 but not Hook3. Hook1 depletion inhibited BDNF trafficking to the soma and blocked downstream BDNF- and TrkB-dependent signaling to the nucleus. Together, these studies support a model in which differential association with cargo-specific effectors efficiently regulates dynein in neurons.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dineínas do Citoplasma/metabolismo , Endossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Receptor trkB/metabolismo , Animais , Transporte Axonal , Sítios de Ligação , Fator Neurotrófico Derivado do Encéfalo/genética , Núcleo Celular/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Endossomos/ultraestrutura , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Neurônios/ultraestrutura , Cultura Primária de Células , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptor trkB/genética , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteína Vermelha FluorescenteRESUMO
The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.