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Parvines and novel Proteins of the a-Actinin/Spectrin-Superfamily

Actin Cell Biology Group - Dr. Elena Korenbaum

Telephone: +49-511-532-8581

e-mail: Elena Korenbaum

Research focus

Our group is interested in the cell biology of the actin cytoskeleton. Currently our research is focused on (1) a novel role of the actin cytoskeleton in controlling the positioning and movement of the nucleus and other large organelles and (2) F-actin binding proteins mediating cell-cell and cell-matrix adhesion. A multifaceted approach is used to analyze intracellular interactions in vivo. We combine biochemical and structural in vitro studies with advanced microscopy based techniques, including digitally enhanced 5D-fluorescence microscopy, Total Internal Reflection Fluorescence Microscopy (TIRFM), Focal Correlation Spectroscopy, confocal and multi-photon confocal microscopy.

Projects



Actin-binding proteins at the nuclear envelope: role of nuance and enaptin in integrating nucleo- and cytoskeleton

We have identified Nuance and Enaptin, large homologous actin-binding proteins of the nuclear envelope. These highly modular proteins with microscopy-image1predicted sizes of about 800 and 1000 kDa respectively, are anchored in the nuclear envelope with their C-terminal transmembrane domain, while the N-terminal actin binding domains face the cyto- or nucleoplasm. The direct physical connection between nucleus and microfilament system implies a role for the actin cytoskeleton in controlling nuclear physiology. Furthermore, we believe that Nuance and Enaptin are candidate genes responsible for the dilated cardiomyopathy syndromes mapped to the gene loci 14q23-q24 (Nuance) and 6q23-q24 (Enaptin).

The project aims at understanding the role of Nuance and Enaptin in integrating cytoskeletal filaments and nuclear scaffolds. Specifically, we are interested in (1) elucidating the role of Nuance and Enaptin in the mitosis-dependent breakdown and reassembly of the nuclear envelope; (2) their potential involvement in the positioning of nucleus, Golgi and mitochondria; (3) functional analysis of Nuance and Enaptin in various cellular systems including differentiated epithelial, muscle and neuronal cells. For this we plan to expand our panel of antibodies against various domains of Nuance and Enaptin and to perform functional analysis by silencing their genes using sequence-specific RNA interference (RNAi). The function of the most prominent modules of Nuance and Enaptin, such as actin-binding domain, spectrin repeats, and the transmembrane domain, will be analyzed in biochemical and cell biological assays.

Parvins and their role in the molecular architecture of cell-matrix adhesions.

Cell locomotion is a complex process essential for embryonic development, differentiation, inflammatory responses and wound healing. It involves continuous formation, maturation and disassembly of the dynamic multiprotein complexes at the cell-matrix contact points. We recently identified a family of microscopy-image2proteins associated with focal adhesions, which we named parvins. alpha-, beta- and the more distantly related gamma-parvin are actin-binding proteins with a molecular mass of 40-43 kDa. Parvins are also components of fibrillar adhesions, elongated structures that segregate from the focal contacts upon fibronectin fibrillogenesis. Parvins are known to play a role in cell spreading, contraction and proliferation.

The project is focused on elucidating a role of parvins in spatial and functional organization of various types of cell-matrix adhesions. The program includes (1) crystallization and structural characterization of alpha- beta- and gamma-parvin, as well as co-crystallization of parvins with their interaction partners; (2) analysis of multiprotein complex formation using analytical centrifugation (in vitro) and FCS (in vivo) to relay structural analysis to intracellular interactions; (3) analysis of the dynamics of parvins and the order of assembly of parvin-containing complexes in focal and fibrillar adhesions of stationary and migrating cells, using confocal two-photon and TIRF microscopy; (4) cellular function of gamma-parvin.

Methods:

  • State-of-the-art methods in molecular biology and protein biochemistry are used.

  • 2D blue native gel electrophoresis and IPs in conjugation with MS are used for the identification of protein-protein interaction

  • X-ray crystallography is used for structural studies

  • RNAi gene silencing in tissue culture cells is used to dissect the function of individual gene products in vivo.

  • Advanced microscopy approaches are used to follow the activity of proteins in vitro and in vivo. The microspopy-based approaches include: multiphoton laser scanning microscopy, super-resolution 4Pi-microscopy, TIRF microscopy, FCS, FRET and FRAP.

Key references:

  • Padmakumar V.C., Braune S., Abraham S., Noegel A., Tunggal B., Karakesisoglou I., Korenbaum E.
    Enaptin, a giant actin-binding protein, is an element of the nuclear membrane and the actin cytoskeleton.
    2004. Exp. Cell Res. 295, 330-339.

  • Korenbaum E, Rivero F. Calponin homology domains at a glance. 2002. J Cell Sci. 115, 3543-3545.

  • Zhen YY, Libotte T, Munck M, Noegel AA, Korenbaum E. 2002. NUANCE, a giant protein connecting the nucleus and actin cytoskeleton.
    J Cell Sci. 115, 3207-3222.

  • Korenbaum E, T. Olski, A. Noegel. Genomic organization and expression profile of the parvin family of focal adhesion proteins in mice and humans. 2001. Gene 279, 69-79.

  • Olski T.M., Noegel A.A., Korenbaum E. 2001. Parvin, a 42 kDa focal adhesion protein, related to the alpha-actinin superfamily. J. Cell Sci. 114 (3), 525-538.