Structural and mechanical roles of spectrin repeat proteins in nucleated cells and implications on muscular dystrophy

This thesis studied the structural and mechanical roles of spectrin repeat (SR) proteins in nucleated cells, specifically focusing on ubiquitously expressed SR protein alphaII-spectrin and nesprin-1alpha. Both of them also associate with Emery-Dreifuss muscular dystrophy causing proteins emerin and lamin A/C. In detailed research of alphaII-spectrin, we explored its effects on both nucleoskelton system and plasma membrane-actin cytoskeleton systems. We showed that alphaII-spectrin is enriched at the nuclear envelope, and that HeLa cells with reduced levels of alphaII-spectrin have unique nuclear phenotypes including enlargement, disorganized A- and B-type lamin networks, reduced emerin and profound failure to 'spring back' after mechanical stretch. These phenotypes were qualitatively and quantitatively different from those caused by reductions in lamin A/C. Mechanical recoil is also reduced in HeLa cells downregulated for emerin. We conclude that alphaII-spectrin is an essential 'spring' component of the nucleoskeleton that is structurally linked to the A- and B-type lamin networks and allows the nucleoskeleton to recoil after deformation. These findings also implicate alphaII-spectrin and recoil defects as contributors to Emery Dreifuss muscular dystrophy. Besides nucleus, alphaII-spectrin was widely recognized as component of plasma membrane in nucleated cells. We found that reduction of alphaII-spectrin influence the organization of actin stress fibers and the force balance of cell cytoskeleton systems, as well as membrane's resistance shear stress and cell mobility. For nesprin-1alpha, we mainly analyzed the structural properties of nesprin-1alpha fragments and extracted the structural information of different domains. We found the spectrin-like repeats in nesprin-1alpha have similar properties of typical spectrin repeats and a central adaptive domain strengthens dimerization and inhibits unfolding.