Apical cell protrusions cause vertical deformation of the soft cancer nucleus

Abstract

Breast cancer nuclei have highly irregular shapes, which are diagnostic and prognostic markers of breast cancer progression. The mechanisms by which irregular cancer nuclear shapes develop are not well understood. Here we report the existence of vertical, apical cell protrusions in cultured MDA-MB-231 breast cancer cells. Once formed, these protrusions persist over time scales of hours and are associated with vertically upward nuclear deformations. They are absent in normal mammary epithelial cells (MCF-10A cells). Microtubule disruption enriched these protrusions preferentially in MDA-MB-231 cells compared with MCF-10A cells, whereas inhibition of nonmuscle myosin II (NMMII) abolished this enrichment. Dynamic confocal imaging of the vertical cell and nuclear shape revealed that the apical cell protrusions form first, and in response, the nucleus deforms and/or subsequently gets vertically extruded into the apical protrusion. Overexpression of lamin A/C in MDA-MB-231 cells reduced nuclear deformation in apical protrusions. These data highlight the role of mechanical stresses generated by moving boundaries, as well as abnormal nuclear mechanics in the development of abnormal nuclear shapes in breast cancer cells.

Keywords: breast cancer; cancer nucleus; lamin A/C; microtubule; nuclear shape; protrusions.

Figure 1.

The presence of apical cell protrusions with associated nuclear deformation in MB-231 cells. Images show representative fluorescent images of x-y and x-z cross-sections of an MCF-10A cell (top) and MB-231 cells (bottom). The MB-231 cell has a ‘Mexican hat’ type appearance due to the local apical cell protrusion in an otherwise well-spread cell.

Figure 2.

Nocodazole treatment enriches apical cell protrusions in MB-231 cells. A) Percentage of fixed cells with apical protrusions under various. Chi-square test was used to test statistical differences. *or #, p<0.05. N > 30 cells for each condition (MCF_ctrl: untreated MCF-10A cells; MB_231_ctrl: untreated MB-231 cells; MCF_noc and MB_231_noc: MCF-10A cells and MB-231 cells respectively treated with 5 μM nocodazole). Error bars indicate SEM. B) Percentage of cells with an apical cell protrusion in which the nuclear shape was vertically deformed into the protrusion. Error bars are SEM. C) Representative fluorescent images of an MB-231 and MCF-10A cell treated with nocodazole for 1 hr, showing the apical cell protrusion and associated vertical nuclear deformation. D) Fluorescent images show confocal images at different planes (marked by horizontal lines) of an MB-231 cell treated with nocodazole. The apical protrusion is present in an otherwise well-spread cell.

Figure 3.

Apical cells protrusion precedes vertical nuclear deformation. A) Reconstructed fluorescent time-lapse images of the vertical cross-section of a live cell expressing NLS-GFP and treated with lipophilic tracer DiI showing the formation of a vertical protrusion in the cell membrane near the nucleus (white arrows) and the consequent deformation of the nucleus (red arrows) after treatment with 5μM nocodazole at time t = 0 min. For reference, vertical dashed lines indicate the initial position of the nucleus and cell membrane. B) Reconstructed fluorescent time-lapse images of the vertical cross-section of a living cell expressing NLS-GFP and RFP-LifeAct showing the formation and retraction of a vertical cell protrusion near the nucleus (white arrows) and the consequent deformation of the nucleus (red arrows) after treatment with nocodazole at t = 0 min. Vertical dashed lines indicate the initial position of the nucleus for reference.

Figure 4.

Apical cell protrusions are NMMII dependent, while vertical nuclear deformation is reduced upon over-expression of GFP-Lamin A. A) An MB-231 cell treated with nocodazole for 1 hr before fixation and stained for pMyosin (orange), F-actin (green) and nucleus (blue). Inset at two z-positions (top and middle planes) shows the distribution of F-actin and pMyosin in the apical cell protrusion. B) Fluorescent images show an example of a MB-231 cell treated with blebbistatin, which abrogated the apical cell protrusion. C) Images show a GFP-Lamin A expressing cell treated with nocodazole for 1 hr before fixation. An apical protrusion is clearly visible, but with no vertically upward deformation. D) Bar plot shows the frequency of cells with apical protrusions of MDA-MB-231 cells (left) and frequency of nuclei deformed into the apical cell protrusions. (MB_231_ctrl: untreated MB-231 cells; MB_231_noc: MB-231 cells treated with 5 μM nocodazole; MB_231_GFPLA_noc: MB-231 cells expressing GFP-Lamin A and treated with 5 μM nocodazole). Error bar is SEM. At least 32 samples from 3 different dishes are analyzed. Chi-square test with Bonferroni correction was used to test statistical differences. *, p<0.05.