| The nuclear envelope forms the boundary between the nucleus and the cytoplasm, and consists of an inner nuclear membrane (INM) and an outer nuclear membrane (ONM), separated by a lumen, called the periplasmic space. The NE is anchored to the actin cytoskeleton via a group of type II transmembrane proteins, the nesprins, which are localized on the ONM of the nucleus. Nesprins are produced by alternative mRNA splicing and initiation of two genes, nesprin-1 and nesprin-2, and contain a variable number of spectrin repeats and a conserved C-terminal Klarsicht-related transmembrane domain. Additionally, full-length nesprin-1 and nesprin-2 contain an N-terminal actin-binding domain (ABD) of the b-spectrin type. Recently, we have isolated a third nesprin that interacts with the ABD of the cytoskeletal linker protein plectin and, like the other two nesprins, nesprin-3 is a component of the ONM. Unlike nesprin-1 and -2, which are giant proteins of 1014 (nesprin-1) and 796 kDa (nesprin-2), nesprin-3 is relatively small, approximately 120 kDa. Moreover, nesprin-3 lacks an ABD and therefore is clearly different from the other nesprins. Since plectin harbors a specific binding site for intermediate filaments (IF?s) of various types, we believe that the interaction of nesprin-3 with plectin helps to anchor the nucleus to the IF system. In this project, we propose to perform siRNA knockdown and protein overexpression experiments to study the function of nesprin-3 in the anchorage of the nucleus to the IF system and determine whether nesprin-3 has a role in the breakdown and re-assembly of the NE during mitosis. Furthermore, we will identify which nesprin-3-binding proteins are important in the recruitment of nesprin-3 to the NE, and acquire information on the dynamics of nesprins and (candidate) binding proteins during the formation and breakdown of the NE. Finally, we will establish whether nesprin-3 can bind to BPAG1 and MACF, two cytoskeletal linker proteins that have an ABD that is highly homologous to that of plectin and that carry a binding site for the microtubule (MT) system and study whether and how the interaction between nesprin-3 and plectin is regulated during mitosis. We anticipate that the results will contribute to our understanding of the role of nesprins in mechanical signaling by linking together the three skeletal networks: the extracellular matrix, the cytoskeleton and the nuclear matrix, as well as in the formation and breakdown of the NE. |
