Functions of the Alpha isoform of lamina associated polypeptide 2 (LAP2-Alpha) in cell cycle-dependent nuclear structure dynamics

In most eukaryotic cells the nucleus is dramatically reorganized in the course of the cell cycle, being disassembled during prophase and reestablished at the end of mitosis. The structural organization of the nucleus is essential for many nuclear activities, such as DNA replication, RNA transcription and RNA processing, but the components and the molecular mechanisms involved are poorly understood. The nuclear lamina, a filamentous meshwork of intermediate filament-type proteins underlying the nuclear membrane, and integral membrane proteins of the inner nuclear membrane, which are tightly associated with the lamina, such as lamina associated polypeptides (LAPs) 1 and 2 and lamin B receptor, have been implicated in chromatin organization and post mitotic nuclear assembly. The inner nuclear membrane protein LAP2 (now called LAP2b ) has recently been shown to be a member of a family of related proteins derived by alternative splicing of a single gene. While most of the LAP2 isoforms are closely related structurally, missing short regions of the nucleoplasmic domain of LAP2b , the largest isoform LAP2a is more distantly related, containing only a 187 amino acid long N-terminus identical to the N termini of the other members and a 506 amino acid long unique C-terminus, which lacks a transmembrane domain. We have recently shown that LAP2a is associated with salt-detergent resistant intranuclear structures during interphase, dissociates from chromosomes during metaphase, and translocates to chromosomal areas very early during post mitotic nuclear reassembly (Dechat et. al. EMBO J., in press), consistant with a role of the protein in nuclear assembly and organization. In the proposed project we plan to study the cell cycle-dependent interactions of LAP2a in detail, and elucidate potential functions of the protein, focusing on the following major topics: (1) Identification of direct interaction partners of LAP2a . While LAP2a has been found to associate with chromosomes and to colocalize transiently with lamin structures, direct interaction partners have not been identified yet. Using yeast two hybrid screening, as well as a series of in vitro binding studies, including cosedimentation, solid phase overlay, affinity chromatography, and co- immunoprecipitation, we plan to identify LAP2a binding proteins. (2) Localization of binding domains within the LAP2a motecule. We will express recombinant proteins, comprising several molecular domains of LAP2a in bacteria and perform in vitro binding assays with purified lamins, chromosomes and identified LAP2a -binding proteins. Similar LAP2a mutant proteins will be expressed in mammalian cells using tetracycline inducible expression systems, and their cellular localization and molecular interactions will be analyzed by confocal microscopy, subcellular fractionation and co-immunoprecipitation assays. (3) Characterization of the cell cycle dependent phosphorylation of LAP2a . In the previous study we provided evidence that LAP2a is phosphorylated in a mitosis specific manner. Here we plan to analyze the state of LAP2a phosphorylation at different cell cycle stages, localize the phosphorylation sites in the molecule, and identify the kinases involved. This will be done by in vivo and in vitro phosphorylation analyses in conjunction with 2D phosphopeptide mapping. The physiological significance of LAP2a phosphorylation will be studied by in vitro binding analyses using phosphorylated proteins, or by expressing LAP2a containing mutated phosphorylation sites in mammalian cells. (4) Analysis of potential functions of LAP2a in nuclear assembly. Using a mammalian in vitro nuclear assembly system we will test the effect of various LAP2a antibodies and (dominant-negative) LAP2a mutant proteins on nuclear envelope formation. Furthermore, cells will be stably transfected with constructs coding for various LAP2a mutant proteins using the tetracycline-inducible expression vector system. Upon induction of protein expression at specific cell cycle stages, we will analyse the effects of exogenous mutant proteins on nuclear disassembly-reassembly.

In the course of this project, we have characterized the dynamics, interactions and potential functions of a structural nuclear protein, termed lamina-associated polypeptide (LAP) 2alpha. These studies revealed that the protein is involved in major processes in living cells, including chromosome organization, cell division and cell proliferation. Most interestingly, our results suggest that the protein may be linked to various types of heritable human diseases, collectively called laminopathies, affecting skeletal muscle, heart, adipose and nerve cells. The molecular mechanisms of these diseases are still unknown. Mammalian nuclei are complex organelles, whose functions depend largely on a spatial, higher order organization of chromatin. The nuclear lamina, the major structural framework of the nuclear envelope of higher eukaryotic cells, internal nuclear lamin structures, and lamin binding proteins in the nuclear membrane and in the nucleoskeleton are major components involved in determining nuclear architecture. The current working model suggests that anchorage of chromatin at the nuclear periphery and its three-dimensional organization within the nuclear interior regulate cell type- and differentiation-specific gene expression. During cell division, the nucleus is completely disassembled and two daughter nuclei have to be newly assembled after mitosis. This process involves the regulated interactions of structural components with chromatin, but the molecular mechanisms are largely unknown. Our research within this project focused on lamina-associated-polypeptides 2 (LAP2), which comprise a family of up to six alternatively spliced isoforms localized in the inner nuclear membrane, such as LAP2beta, and in the nuclear interior, such as LAP2alpha. Both proteins bind to chromatin during interphase, but dissociate from chromosomes in metaphase. While LAP2alpha redistributes to chromosomes at very early stages of nuclear assembly providing a scaffold for chromosome decondensation, LAP2beta targets membranes to chromosomes and helps to assemble the nuclear lamina in subsequent stages of nuclear reorganization. We therefore suggest that a tightly coordinated sequential interaction of LAP2alpha LAP2beta and lamins with chromosomal components help to build up nuclear structure after mitosis and to organize chromatin structure in higher eukaryotes. In the course of the project, we have characterized the interaction of LAP2alpha with chromosomes. We have narrowed down the interaction domain in the molecule by in vitro binding studies and transient and stable expression analyses in living cells, we have identified chromosomal proteins as potential binding partners by yeast two hybrid analysis, and we have studied the cell cycle-specific regulation of this interaction by phosphorylation using site directed mutagenesis. Expression of the LAP2alpha chromosome-binding domain interfered with cell proliferation and nuclear reassembly after mitosis, confirming the important role of LAP2alpha for these processes. We also found that LAP2alpha interacts specifically with a subtype of lamins, which have recently been shown to be mutated in various heritable human diseases (laminopathies) involving muscle, fat, and nerve tissue. It is completely unknown how lamin mutations can cause the diverse disease phenotypes in humans. Our studies suggest that lamin-LAP2alpha complexes form functional units in the nucleus involved in nuclear structure organization, transcriptional regulation and cell proliferation. Lamin mutations may inhibit lamin-LAP2alpha interaction and affect specific functions of LAP2alpha, leading to disease. Thus, our results on the functions of LAP2alpha may help to explain the molecular mechanisms of disease. This hypothesis is currently tested in the laboratory.