Fig. 3: HSPB3 displaces LBR from the nuclear envelope to the nucleoplasm.
From: Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor
A Immunofluorescence pictures showing the distribution of LBR1-238-GFP in HeLa cells 48 h after transfection. Co-expression of myc-HSPB1 or V5-HSPB7 does not affect LBR1-238-GFP distribution compared to control cells, expressing LBR1-238-GFP alone. Myc-HSPB3 displaces LBR1-238-GFP from the NE to the nucleoplasm. Scale bar = 10 µm. B Quantification of transfected cells from A and showing LBR1-238-GFP at the NE or displaced in the nucleoplasm. n = 3 independent experiments, ± s.e.m. P < 10−5 between control and myc-HSPB3; P = n.s. between control and myc-HSPB1 or V5-HSPB7. Total number of cells analyzed: LBR1-238-GFP (273); + myc-HSPB3 (149); + myc-HSPB1 (226); + V5-HSPB7 (129). C Immunofluorescence showing the distribution of LBR1-238-GFP in HeLa cells transfected for 48 h with vectors coding for LBR1-238-GFP alone or with a deletion mutant of HSPB3 that accumulates in the cytoplasm (HSPB3-dN). The blue arrowhead points to a cell with nuclear HSPB3-dN that displaces LBR1-238-GFP; the white arrowhead points to a cell with cytoplasmic HSPB3-dN that does not displace LBR1-238-GFP from the NE. Quantitation of LBR1-238-GFP distribution is reported. n = 3 independent experiments, ± s.e.m.; P = 0.004. The total number of cells analyzed: cytosolic;74 nuclear.84 Scale bar = 10 µm. D HeLa cells overexpressing LBR1-238-GFP alone, with mCherry or with mCherry-HSPB3 + myc-HSPB3 (at a 1:8 ratio) were subjected to fluorescence recovery after photobleaching (FRAP). Pre-bleach, bleach and post-bleach images of LBR1-238-GFP inserted at the NE and diffusely distributed in the nucleoplasm are shown. E Quantitation of the fluorescence intensity recovery after bleach of cells treated as described in D. The mean of 12–14 FRAP curves and the fitting curves are shown. sem is shown in gray. F HeLa cells co-expressing LBR1-238-GFP and myc-HSPB3 were subjected to proximity ligation assay (PLA) using antibodies specific for GFP and HSPB3. GFP-positive cells were segmented and PLA foci/cell were automatically quantified using ScanR. The average number of PLA foci in cells incubated with GFP or HSPB3 antibody (used as controls) or with both antibodies is shown. The PLA foci number was normalized for cells incubated with GFP antibody alone. n = 4 independent experiments, ± s.e.m.; total number cells analyzed/sample: 78–90, P < 0.01. Scale bar = 10 µm. G Left panel: automated segmentation of the nucleus (using DAPI staining), NE (using LMNB1), and nucleoplasm with ScanR. Scale bar = 10 µm. Right panel: automated quantification of LBR1-238-GFP NE:nucleoplasm signal ratio in cells expressing LBR1-238-GFP alone (control) or with myc-HSPB3 (+ HSPB3) is shown. n = 3, ± s.e.m.; P = 0.0018. H Schematic representation of the putative effect of HSPB3 on the LBR-tether, with potential implications on myogenic gene expression. Related to Supplementary Fig. S3.
