Transfection-mediated re-expression of GFPCK17 WT, but not GFPCK17NLS, restored the nuclear dimensions in keratinocytes in primary culture (Fig.?2H). cells. Numbers above or below each box-and-whisker plot designate the means.e.m. In the box-and-whisker plots, the box represents the 25C75th percentiles, and the median is usually indicated. The whiskers show the minimum and maximum values. WT and KO HeLa cells, immunostained with antibodies against nuclear lamina-associated proteins (green). Scale bars: 10?m. We hypothesized that K-Ras(G12C) inhibitor 12 differences in nuclear shape and surface area may entail changes in inner nuclear membrane (INM) K-Ras(G12C) inhibitor 12 proteins, including lamins A, C and B1/B2 and LEM-domain-containing proteins [LAP2, LAP2 and LAP2 (all encoded by WT and KO cells revealed significant increases in the steady-state levels of these proteins, except for LAP2, in cells lacking K17 (Fig.?S1E,F). The observed increases (15C50%) were proportional to the increase in surface area (10C20%) in KO nuclei relative to control (Fig.?1BCD). Consistent with these findings, indirect immunofluorescence indicated a Rabbit polyclonal to Prohibitin significant increase in signal intensity for each analyzed protein in KO cells compared to WT (Fig.?1E; quantification in Fig.?S1G). Again, LAP2 behaved differently (see below). Impact of K17 on nuclear morphology requires an intact NLS We next assessed whether nuclear-localized K17 impacts nuclear dimensions. We transiently transfected KO cells (HeLa and A431) with either GFP (control), GFPCK17 WT or GFPCK17NLS (in which K399A and K400A mutations impair NLS function; Escobar-Hoyos et al., 2015; Hobbs et al., 2015) (Fig.?2A) and assessed the size and shape of nuclei from 2D and 3D images. Expression of GFPCK17 WT, but not GFPCK17NLS, restored normal dimensions to the nucleus, including decreased area (2D; Fig.?2B,C), increased sphericity (3D; Fig.?S2A,B), and decreased surface area (3D; Fig.?2D,E). Expression of GFPCK17 NES, in which L194A, L197A and L199A mutations impair the nuclear export sequence (NES) function of K17 (thus favoring nuclear retention; Escobar-Hoyos et al., 2015) (Fig.?S2C), also rescues nuclear shape defects in KO A431 keratinocytes. Expression of GFPCK14 WT (Fig.?S2D) is ineffective in doing so, suggesting specificity for K17. Next, we used proximity ligation assays (PLAs) with two different host-species antibodies against K17 [allows the visualization of the K17 nuclear pool without Leptomycin B treatment (Hobbs et al., 2015)] in HeLa KO cells expressing GFPCK17 WT or GFPCK17NLS. Both constructs gave rise to well-developed filamentous arrays in the cytoplasm of transfected cells (Fig.?2A). However, GFPCK17NLS gave rise K-Ras(G12C) inhibitor 12 to a markedly reduced nuclear signal relative to GFPCK17 WT (Fig.?S2E; quantification in Fig.?S2F). These data suggest that, in transfected human A431 and HeLa cells, a nuclear pool of K17 is responsible for the impact on nuclear morphology, independently of the property of fostering a dense network of keratin filaments in the perinuclear cytoplasm. Open in a separate windows Fig. 2. Impact of K17 on nuclear morphology requires an intact NLS. (A) Confocal micrograph MIPs of HeLa KO cells transiently transfected (48?h) with GFP, GFPCK17 WT or GFPCK17NLS (green). Nuclei are stained with DAPI (blue). Scale bars: 10?m. (B,C) Quantified area (2-dimensional; 2D) of nuclei from (B) HeLa KO and K-Ras(G12C) inhibitor 12 (C) A431 KO cells expressing GFP, GFPCK17 WT or GFPCK17NLS. (D,E) Quantified surface area (3-dimensional; 3D) of nuclei from (D) HeLa KO and (E) A431 KO cells expressing GFP, GFPCK17 WT or GFPCK17NLS. (F) Confocal MIPs for K17 (red) in MEKs from and mice. Nuclei are stained with DAPI (blue). Scale bars: 10?m. (G) Quantified area of nuclei from primary MEKs from mice. (H) Quantified area of nuclei from MEKs untransfected or transfected with GFP-K17 WT. For BCE, G, H, numbers above or below each box-and-whisker plot represent the means.e.m. In the box-and-whisker plots, the box represents the 25C75th.