Abstract
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 dynamics 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 NIH 3T3 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 dynamics 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 that may represent targets for regulation. Cain et al. test the function of mutant SUN and KASH proteins in C. elegans nuclear positioning, NIH 3T3 fibroblast polarization, and simulations of LINC complexes under mechanical strain to gain mechanistic insights into how SUN-KASH interactions might be regulated to transfer forces from the cytoskeleton to the nucleus.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3086-3097.e4 |
| Journal | Current Biology |
| Volume | 28 |
| Issue number | 19 |
| DOIs | |
| State | Published - Oct 8 2018 |
Bibliographical note
Funding Information:We thank Thomas Schwartz, Ulrike Kutay, and members of the Starr, Mofrad, and Luxton laboratories for helpful discussions. This work was supported by the NIH ( R01GM073874 to D.A.S.; R21NS095109 to G.W.G.L.; and AR007612 to N.J.H.), National Science Foundation (CAREER award CBET-0955291 to M.R.K.M.), Natural Sciences and Engineering Research Council of Canada (to Z.J.), Dystonia Medical Research Foundation (to G.W.G.L.), and American Cancer Society Illinois Division ( PF-13-094-01-CGC to N.E.C.).
Funding Information:
Caenorhabditis elegans were cultured on nematode growth medium plates spotted with OP50 bacteria and maintained at 15°C or room temperature (approximately 22°C). N2 was used as the wild-type control strain [ 49 ]. Some strains were provided by the Caenorhabditis Genetics Center, funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). Only healthy animals that were not used in previous procedures and were naive to testing were used. See the Key Resources Table for strain list.
Funding Information:
We thank Thomas Schwartz, Ulrike Kutay, and members of the Starr, Mofrad, and Luxton laboratories for helpful discussions. This work was supported by the NIH (R01GM073874 to D.A.S.; R21NS095109 to G.W.G.L.; and AR007612 to N.J.H.), National Science Foundation (CAREER award CBET-0955291 to M.R.K.M.), Natural Sciences and Engineering Research Council of Canada (to Z.J.), Dystonia Medical Research Foundation (to G.W.G.L.), and American Cancer Society Illinois Division (PF-13-094-01-CGC to N.E.C.).
Publisher Copyright:
© 2018 Elsevier Ltd
Keywords
- KASH proteins
- LINC complex
- Nesprin
- SUN proteins
- nuclear envelope
- nuclear positioning