Figure 3. Confinement induces elevated nuclear influx that correlates with nuclear blebbing. (a) Time-dependent image sequence of PA-GFP influx into the nucleus from the posterior (top image sequence) or anterior (middle image sequence) compartment, or efflux from the nucleus (bottom image sequence) upon UV excitation in confinement. Red “X” symbols represent points of excitation. Scale bars, 20 µm. (b) Quantification of transport of PA-GFP into the nucleus of HT-1080 cells on 2D or inside confined channels following UV illumination in the anterior or posterior compartments, reported as the t1/2 required for the signal (PA-GFP) to reach maximum intensity in the nucleus (n ≥ 27 cells from three or more independent experiments). (c) t1/2 of PA-GFP nuclear influx from the cell posterior for HT-1080 cells inside 10-µm- and 50-µm-wide channels with a fixed height of 3 µm (n ≥ 24 cells from three independent experiments). (d) Schematic (left) and quantification (right) of t1/2 of PA-GFP nuclear influx for confined HT-1080 cells exposed to a hypotonic shock (165 mOsm) at their leading edge (n ≥ 16 cells from three independent experiments). (e) Schematic (left) and quantification (right) of t1/2 of PA-GFP nuclear influx for confined HT-1080 cells exposed to a hypertonic shock (620 mOsm) at their trailing edge (n ≥ 27 cells from five independent experiments). (f) t1/2 of PA-GFP nuclear influx for confined cells with and without nuclear blebs, as well as Y27632-treated (10 µM) HT-1080 cells (n ≥ 15 cells from four or more independent experiments). (g) t1/2 of PA-GFP nuclear influx for control versus actin cortex–ablated HT-1080 cells. Simultaneous photoablation and photoactivation occured at the cell trailing edge (n ≥ 26 cells from four independent experiments). (h) Time-dependent image sequence of simultaneous actin cortex ablation (top) and PA-GFP influx into the nucleus (bottom) at the cell posterior. Yellow arrow indicates ablation. Blue arrow indicates photoactivation. White arrows indicate bleb formation as a result of ablation. Scale bar, 10 µm. (i) Nuclear position of scramble control versus SUN1-KD HT-1080 cells (n ≥ 33 cells from three independent experiments). (j) t1/2 of PA-GFP nuclear influx for confined scramble control cells and SUN1-knockdown HT-1080 cells (n ≥ 31 cells from three independent experiments). Values represent mean ± SD. **, P < 0.01 relative to 2D cytoplasmic, confinement anterior; $$, P < 0.01 relative to 10 µm width; ##, P < 0.01 relative to confinement nuclear bleb-bearing; §, P < 0.05; §§, P < 0.01 relative to scramble/isotonic/control.
Figure 3.

Confinement induces elevated nuclear influx that correlates with nuclear blebbing. (a) Time-dependent image sequence of PA-GFP influx into the nucleus from the posterior (top image sequence) or anterior (middle image sequence) compartment, or efflux from the nucleus (bottom image sequence) upon UV excitation in confinement. Red “X” symbols represent points of excitation. Scale bars, 20 µm. (b) Quantification of transport of PA-GFP into the nucleus of HT-1080 cells on 2D or inside confined channels following UV illumination in the anterior or posterior compartments, reported as the t1/2 required for the signal (PA-GFP) to reach maximum intensity in the nucleus (n ≥ 27 cells from three or more independent experiments). (c) t1/2 of PA-GFP nuclear influx from the cell posterior for HT-1080 cells inside 10-µm- and 50-µm-wide channels with a fixed height of 3 µm (n ≥ 24 cells from three independent experiments). (d) Schematic (left) and quantification (right) of t1/2 of PA-GFP nuclear influx for confined HT-1080 cells exposed to a hypotonic shock (165 mOsm) at their leading edge (n ≥ 16 cells from three independent experiments). (e) Schematic (left) and quantification (right) of t1/2 of PA-GFP nuclear influx for confined HT-1080 cells exposed to a hypertonic shock (620 mOsm) at their trailing edge (n ≥ 27 cells from five independent experiments). (f) t1/2 of PA-GFP nuclear influx for confined cells with and without nuclear blebs, as well as Y27632-treated (10 µM) HT-1080 cells (n ≥ 15 cells from four or more independent experiments). (g) t1/2 of PA-GFP nuclear influx for control versus actin cortex–ablated HT-1080 cells. Simultaneous photoablation and photoactivation occured at the cell trailing edge (n ≥ 26 cells from four independent experiments). (h) Time-dependent image sequence of simultaneous actin cortex ablation (top) and PA-GFP influx into the nucleus (bottom) at the cell posterior. Yellow arrow indicates ablation. Blue arrow indicates photoactivation. White arrows indicate bleb formation as a result of ablation. Scale bar, 10 µm. (i) Nuclear position of scramble control versus SUN1-KD HT-1080 cells (n ≥ 33 cells from three independent experiments). (j) t1/2 of PA-GFP nuclear influx for confined scramble control cells and SUN1-knockdown HT-1080 cells (n ≥ 31 cells from three independent experiments). Values represent mean ± SD. **, P < 0.01 relative to 2D cytoplasmic, confinement anterior; $$, P < 0.01 relative to 10 µm width; ##, P < 0.01 relative to confinement nuclear bleb-bearing; §, P < 0.05; §§, P < 0.01 relative to scramble/isotonic/control.

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