RESUMO
During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a single-molecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.
Assuntos
DNA de Cadeia Simples/química , Rad51 Recombinase/química , Núcleo Celular/metabolismo , Corantes Fluorescentes/química , Humanos , Funções Verossimilhança , Microfluídica , Microscopia de Fluorescência , Pinças Ópticas , RNA Interferente Pequeno/metabolismo , Recombinação Genética , Reprodutibilidade dos Testes , Processos Estocásticos , Especificidade por SubstratoRESUMO
Microtubule-based transport is essential for neuronal function because of the large distances that must be traveled by various building blocks and cellular materials. Recent studies in various model systems have unraveled several regulatory mechanisms and traffic rules that control the specificity, directionality and delivery of neuronal cargos. Local microtubule cues, opposing motor activity and cargo-adaptors that regulate motor activity control microtubule-based transport in neurons. Impairment of intracellular transport is detrimental to neurons and has emerged as a common factor in several neurological disorders. Genetic approaches have revealed strong links between intracellular transport processes and the pathogenesis of neurological diseases in both the central and peripheral nervous system. This Commentary highlights recent advances in these areas and discusses the transport defects that are associated with the development of neurological diseases.
Assuntos
Doenças do Sistema Nervoso Central/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Transporte Biológico Ativo/genética , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Humanos , Microtúbulos/genética , Neurônios/patologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologiaRESUMO
Development, polarization, structural integrity, and plasticity of neuronal cells critically depend on the microtubule network and its dynamic properties. SLAIN1 and SLAIN2 are microtubule plus-end tracking proteins that have been recently identified as regulators of microtubule dynamics. SLAINs are targeted to microtubule tips through an interaction with the core components of microtubule plus-end tracking protein network, End Binding family members. SLAINs promote persistent microtubule growth by recruiting the microtubule polymerase ch-TOG to microtubule plus-ends. Here, we show that SLAIN1/2 and ch-TOG-proteins are highly enriched in brain and are expressed throughout mouse brain development. Disruption of the SLAIN-ch-TOG complex in cultured primary rat hippocampal neurons by RNA interference-mediated knockdown and a dominant-negative approach perturbs microtubule growth by increasing catastrophe frequency and inhibits axon extension during neuronal development. Our study shows that proper control of microtubule dynamics is important for axon elongation in developing neurons.
Assuntos
Axônios/fisiologia , Hipocampo/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Hipocampo/embriologia , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , RatosRESUMO
The development and homeostasis of neurons relies heavily on the selective targeting of vesicles into axon and dendrites. Microtubule-based motor proteins play an important role in polarized transport; however, the sorting mechanism to exclude dendritic cargo from the axon is unclear. We show that the dynein regulator NDEL1 controls somatodendritic cargo transport at the axon initial segment (AIS). NDEL1 localizes to the AIS via an interaction with the scaffold protein Ankyrin-G. Depletion of NDEL1 or its binding partner LIS1 results in both cell-wide and local defects, including the non-polarized trafficking of dendritic cargo through the AIS. We propose a model in which LIS1 is a critical mediator of local NDEL1-based dynein activation at the AIS. By localizing to the AIS, NDEL1 facilitates the reversal of somatodendritic cargos in the proximal axon.
Assuntos
Axônios/metabolismo , Proteínas de Transporte/metabolismo , Dineínas/metabolismo , Animais , Anquirinas/metabolismo , Proteínas de Transporte/genética , Citoesqueleto/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico , Vesículas Sinápticas/metabolismoRESUMO
Kinesin and dynein motors drive bidirectional cargo transport along microtubules and have a critical role in polarized cargo trafficking in neurons [1, 2]. The kinesin-2 family protein KIF17 is a dendrite-specific motor protein and has been shown to interact with several dendritic cargoes [3-7]. However, the mechanism underlying the dendritic targeting of KIF17 remains poorly understood [8-11]. Using live-cell imaging combined with inducible trafficking assays to directly probe KIF17 motor activity in living neurons, we found that the polarized sorting of KIF17 to dendrites is regulated in multiple steps. First, cargo binding of KIF17 relieves autoinhibition and initiates microtubule-based cargo transport. Second, KIF17 does not autonomously target dendrites, but enters the axon where the actin cytoskeleton at the axon initial segment (AIS) prevents KIF17 vesicles from moving further into the axon. Third, dynein-based motor activity is able to redirect KIF17-coupled cargoes into dendrites. We propose a three-step model for polarized targeting of KIF17, in which the collective function of multiple motor teams is required for proper dendritic sorting.
Assuntos
Axônios/metabolismo , Dendritos/metabolismo , Cinesinas/metabolismo , Animais , Células Cultivadas , Microtúbulos/metabolismo , Transporte Proteico , RatosRESUMO
Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development.