Defining the role and essential interactions of the centrosomal protein pericentrin in microtubule formation during myogenesis

Lead Research Organisation: University of Leicester
Department Name: Molecular and Cell Biology

Abstract

Muscle fibers are long multinucleated cells formed from the fusion of myoblasts in a process called myogenesis. Nuclei are arranged regularly along the periphery of the myofiber. Correct nuclear positioning involves interactions between nuclear envelope proteins, centrosomal proteins and motor proteins. Aberrant positioning such as nuclear clustering is associated with muscular dystrophies, suggesting that correct nuclear postioning is important for the formation of healthy functioning skeletal muscle. Yet, the details of the molecular mechanisms and the precise roles of proteins involved in nuclear positioning are unclear.
In myoblasts, proteins of the microtubule organising centre (MTOC) are localised at the centrosome. It has been shown that just hours after differentiation, markers of the MTOC are released from the centrosome and instead localise at the perinuclear surface. These proteins retain their role as a microtubule nucleation site. Our hypothesis is that centrosomal proteins localise to the perinuclear surface by binding to nesprin-1, a nuclear envelope protein that is part of the LINC complex that physically links the nucleus to cytoskeletal components. This is supported by the fact that mutations in LINC complex components can cause nuclear clustering. An explanation could be because recruitment of MTOC proteins to the nuclear envelope and subsequent microtubule reorganisation becomes impaired. Since motor proteins use microtubules to move cargo, nuclear movement to the cell periphery becomes affected.
This project will test our hypothesis, leading to a comprehensive understanding of the roles of centrosomal proteins in microtubule nucleation during myogenesis. We will identify the function of specific protein involved in microtubule nucleation and nuclear positioning, and define the protein interactions using a range of cell biology and structural techniques. This will build upon our current molecular model of myogenesis.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1645609 Studentship BB/M01116X/1 05/10/2015 30/09/2019 Caroline Shak
 
Description The results show that the N-terminal region of nesprin-1alpha2 is responsible for the recruitment of at least two centrosomal proteins, PCM1 and AKAP450, to the nuclear envelope in myotubes. This region contains a motif also known to interact with kinesin light chains (Wilson and Holzbaur, 2015; Zhou et al., 2017). A major question is whether other centrosomal proteins also require the same nesprin-1alpha2 region. This would indicate similarities or differences in the recruitment of centrosomal proteins to the nuclear envelope.

It is well established that at the centrosome, PCM1 is required for the localisation of pericentrin. This has also been suggested to be the case at the nuclear microtubule organising centre (MTOC). The results in this study confirms these results using quantitative analysis. This shows functions of centrosomal proteins at the NE may be linked to their role at the centrosome. The results of this study also suggests, for the first time that PCM1 plays a role in the recruitment of AKAP450 to the nuclear envelope in myotubes. This suggests that the nuclear MTOC may be formed in order, similar to the centrosomal MTOC, where specific layers of proteins form around the mother centriole. Further research to delineate how the nMTOC forms will involve exploration of functions of other centrosomal proteins.

The recruitment of centrosomal proteins to the myotube nuclear envelope likely involves muscle-specific factors. Overexpression of nesprin-1alpha2 alone in a non-muscle cell does not affect centrosomal protein localisation. Co-expression of nesprin-1alpha2 and centrosomal proteins in non-muscle cells in attempt to saturate centrosomal protein localisation at the centrosome, also does not lead to their co-localisation with nesprin-1alpha2. The same results occur in cells treated with centrinone, a drug that leads to centriole-depleted cells. Thus, this research opens up questions to the muscle-specific factors required. It may be the expression of a protein bridging nesprin-1alpha2 and centrosomal proteins. Alternatively, muscle-specific phosphorylations may be required for nuclear MTOC formation.

Altogether, these results reveal molecular details of nuclear MTOC formation at the nesprin-1alpha2 interface. It is important to note that AKAP450, is localised at the centrosome and Golgi, in centrosome containing cells. It would be of interest to consider whether AKAP450 localisation at the NE in myotubes is by associating with Golgi protein elements. This would open up questions into the interplay between nMTOC and Golgi proteins in microtubule nucleation in myotubes.
Exploitation Route For the first time, this project investigated the role of each nesprin-1alpha2 domain in the recruitment of centrosomal proteins to the NE. This narrows down the nesprin-1alpha2 domains involved in direct or indirect interaction with centrosomal proteins, allowing structural studies or detailed interaction studies to be pursued.

This information also tells us whether a muscle-disease causing nesprin-1alpha2 mutation has the potential to affect centrosomal protein recruitment to the nuclear MTOC, ultimately affecting the arrangement of the microtubule cytoskeleton. This information, together with results showing a relationship between centrosomal proteins at the nuclear MTOC, can be used to draw a simple diagram intended for members of the public, to understand how disruption of different proteins in muscle cells can lead to muscle dystrophy.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Participation in an open day at my department 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Pupils looking at prospective universities attended the University open day. I was involved in outreach activities for biological sciences and also was at a stand to answer any questions about my department. This helped prospective students understand what studying biological sciences at university is like.
Year(s) Of Engagement Activity 2016
 
Description Scientific article for university website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Writing an article about the background and importance of my PhD project to inform the public.
Year(s) Of Engagement Activity 2016
URL https://www2.le.ac.uk/offices/press/think-leicester/health-and-medicine/2016/looking-inside-cells-to...