title: Dynamic interaction between actin and nesprin2 maintain the cell nucleus in a prestressed state.
abstract: Mechanical coupling between the nucleus and the cytoskeleton is indispensable for direct force transduction from the extra cellular matrix (ECM) to the chromatin. Although this physical coupling has been shown to be crucial for nuclear positioning and its function, the quantification of nuclear-cytoskeleton interaction has been lacking. In this paper, using various quantitative fluorescence spectroscopy techniques, we investigate the nature of this connection. High-resolution 3D imaging shows that nesprin2G forms short linear structures along actin stress fibers (ASFs) in the apical region of the nucleus. Fluorescence recovery after photobleaching (FRAP) revealed that the alignment of nesprin2G becomes heterogeneous when cell shape is engineered from elongated rectangular shape to square using micropatterned substrates. Further, fluorescence cross-correlation spectroscopy (FCCS) revealed that actin interacts transiently with outer nuclear membrane protein nesprin2G with a time scale of 12 ms. In addition, fluorescence resonance energy transfer (FRET) experiments show that the apical ASFs and nesprin2G are in close physical proximity. This interaction is spatially heterogeneous with high FRET along the ASFs. Lastly, we show that the disruption of actin to nuclear connection by over-expression of Dominant Negative Klarsicht, ANC-1, Syne Homology (DNKASH) leads to an increase in nuclear height. These results not only reveal the characteristics of actin-nesprin2G interaction and its significance in regulating nuclear morphology, but also validate the utility of quantitative fluorescence techniques in deciphering physical connections that are essential for mechanotransduction.
authors.affiliation: Mechanobiology Institute and Department of Biological Sciences, National University of Singapore, Singapore 117411, Singapore. Current address: Department of Internal Medicine, Yale Cardiovascular Research Center, Cardiovascular Medicine, Yale School of Medicine, Yale University, New Haven, CT 06511, USA.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Kumar
authors.forename: G V
authors.initials: GV
authors.lastname: Shivashankar
mesh_terms: D008841:Actin Cytoskeleton; D000199:Actins; D002467:Cell Nucleus; D003599:Cytoskeleton; D040542:Mechanotransduction, Cellular; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D022502:Stress Fibers
chemical_list: D000199:Actins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins