Higginson, Jennifer R.
An investigation into dystroglycan deficiency in fibroblasts and a role for dystroglycan in cytokinesis.
PhD thesis, University of Glasgow.
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Dystroglycan is a heterodimeric cell adhesion molecule consisting of alpha and beta subunits, which links the actin cytoskeleton to the extracellular matrix. Dystroglycan has an important structural role and is integral for maintaining tissue integrity, but new avenues have research have implicated it in other roles within the cell due to its ability to bind a number of interacting proteins including cytoskeletal components and proteins involved in signal transduction. To elucidate the functions of dystroglycan at the cellular level, fibroblasts were differentiated from dystroglycan null embryonic stem cells. Analysis of these cells by western blot, however, showed them to be expressing dystroglycan and this was confirmed by RT-PCR, genomic PCR and northern blot. Subsequently, Swiss 3T3 fibroblasts with greater than 60% reduction in dystroglycan expression were generated by stable retroviral infection of a shRNA construct. Dystroglycan deficient cells were smaller and were found to have a reduction in cell-substrate adhesions, when compared to a control cell line. Dystroglycan deficiency did not affect cell motility or polarity in fibroblasts, but did result in a reduction in the rate of cell proliferation, which was thought to be the consequence of an increase in apoptosis. Dystroglycan deficiency also inhibited the formation of filopodia and lamellipodia in response to dominant active Cdc42 or Rac1 respectively, suggesting that it an important mediator of Rho GTPase-mediated cytoskeletal rearrangements. This study has also identified a possible new function for dystroglycan during cytokinesis. Endogenous dystroglycan was localised to the cleavage furrow and midbody of dividing fibroblasts, where it was found to co-localise with the ERM family member, ezrin. Expression of mutated and truncated dystroglycan-GFP constructs in dividing HeLa cells revealed that the cytoplasmic domain is required for cleavage furrow localisation, but ezrin-binding is not. A model for dystroglycan function at the cleavage furrow is presented whereby dystroglycan recruits ezrin to the site of furrow ingression and together they serve to tether the actomyosin contractile ring to the plasma membrane during cytokinesis. These findings provide further insight into the multifaceted functions of dystroglycan within the cell and also raised interesting questions about possible new roles for dystroglycan.
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