RESUMO
Bidirectional cargo transport in neurons can be explained by two models: the "tug-of-war model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons.
Assuntos
Transporte Axonal/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Complexo Dinactina/fisiologia , Dineínas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Animais , Transporte Axonal/genética , Axônios/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Ensaios de Migração Celular , Complexo Dinactina/genética , Dineínas/genética , Cinesinas , Proteínas Associadas aos Microtúbulos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
In the mammalian central nervous system transcripts of certain synaptic components are localized near the synapse, allowing for rapid regulation of protein levels. Here we test whether an mRNA localization mechanism also exists in the postsynaptic specialization induced by agrin in C2C12 myotubes. RT-PCR showed that Chrna1 was co-purified with nicotinic acetylcholine receptor (AChR) isolated by affinity column or by ultracentrifugation. In addition, Stau1 was found to interact with Chrna1 mRNA, and knocking down of Stau1 by RNAi resulted in defective AChR clustering. These results suggest that mRNA localization also participates in the formation of mammalian neuromuscular junction (NMJ).
