Functional analysis of IMPs
The aim of our studies is to gain insight into interactions of nuclear envelope proteins in vivo in single cells and tissues. We are analysing in vivo and in vitro the functions of the following integral membrane proteins of the inner nuclear membrane. These are the lamin B receptor (LBR) of Drosophila (Wagner et al., 2004b), Xenopus (Gajewski et al., 1999), and zebrafish, and the family of lamina associated polypeptide 2 (LAP2) of zebrafish (Prüfert et al., 2004a; Schoft et al., 2003) and Xenopus (Lang et al., 2003; Brown et al., 2002; Del-Pino et al., 2002; Lang et al., 1999).
LEM-domain proteins of Drosophila melanogaster
LEM domain proteins present a family of non-related inner nuclear membrane and soluble proteins, including LAP2, emerin, and MAN1.
They are involved in several cellular processes including membrane-chromatin attachment via interaction with BAF, a conserved protein, cell division and nuclear reconstitution, chromosome segregation and the regulation of transcription. Despite their binding to BAF, LEM domain proteins are targeted to the inner nuclear membrane by binding to lamins, which present the major structural elements of the lamina.
Compared to the complexity of the inner nuclear membrane in higher eukaryotic cells (Fig. A), the nuclear envelope of Drosophila (Fig. B) seems to be more simple and therefore particularly suitable to understand fundamental questions concerning the structure, organization, interactions and basic functions of inner nuclear membrane proteins (Wagner et al., 2004a).
Lamins and nuclear membrane growth
Lamins are type V intermediate filament proteins, they are essential and major structural components of the metazoan nuclear envelope. Two sequences have been previously identified in lamins that enable their targeting to the inner nuclear membrane. The first sequence is the C-terminal CxxM motif (C = cysteine, x = any amino acid, M = methionine) of B-type lamins and vertebrate lamin A, and the second motif is the N-terminal GNAEGR hexapeptide of lamin C2 that is expressed in mammalian spermatocytes. The CxxM motif is posttranslationally modified by the attachment of a farnesyl moiety to the cysteine and the aminoterminal glycine of the GNAEGR sequence is myristoylated. Both modifications confer a higher hydrophobicity of the C-terminal or N-terminal end of the lamin molecule and are essential for the correct targeting to the inner nuclear membrane. By the overexpression of wild type and mutant lamins in cultured vertebrate cells we have identified additional functions of these targeting motifs. The electron microscopical analysis of cells overexpressing lamins revealed that both motifs promote the growth of the nuclear membrane. Nuclear membrane growth was visible by the formation of highly folded nuclear envelopes (typical for the CxxM motif), nuclear envelope protuberances (GNAEGR sequence) and the formation of intranuclear membranes (both motifs).
Currently we are investigating whether or not lamins are the main regulators of nuclear envelope growth (Prüfert et al., 2004b).
As methods we are using immunofluorescence microscopy, GFP-fusion proteins, transfections, and co-immunoprecipitations. One main method is the down-regulation of mRNAs and the encoded proteins by the microinjection of antisense morpholino modified oligonucleotides (zebrafish embryos), and by RNA interference (RNAi) in Drosophila cultured cells and embryos. As an important tool for all investigations we have generated antibodies against all proteins of interes
Other Research projects
Maurer's clefts in the cytoplasm of erythrocytes infected by Plasmodium falciparum
This project is a collaboration with the laboratory of Michael Lanzer (Hygiene Institut, Abt. Parasitologie, Universitätsklinikum Heidelberg).
Maurer's clefts are single-membrane-limited structures in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum (Przyborski et al., 2003; Wickert et al., 2003a; Wickert et al., 2003b). The currently accepted model suggests that Maurer's clefts act as an intermediate compartment in protein transport processes from the parasite across the host cell cytoplasm to the erythrocyte surface, by receiving and delivering protein cargo packed in vesicles. This model is mainly based on two observations. Firstly, single section electron micrographs have shown, within the cytoplasm of infected erythrocytes, stacks of long slender membranes in close vicinity to round membrane profiles considered to be vesicles. Secondly, proteins that are transported from the parasite to the erythrocyte surface as well as proteins facilitating the budding of vesicles have been found in association with Maurer's clefts (Wickert et al., 2004). To get a better understanding of the morphology, dimensions and origin of the Maurer's clefts, we performed immunolocalisations and made three-dimensional reconstructions of serial ultrathin sections covering segments of P. falciparum-infected erythrocytes of more than 1 µm thickness. Our results indicate that Maurer's clefts are often complex membrane networks heterogeneous in structure and size that can bridge most of the distance between the parasite and the surface of the erythrocyte. We have not seen clearly discernible isolated vesicles in the analysed erythrocyte segments suggesting that the current view of how proteins are transported within the Plasmodium-infected erythrocyte may need reconsideration.