Figure 5.
Figure 5. BAF is required to functionally repair the ruptured NE via recruitment of multiple LEM-domain proteins. (A) Representative images of BJ-5ta human fibroblast cells expressing GFP-NLS transfected with siControl, siBAF, or a combination of siLEMD2, siEmerin, and siANKLE2 for 96 h before laser-induced NE rupture (purple arrowheads). Bar, 10 µm. (B) Quantification of the cytoplasmic/nucleoplasmic ratio of GFP-NLS following laser-induced nuclear rupture in BJ-5ta cells transfected with siControl, siBAF, siLEMD2, siChmp7, or a combination of siLEMD2, siEmerin, and siANKLE2 for 96 h. The graph represents mean values ± SEM (n = 17, 10, 12, 12, 11 cells, respectively; **, P < 0.0001 from siControl by a mixed-effects model with Tukey’s post hoc comparison). (C) Quantification of cytoplasmic/nucleoplasmic ratio of GFP-NLS following laser-induced nuclear rupture in BJ-5ta cells transfected with siControl, siEmerin, siANKLE2, siLEMD2, or a combination of siLEMD2, siEmerin, and siANKLE2 for 96 h. The graph represents mean values ± SEM (n = 17, 14, 16, 12, 11 cells, respectively; **, P < 0.0001 from siControl by a mixed-effects model with Tukey’s post hoc comparison). (D) A model for NE rupture repair. In control cells (siControl), cytosolic BAF is recruited to the nuclear rupture via DNA binding, followed by the subsequent mobilization of LEM-domain proteins, ESCRT-III, and membranes to the rupture. Loss of BAF (siBAF) results in failure to functionally recruit ESCRT-III, LEM-domain proteins, and membranes, resulting in a failure to repair the NE rupture. The combinatorial loss of NE- and ER-resident LEM-domain proteins (siLEMD2 + siEmerin + siAnkle2), also results in a failure to actively repair the NE rupture. The loss of either LEMD2 or CHMP7 (siLEMD2 or siCHMP7) does not significantly impair NE rupture repair, suggesting that the ESCRT-III complex may facilitate, but is not required for, rupture resealing.

BAF is required to functionally repair the ruptured NE via recruitment of multiple LEM-domain proteins. (A) Representative images of BJ-5ta human fibroblast cells expressing GFP-NLS transfected with siControl, siBAF, or a combination of siLEMD2, siEmerin, and siANKLE2 for 96 h before laser-induced NE rupture (purple arrowheads). Bar, 10 µm. (B) Quantification of the cytoplasmic/nucleoplasmic ratio of GFP-NLS following laser-induced nuclear rupture in BJ-5ta cells transfected with siControl, siBAF, siLEMD2, siChmp7, or a combination of siLEMD2, siEmerin, and siANKLE2 for 96 h. The graph represents mean values ± SEM (n = 17, 10, 12, 12, 11 cells, respectively; **, P < 0.0001 from siControl by a mixed-effects model with Tukey’s post hoc comparison). (C) Quantification of cytoplasmic/nucleoplasmic ratio of GFP-NLS following laser-induced nuclear rupture in BJ-5ta cells transfected with siControl, siEmerin, siANKLE2, siLEMD2, or a combination of siLEMD2, siEmerin, and siANKLE2 for 96 h. The graph represents mean values ± SEM (n = 17, 14, 16, 12, 11 cells, respectively; **, P < 0.0001 from siControl by a mixed-effects model with Tukey’s post hoc comparison). (D) A model for NE rupture repair. In control cells (siControl), cytosolic BAF is recruited to the nuclear rupture via DNA binding, followed by the subsequent mobilization of LEM-domain proteins, ESCRT-III, and membranes to the rupture. Loss of BAF (siBAF) results in failure to functionally recruit ESCRT-III, LEM-domain proteins, and membranes, resulting in a failure to repair the NE rupture. The combinatorial loss of NE- and ER-resident LEM-domain proteins (siLEMD2 + siEmerin + siAnkle2), also results in a failure to actively repair the NE rupture. The loss of either LEMD2 or CHMP7 (siLEMD2 or siCHMP7) does not significantly impair NE rupture repair, suggesting that the ESCRT-III complex may facilitate, but is not required for, rupture resealing.

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