Salpingidou, Georgia (2005) The role of emerin and LEM domain proteins in nuclear envelope assembly and cytoskeleton organisation. Doctoral thesis, Durham University.
The nuclear envelope (NE) plays a fundamental role in the cell by separating nuclear from cytoplasmic activities, and mutations in NE proteins have been associated with a diverse array of diseases. In the present study the Xenopus cell-free system was used to investigate the function of the inner nuclear membrane protein, emerin, which is associated with the Emery-Dreifuss muscular dystrophy (X-EDMD).Initially, the order and dynamics of NE assembly in Xenopus egg extracts have been investigated. Using a panel of antibodies it was shown that NE assembly proceeds by the ordered recruitment of two membrane populations, Nuclear Envelope Precursor vesicles -A and -B (NEP-A and NEP-B), to chromatin. As shown by immunofluorescence NEP-B vesicles, together with nucleoporins (Nups), appear first around chromatin at about ten minutes after initiation of NE assembly while NEP-A vesicles appear at a later stage, at about twenty minutes. To investigate the role of different emerin domains in this process, four human emerin peptides consisting of amino acids (aa) 1-70, 1-176, 1-220 and 73-180 were added individually to Xenopus nuclear assembly reactions at different concentrations and the effect on nuclear vesicle recruitment and NPC formation was monitored. Immunofluorescence analysis showed that peptides containing the LEM domain of emerin interfere with a correct NE assembly by inhibiting chromatin decondensation and recruitment of membranes to chromatin. This inhibitory effect was shown to be exerted mainly on NEP-A membranes and on Nup62 and Nupl53. By the use of two antibodies, raised against the LEM domain of human emerin and LAP2ß, two proteins of 30 and 36 kD, respectively, were identified in Xenopus. Both proteins were shown to reside in the NEP-A membrane population providing an explanation for the preferential inhibition of NEP-A recruitment to chromatin by exogenously added LEM domain containing emerin peptides. To further investigate whether the domain specific inhibitory effects of emerin on nuclear assembly correlate with specific interacting proteins, co-precipitation experiments were performed to identify emerin binding proteins in the Xenopus cytosol. From these experiments ß -tubulin was identified as a protein able to interact with emerin peptides 1-70 and 73-180. Staining of X-EDMD cells, which lack emerin, with a ß -tubulin antibody revealed no alterations in the organisation of the microtubule (MT) network. The most prominent effect of emerin mutations regarding MTs was the position of the Microtubule Organising Centre (MTOC) relative to the NE. Staining for the centrosomal protein pericentrin revealed a mis-localisation of the MTOC away from the NE in X-EDMD cell lines at distances at least double compared to control cells.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:27|