| The structural proteins that are required for maintaining the integrity of the mammalian cell nucleus also regulate cell function. The last several decades have revealed a large set of proteins at the nuclear envelope, the tightly regulated barrier between the cytoplasm and the nucleus, which interact with the genetic material within and are also critical for proper cell behavior. A significant subset of these nuclear envelope proteins are implicated in a host of human diseases, collectively known as laminopathies, that result when mutations impair their ability to function properly. The gene lmna encodes the proteins lamin A/C, two of the most well known and critical structural proteins of the somatic cell nuclear envelope. They have long been suspected to play an important role in nuclear assembly, shape, and integrity and defects lead to laminopathies. In this dissertation, physicochemical methods are developed and/or used to study these proteins in situ. At the supramolecular, whole-organelle level, the perturbations of nuclear structure associated with cell differentiation and disease processes are assessed by measuring the rheological properties of nuclei, using Fluorescence Imaged MicroDeformation via micropipette aspiration. The results generally show that lamin A/C stiffens the somatic cell nucleus by at least two-fold. A key domain in lamin A, the c-terminal Ig like fold, was then studied in vitro. The destabilizing effect of a muscular dystrophy causing mutation was assessed through development of a temperature-dependant Cysteine Protection Kinetic (CPK(T)) assay. Working towards in situ assays, studies were then conducted on soluble cell extracts, leading to the identification of a set of prominently labeled bands that were identified by mass spectrometry. The CKP(T) profiles real a range of transition points from protected to unprotected. Using the in vitro results as a foundation, the method was applied in situ to study the same set of proteins. In addition, preliminary studies of immunoprecipitated lamin A/C demonstrate in situ labeling that progressively increases with time of exposure. Overall the studies demonstrate that the CPK(T) provides a robust measure of structural transitions that is applicable both in vitro as well as in situ. |
