Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments

Abstract

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.

Mechanical control of mitotic progression in single animal cells

“…By applying a range of feedback-controlled confinement forces, we confirmed several of their observations including spindle widening and flattening (28,29), accelerated mitotic progression with small deformations (23), asymmetric spindle positioning (10), and delayed mitotic progression due to ectopic spindle function below a critical height (2). We identified several force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces (50-100 nN) where increased intracellular pressure was related to resistance to confinement, and, finally, yield forces (>100 nN) where mitotic progression was inhibited by mechanical perturbation of spindle geometry.…”

“…In mitosis, cells generate actomyosindependent (1) intracellular pressure to round up and optimize geometry for proper function of the mitotic spindle, the machinery that organizes and segregates chromosomes (2,9,10,22). Restricting cell rounding height below 5-8 μm with microfabricated PDMS chambers perturbs mitotic progression in several cell types (2, 10), but the forces that cells can withstand remain unquantified.…”

“…Despite the importance of cell rounding in mitotic progression and tissue organization, the mechanical robustness of mitotic cells remains poorly investigated even in vitro, probably due to a paucity of suitable experimental tools that can apply precise forces to poorly adherent cells. Confining cell rounding below 5-8 μm with microfabricated polydimethylsiloxane (PDMS) chambers perturbs mitotic progression in several cell types (2,10), but the forces required to do this remain unquantified. Thus, the magnitude of confinement force required to prevent mitosis and the mechanisms of cell response to such forces are not known.…”

Biological effects of in vitro THz radiation exposure in human foetal fibroblasts

“…Many reports have described that actin and actin-binding proteins function at the heart of the spindle [61] and several groups have shown defects in spindle formation following perturbations of the actin cytoskeleton [62,63]. Such observations suggested the possibility that the actin cytoskeleton might play a direct role in spindle formation itself [61,64,65]. During mitosis, accurate chromosome segregation requires precise coordination of the microtubule-based spindle and the actin-based cell cortex.…”

HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution

“…293 daughter cells resulting from the rare multipolar divisions and incomplete cytokinesis, which are accompanied by an unequal segregation of chromosomes (Gisselsson et al, 2008(Gisselsson et al, , 2010, are presumably unviable undergoing mitotic cell death, cell-cycle arrest or senescence as it was demonstrated for other cell lines (Ganem et al, 2009;Marthiens et al, 2012;Stewénius et al, 2005). Nevertheless, it was observed that a few karyotypes of multipolar mitosis daughter cells could survive, underwent clonal evolution and expanded in culture (Tse et al, 2012;Yang et al, 2012 and refs herein). Thereby, the rare multipolar mitoses can potentially contribute to karyotype diversification of 293 cells.…”