Conserved SUN-KASH Interfaces Mediate LINC Complex-Dependent Nuclear Movement and Positioning

Abstract

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.

SUN/KASH interactions facilitate force transmission across the nuclear envelope

“…When an intermolecular disulfide bond was present, the pulling forces were transmitted effectively through the LINC complex to the coiled-coil regions (Figure 1b). However, in the cysteine mutant, the SUN/KASH interactions were not strong enough to withstand the forces and the KASH peptide and KASH lid of the SUN protein underwent significant stretching (Figure 1f) [1]. Taken together, our cellular and modeling data support the hypothesis that an intermolecular disulfide bridge is needed to stabilize SUN-KASH interactions to transmit maximal forces through LINC complexes.…”

“…We, therefore, tested the role of the −23 cysteine in nuclear anchorage in C. elegans and nuclear rearward movement in polarizing NIH 3T3 fibroblasts. In support of our model, mutating the −23 cysteine in ANC-1, Nesprin-2, or their partner cysteines in UNC-84 or SUN2, disrupted nuclear positioning [1]. Since the cysteine is not required for the SUN/KASH interaction in the in vitro pulldown assay [8], we proposed that the nuclear positioning defects might be caused by insufficient force transmission across the LINC complex (Figure 1e-f).…”

“…In our recent study [1], we tested the ability of conserved residues for SUN-KASH interactions and force transmission. To examine these processes in cells, we used C. elegans developing hypodermal tissues.…”

“…To test the function of conserved residues of SUN-KASH interfaces in live cells, our collaborators, Natalie Cain and Gant Luxton, led teams that tested predictions from the crystal structure and in vitro assays in the context of nuclear positioning in developing C. elegans hypodermal cells and polarizing NIH 3T3 fibroblasts, respectively [1]. We first showed that extending the C terminus of the UNC-83 KASH domain by a single alanine completely disrupts nuclear migration during C. elegans embryonic hypodermal development.…”

“…We developed nuclear positioning assays in adult C. elegans hypodermal syncytia as a model to test the function of longer KASH domains [25]. Interestingly, mutating the phenylalanine residue at −7 of the ANC-1 KASH domain did not cause nuclear anchorage defects [1]. This indicates that nuclear anchorage in C. elegans hyp7 syncytia might not require as high of forces to be transmitted through LINC complexes as nuclear migration does.…”