In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, orienting the centrosome toward the leading edge. The nucleus engages moving dorsal actin cables through linear arrays of nesprin-2G and SUN2 called TAN lines. In this study, Saunders et al. report that the nuclear envelope–localized AAA+ ATPase torsinA and its activator, LAP1, are required for TAN line assembly and retrograde dorsal actin cable flow.
Summary Many nuclear positioning events involve linker of nucleoskeleton and cytoskeleton (LINC) complexes, which transmit forces generated by the cytoskeleton across the nuclear envelope. LINC complexes are formed by trans-luminal interactions between inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, but how these interactions are regulated is poorly understood. We combine in vivo C. elegans genetics, in vitro wounded fibroblast polarization, and in silico molecular dynamic simulations to elucidate mechanisms of LINC complexes. The extension of the KASH domain by a single alanine residue or the mutation of the conserved tyrosine at −7 completely blocked the nuclear migration function of C. elegans UNC-83. Analogous mutations at −7 of mouse nesprin-2 disrupted rearward nuclear movements in NIH3T3 cells, but did not disrupt ANC-1 in nuclear anchorage. Furthermore, conserved cysteines predicted to form a disulfide bond between SUN and KASH proteins are important for the function of certain LINC complexes and might promote a developmental switch between nuclear migration and nuclear anchorage. Mutations of conserved cysteines in SUN or KASH disrupted ANC-1 dependent nuclear anchorage in C. elegans and Nesprin-2G dependent nuclear movements in polarizing fibroblasts. However, the SUN cysteine mutation did not disrupt nuclear migration. Moreover, molecular dynamic simulations showed that a disulfide bond is necessary for the maximal transmission of cytoskeleton-generated forces by LINC complexes in silico. Thus, we have demonstrated functions for SUN-KASH binding interfaces, including a predicted intermolecular disulfide bond, as mechanistic determinants of nuclear positioning and may represent targets for regulation.
Desmosomes are intercellular junctions found in epithelia and cardiac muscle that resist mechanical stress by linking the intermediate filaments of neighboring cells. Disruption of the desmosome-intermediate filament complex (DIFC) can cause a loss of tissue integrity as well as abnormalities in tissue differentiation, leading to a variety of diseases. Understanding these diseases will require expanding our knowledge of the relationship between structure and function in desmosomes. Of particular interest is the arrangement, or order, of the desmosomal cadherins. Cadherins are calciumdependent, transmembrane adhesion proteins. In a single desmosome, many copies of these proteins engage in trans-binding with the cadherins of neighboring cells to form the adhesive interface. Large macromolecular complexes and membrane-associated proteins are difficult to study using traditional structural techniques. To overcome this, we pioneered excitation-resolved fluorescence polarization microscopy (FPM) to study
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