Nesprins form a novel class of nuclear envelope-anchored spectrin-repeat proteins. We show that a direct association of their highly conserved C-terminal luminal domain with the inner nuclear membrane protein Sun1 mediates their nuclear envelope localisation. In Nesprin-1 and Nesprin-2 the conserved C-terminal amino acids PPPX are essential for the interaction with a C-terminal region in Sun1. In fact, Sun1 is required for the proper nuclear envelope localisation of Nesprin-2 as shown using dominant-negative mutants and by knockdown of Sun1 expression. Sun1 itself does not require functional A-type lamins for its localisation at the inner nuclear membrane in mammalian cells. Our findings propose a conserved nuclear anchorage mechanism between Caenorhabditis elegans and mammals and suggest a model in which Sun1 serves as a `structural bridge' connecting the nuclear interior with the actin cytoskeleton.

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns.

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NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.