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Title: Investigating the recruitment of centrosomal proteins to the nuclear envelope during myogenesis
Author: Shak, Caroline
ISNI:       0000 0004 8506 9612
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2020
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Several stages of nuclear movement and positioning occur during the differentiation of myoblasts into myotubes and myofibres, allowing nuclei to be spread along the cell length and periphery. In a developing myotube, the nuclear envelope (NE) spanning linker of nucleoskeleton to cytoskeleton (LINC) complex recruits centrosomal proteins such as PCM1, pericentrin and AKAP450 to form the nuclear microtubule organising centre (nMTOC). Microtubules nucleated from the nMTOC are used by motor proteins to position myonuclei, failure of which leads to myonuclear clustering and muscle disease. This study explored how the LINC complex component nesprin-1 acts as a centrosomal protein receptor, and how centrosomal proteins relocate to the NE. The muscle-specific isoform nesprin-1α2 recruited PCM1 and AKAP450 to the NE in nesprin-1 null myotubes, through its Nterminal region comprising a 31 residue isoform-specific sequence, 3 spectrin repeats and the adaptive domain. PCM1 residues 1-331 localised to the NE and interacted with nesprin-1α2. In contrast, residues 302-573 localised to the centriolar satellites, suggesting the mode of PCM1 binding, and potentially, function at the two sites is different. GFP nesprin 1α2 expression in non-myotube cells was not sufficient to recruit PCM1 or pericentrin to the NE. Yet, forced tethering of PCM1 residues 1-1089 to the NE appeared to be able to weakly recruit pericentrin to the NE. This suggests that upon myogenesis, myogenic events such as phosphorylation, mediate PCM1 transfer to the NE, where it readily functions as a scaffolding protein. In nesprin-1 null myotubes, NE tethered PCM1 appeared to recruit AKAP450 to the NE. However, depletion of PCM1 in myotubes only partially reduced the NE localisation of pericentrin, whereas AKAP450 was unaffected. This suggests that at the NE, PCM1 functions as part of a larger protein scaffold to recruit pericentrin and AKAP450 to the NE. This study starts to delineate nMTOC formation at the nesprin-1α2 interface.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Centrosomal Proteins ; nuclear envelope ; myogenesis