Investigating the recruitment of microtubule motor proteins to the nuclear envelope in myogenesis

Myotubes are syncitial cells which are formed by the fusion of myoblasts in a process termed myogenesis. During myogenesis, the multiple nuclei of the myotube move to be roughly equidistant from one another before proceeding to the cell periphery. Nesprin-1α2 is a short, muscle specific isoform of nesprin-1. Nesprins are nuclear envelope (NE) proteins that form part of the LINC complex, a bridge between the cytoskeleton and nuclear matrix. Nesprin-1α2 and the microtubule organising centre (MTOC) protein PCM1 have been implicated in the recruitment of motor proteins, kinesin-1 and dynein/dynactin, to the NE during myogenesis. This is thought to assist nuclear repositioning by facilitating nuclear movement along the microtubules. This study aimed to identify novel regions of nesprin-1α2, besides the previously described adaptive domain ‘LEWD’ motif, required for kinesin-1 recruitment to the NE. The role of PCM1 in the recruitment of kinesin-1 to the NE during myogenesis was also investigated. In addition to the ‘LEWD motif’, a novel N-terminal region of nesprin-1a2 was revealed to be essential for kinesin localisation to the NE. The region required for the recruitment of kinesin-1 to the NE overlapped substantially with that required for PCM1 localisation at the NE and co-immunoprecipitation studies indicated an interaction between PCM1 and KLC1/2. A PCM1 knock-down time-course, along with dynein inhibition and nocodazole experiments, suggested that kinesin-1 is trafficked along the microtubules to the NE in the early stages of myogenesis in a dynein/dynactin and PCM1 dependent manner. My data supports a model in which the N-terminal portion of nesprin-1α2, in combination with PCM1, is responsible for the early recruitment of kinesin-1 and that, once at the NE, kinesin-1 is anchored there via KLC1/2 interaction with the ‘LEWD’ motif.