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. Author manuscript; available in PMC: 2018 Jul 10.
Published in final edited form as: Curr Biol. 2017 Jun 15;27(13):2014–2022.e6. doi: 10.1016/j.cub.2017.05.066

Figure 3. Recombinant rootletin filaments recruit multiple isoforms of Nesprin1.

Figure 3

A. Nesprin1 and Myc colabelling of NIH3t3 cells transfected with Myc-root. Note the NE aggregation and centrosome recruitment of Nesprin1 by Myc-root filaments. See also Figure S2A,B. B, C, D. Same colabelling experiment with antibodies against Nesprin2 (B), Sun1 (C) or Sun2 (D) at the NE. Note that whereas the NE localization of Nesprin2 and Sun1 is not affected by Myc-root filaments, Sun2 aggregates into NE Myc-root/Nesprin1 filaments. See also Figures S2D–G. E. Model of Sun2/Nesprin1 LINC complexes that dock perinuclear rootletin filaments at the nuclear surface of rods and of NIH3t3 cells transfected with Myc-root. Red dots: KASH domains. F. Nesprin1 and Sun2 colabelling of the ONL of C57/Bl6 retinas. Note the discrete colocalization of Sun2 with Nesprin1 filaments (arrows). G. Myc and Nesprin1 coimmunolabeling of NIH3t3 cells transfected either with control SiHPRT (top) or with SiNes1 (bottom) SiRNAs for 36h and subsequently with Myc-root for 12h. Note the lack of rootletin filaments at the nuclear surface in the absence of Nesprin1 expression and their accumulation in the cytoplasm. See also Figure S2H. H, I: Basal views of NIH3t3 cotransfected with Myc-root and either EGFP-Nes1α (H) or EGFP-Nes1αΔKASH (I). Note that whereas EGFP-Nes1α and rootletin filaments essentially colocalize at the NE, EGFP-Nes1αΔKASH colocalizes with rootletin filaments in the cytoplasm. J: Volume to volume partition into pellets (P) and supernatant (S) of Myc-Root deletion mutants extracted with RIPA buffer from transfected NIH3t3 cells. Bottom: depiction of the coiled-coil domains of Myc-root (C1 to C6) and of each deletion mutant. K: Nesprin1 and rabbit immunoglobulins (IgG, used as a negative control) immunoprecipitations (IP) of RIPA lysates from NIH3t3 cells cotransfected with EGFP-Nes1αΔKASH and indicated Myc-Root deletion constructs. Immunoprecipitates were immunoblotted (IB) either with Myc (top panel) or with EGFP (bottom panel) antibodies to detect deletion mutants of Myc-Root and EGFP-Nes1αΔKASH, respectively. See also Figure S2I.