P4: Analysis of nucleocytoplasmic transport in muscle disorders caused by mutations in lamins A/C
Uwe Vinkemeier
Summary
This research proposal is based on the hypothesis that specific mutations in lamins A/C cause partial lipodystrophy syndromes due to the impaired nucleocytoplasmic transport of cytokine responsive transcription factors. Because of the striking similarities between myostatin deficient animals and patients affected by certain lamin mutations, our analysis will concentrate on the myostatin/SMAD signaling system. We will adopt the established analyses of nucleocytoplasmic transport for myoblasts and muscle fibers. Subsequently, we will analyze the translocation of signal transducers in healthy and diseased cells. Besides histocytochemistry, we will use confocal fluorescence microscopy, the microinjection of recombinant proteins, and in-vitro-transport assays with permeabilized cells, and the transfection of cells with recombinant DNA. For these experiments, we have access to an immortalized muscle cell line, to material from normal control individuals, and from patients with molecularly characterized laminopathies. The relevant cDNAs (lamins, SMADs, STATs) are available.
Co-localization of laminA/C (red) and phosphorylated SMAD2 (green) in cryosections of frozen biopsies as revealed by 2-photon confocal microscopy. A, control. B, LMNA p. R482Q. C, 3-D rendering of the cell nucleus and the position of phosphorylated SMAD2 as observed in B (done with the program Amira 3.0, http://www.zib.de)
