FIGURE 3:

Constricted migration of myoblasts causes nuclear damage. (A) Confocal 3D sections of C2C12 myoblast nuclei exiting large 8 μm– and constrictive 3 μm–diameter pores. Yellow lines indicate positions of orthogonal sections, and dashed pink lines indicate pore diameters and edges of the membranes. Scale bar = 10 μm. (B) Number of migrated C2C12 myoblasts on the bottom of a transwell membrane, normalized to number of nonmigrated cells on top of the membrane. Significantly fewer cells migrate through 3-μm than 8-μm pores. N = 3 samples. (C) Representative images of nuclear blebs following constricted migration through 3-μm pores. Blebs (white arrows) are lamina protrusions deficient in lamin-B and enriched in lamin-A/C. Scale bar = 10 μm. (D) The frequency of nuclear blebbing is low before migration and after migration through large 8-µm pores, but it increases significantly after migration through 3-µm pores. N = 219–598 cells. (E) Nonmigrated and migrated C2C12 myoblast nuclei are shown on the top and bottom, respectively, of transwell membranes. Nuclei exhibit excess DNA damage, as indicated by foci of γH2AX, after migration. Scale bar (main) = 50 μm. Scale bar (expanded) = 10 μm. (F) Number of γH2AX foci per cell, normalized to number of foci per nonmigrated (Top) cell. Migration causes excess DNA damage, with greater damage accrued during migration through constricting vs. large pores. N = 257–358 cells. (G) Superresolution imaging of a C2C12 myoblast after constricted migration shows γH2AX foci are not enriched at sites of nuclear blebs. Multiple blebs are sometimes visible and tend to occur at the high curvature end(s) of a nucleus. Scale bar = 10 μm. (H) C2C12 myoblasts show nuclear bleb formation and excess DNA damage foci after three rounds of constricted migration. DNA damage is more severe on the bottom but is also evident on the top after successive migrations. Nuclear blebs are observed on the bottom only. Scale bar = 10 μm. (I) Numbers of γH2AX foci per C2C12 cell before (Top) and after (Bot.) each of three rounds of migration (TW1 = round 1, etc.). A single round of migration—especially through constricting 3-μm pores—leads to excess DNA damage; additional rounds of migration do not further increase the damage level on transwell Bot. However, the level of DNA damage on transwell Top increases with serial migration, indicating that damage is not fully repaired during the recovery period between migrations. N = 126–203 cells. * indicates significant (p < 0.05) difference from nonmigrated or † from 8 μm.