This research will be done primarily in Chile at the Catholic university of Chile in collaboration with Alfredo Celedon, as an extension of NIH Grant No. R01GM08420401, 09/30/08 - 08/31/12." The nuclear envelope has an internal shell called nuclear lamina, a thin meshwork of intermediate filaments composed of A- and B-type lamins. Mutations scattered along Lmna, which encodes A-type lamins, have been associated with a broad range of human diseases, collectively called laminopathies. In mammalian cells, the recent characterization of the LINC complex, an evolutionary-conserved protein complex that interacts both with the nuclear lamina and the cytoskeleton of mammalian cells suggests that nucleus and cytoskeleton are intimately connected. Our main hypothesis is that the nuclear envelope is mechanically connected to the actin filament network and the MTOC directly through specific linker proteins, including emerin and the LINC complexes, and that these connections are disrupted in laminopathies. Recent results obtained in the PI's lab suggest that the depletion of lamin A/C, as well as the specific rupture of the LINC complexes negatively affects both cell motility and intracellular mechanics. Moreover, results from a shear flow assay combined with immuno-fluorescence show that the distance between MTOC and nuclear envelope is greatly increased following emerin depletion and that this loosening of the MTOC correlates with the inability of emerin- deficient cells and lamin A/C-deficient cells to polarize in the flow direction. However, a direct demonstration that the actin cytoskeleton and the MTOC are actually both connected to the nuclear envelope and that these physical connections are mediated by the LINC complexes and emerin, respectively, is lacking. Here, we propose to use magnetic tweezers and magnetic nanorods to demonstrate the existence of these molecular connections, decipher the role of emerin and the LINC complexes in these connections and determine the mechanical strength of the links between the nucleus and both the actin filament network and the MTOC. We also use magnetic tweezers to measure for the first time the micromechanical properties of nucleus in live cells.
Mutations scattered along the Lmna gene, which encodes A-type lamins, are associated to a broad range of human diseases, collectively called laminopathies. Using a new, single cell magnetic tweezers strategy, the proposed research may help establish a biophysical basis for the wide variety of disease phenotypes associated to human laminopathies.
|Castillo, Matias; Ebensperger, Roberto; Wirtz, Denis et al. (2014) Local mechanical response of cells to the controlled rotation of magnetic nanorods. J Biomed Mater Res B Appl Biomater 102:1779-85|
|Celedon, Alfredo; Hale, Christopher M; Wirtz, Denis (2011) Magnetic manipulation of nanorods in the nucleus of living cells. Biophys J 101:1880-6|