The Role of the LAP2alpha-Lamin Interaction in Laminopathies

Lamins are the major components of the nuclear lamina, a two-dimensional network underlying the inner nuclear membrane and providing nuclear shape and stability. Beside their structural functions, nuclear lamins participate also in chromatin organization, DNA replication, transcription, and nuclear assembly. Lamin-binding proteins in the nuclear envelope and the nucleoplasm play important roles for the organization and functionality of lamins . Among those, lamina-associated polypeptide (LAP) 2alpha interacts specifically with the C-terminus of the nucleoplasmic fraction of A-type lamins. LAP2alpha`s function is not yet clear, but it may play a role in chromatin organization and in mitotic nuclear reformation. The importance of lamins has become more apparent over the past few years as a still increasing number of human diseases, which are collectively termed laminopathies, are linked to mutations in the gene coding for A-type lamins (LMNA) and affect skeletal and heart muscle, fat tissue, peripheral nerves, or cause premature aging (progeria). The reason for this divergence is not clear, but it has been assumed that the different lamin-binding proteins play an important role for the divergent consequences of the single mutations. Although mutations in LMNA are distributed all over the gene, mutations affecting the fat tissue or leading to progeria are predominantly located within the lamin C-terminus responsible for the interaction with LAP2alpha, suggesting that the lamin LAP2alpha interaction may be affected in some laminopathies and contribute to the clinical phenotype. The aim of this project is the detailed characterization of the interaction between LAP2alpha and A-type lamins in vitro and in vivo and to test specific effects of disease-linked mutations in lamins. The binding-sites will be narrowed down on both proteins by several in vitro interaction assays and by site directed mutagenesis. By 4D multicolour live cell-imaging I will study the interaction of the wild type and mutated proteins throughout the cell cycle and investigate binding dynamics, localization throughout the nucleus in interphase and during mitotic nuclear reformation, and the relation to DNA replication sites and factors. The use of wild type proteins as well as of binding-deficient and/or disease-specific mutants will allow to unravel the functional significance of the lamin A-LAP2alpha interactions in cells and will help to shed light on the potential molecular mechanisms of laminopathic diseases.