Mechanisms and regulation of emerin self-assembly at the nuclear envelope: Implication for Emery-Dreifuss Muscular Dystrophy - ABSTRACT Emerin is a largely disordered integral protein of the inner nuclear envelope (INE) with a short LEM-domain. It is a major contributor to the maintenance of the nuclear architecture and to mechanotransduction processes at the NE. When mutated, emerin causes Emery-Dreifuss muscular dystrophy (EDMD), an envelopathy whose underlying mechanisms and muscle specific effects are not fully understood. How emerin participates in molecular scaffolding at the INE and helps protect the nucleus against mechanical strains has remained largely elusive. Recently, we discovered that emerin monomers self- assemble into INE oligomeric nanodomains to coordinate NE mechanics. Compared to wild-type emerin, EDMD-inducing emerin mutants display either insufficient or excessive self-assemblies, both of which result in defective nuclear shape adaptations against mechanical challenges, as typically observed in EDMD patients. Defining the pathogenesis of EDMD therefore hinges on understanding how the oligomerization of emerin is regulated in response to forces exerted at the NE. Here, we propose innovative approaches that integrate quantitative proteomics, super-resolution microscopy in cells, nuclear biomechanics, and single-molecule biophysical studies: (i) to establish the proteomic environments and the mechanisms governing the self-assembly of emerin in muscle and non-muscle cells and (ii) to uncover the molecular steps and conformational changes of emerin allowing its oligomerization to promote adequate nuclear compliance against forces and prevent abnormal NE mechanics in the context of EDMD. Building on our previous work and preliminary data, we hypothesize that the nanoscale self-assembly of emerin is regulated by competitive interactions with a meshwork of INE-proximal proteins that modulate its structural conformations and the ability of the LEM-domain to bind the disordered region of other emerin for emerin:emerin contacts. We also hypothesize that tuning those emerin self-assemblies and the mechanics of the nucleus during myogenesis and responses to force involves adaptive changes in this interaction meshwork, which do not occur correctly in EDMD. These hypotheses will be tested in three aims. In Aim 1, we will employ in situ spatial proximity labeling and quantitative proteomics to establish changes in the local proteomic environments of emerin monomers or oligomers within the NE as a function of: (i) EDMD- inducing emerin mutations, (ii) cell myogenesis, and (iii) mechanical forces on nuclei. In Aim2, we will define how interactions of the emerin LEM-domain with the disordered region of other emerin, and competition from other LEM- domain proteins at the INE, allow for the adaptive formation of emerin oligomers in response to forces. This will be done using functional genomics, super-resolution imaging and single molecule tracking in cells and biomechanic assays. In Aim3, we will map conformational changes as the emerin LEM-domain makes in-cis or in-trans molecular contacts with various parts of the disordered region. We will also define how interacting partners of emerin modulate those conformations to promote or impede its self-assembly. These studies will be done by in vitro single molecule FRET on recombinant emerin. This work will provide novel mechanistic insights into emerin’s function and the general principles of NE mechanics, laying a groundwork to develop remedial strategies for EDMD and other diseases involving impaired nuclear responses to forces.
