Figure 2. Confined migration leads to chromatin protrusions, nuclear fragmentation, and DNA damage.

(A) Representative image of an MDA-MB-231 cell co-expressing GFP-lamin C and H2B-RFP developing chromatin protrusion (arrowhead) during migration through a microfluidic constriction. Scale bar: 5 µm. (B) HT1080 cell in a collagen matrix (2.5 mg/mL + GM6001) with chromatin protrusion (arrowheads) across the nuclear lamina, stained for lamin A/C (green), DNA (red), and F-actin (turquoise). Scale bars: 10 µm, 2 µm (bottom inset). (C) Percentage of cells with chromatin protrusions as a function of collagen matrix pore size. 2D, unconfined migration on glass slide. **, p < 0.01; ***, p < 0.0001; n = 50–146 cells per condition. (D) Representative image sequence of the formation of chromatin-filled nuclear membrane blebs (white arrowheads) and subsequent nuclear fragmentation in an MDA-MB-231 breast cancer cell co-expressing NLS-GFP (green) and H2B-RFP (red) during migration through consecutive 2 × 5 µm² constrictions. Insets are indicated by dashed lines. See also Movie S6. (E) Percentage of cells with fragmented nuclei before entry into constriction channel, inside the channel, and after exit. ***, p < 0.001; n = 9775, 1376, and 3072, respectively. (F) Example of an intact nucleus (left top), a nucleus with a small fragment (arrowhead) (left middle), and a nucleus with a γ-H2AX-positive fragment (left bottom). Scale bar: 10 µm. Percentage of cells with γ-H2AX-positive nuclear fragments before, inside, and after migration through constriction channels (right). ***, p < 0.001; n = 1376–3072 cells per condition. (G) Percentage of HT1080 cells migrating through 2 × 5 µm² or 1 × 5 µm² constrictions that formed 53BP1-RFP foci as a function of nuclear rupture. **, p < 0.01 n = 35 cells total. (H) Representative example of formation of 53BP1 foci (arrowheads) in U2OS cell co-expressing NLS-GFP and 53BP1-RFP during migration through 2 × 5 µm² constriction and NE rupture. Scale bar: 10 µm. Error bars in figure: mean ± s.e.