Mechanical Strain Alters Cellular and Nuclear Dynamics at Early Stages of Oligodendrocyte Differentiation

Abstract

Mechanical and physical stimuli including material stiffness and topography or applied mechanical strain have been demonstrated to modulate differentiation of glial progenitor and neural stem cells. Recent studies probing such mechanotransduction in oligodendrocytes have focused chiefly on the biomolecular components. However, the cell-level biophysical changes associated with such responses remain largely unknown. Here, we explored mechanotransduction in oligodendrocyte progenitor cells (OPCs) during the first 48 h of differentiation induction by quantifying the biophysical state in terms of nuclear dynamics, cytoskeleton organization, and cell migration. We compared these mechanophenotypic changes in OPCs exposed to both chemical cues (differentiation factors) and mechanical cues (static tensile strain of 10%) with those exposed to only those chemical cues. We observed that mechanical strain significantly hastened the dampening of nuclear fluctuations and decreased OPC migration, consistent with the progression of differentiation. Those biophysical changes were accompanied by increased production of the intracellular microtubule network. These observations provide insights into mechanisms by which mechanical strain of physiological magnitude could promote differentiation of progenitor cells to oligodendrocytes via inducing intracellular biophysical responses over hours to days post induction.

Keywords: cell migration; microtubules; nuclear dynamics; oligodendrocyte differentiation; strain.

Figure 1

Experimental setup. (A) Cartoon of the plastic mold and the PDMS plate. (B) OPCs transfected with H2B-GFP are seeded in the thin well on the PDMS plate. (C) PDMS plate with cells mounted on the stretcher for imaging on the inverted microscope. (D) Table summarizing the experimental conditions (details described in section Cell Culture and Media).

Figure 2

Mechanically strained OPCs exhibit faster dampening of Nuclear Projected Area Fluctuations during differentiation. (A) Typical image of OPC showing cell body and processes in the bright field (gray), and nucleus in the GFP fluorescence (green) captured using 40X objective. Scale bar 10 μm. (B,C) Fluctuations in nuclear projected area, calculated from time-lapse imaging (60 images at 30 s interval) of OPCs undergoing differentiation without (B) or with (C) 10% strain, for 1, 24, and 48 h post-induction (see Figures S4A,B for calculation of area fluctuations). Dotted horizontal green lines represent average variance of the fluctuations. Unstrained n = 59 (1 h), 46 (24 h), 13 (48 h); Strained n = 38 (1 h), 35 (24 h), 12 (48 h). (D,E) Standard deviations of time series plotted in (B,C) to compare amplitude of nuclear area fluctuations. Solid black arrow lines drawn manually to highlight the differential decreasing trend without and with 10% strain. Error bars represent standard errors. ^**p < 0.05.

Figure 3

Mechanical strain decreases migration of OPCs undergoing differentiation. (A) Left column shows typical field-of-view images of OPCs undergoing differentiation without (top row) or with 10% strain (bottom row), at 1 h post-induction (using 20X objective). Scale bar 50 microns. Right column shows corresponding time-stacked images obtained using minimum-intensity-projections of time lapse images (100 images at 36 s interval) captured starting at 1 h post-induction. Green lines drawn manually in ImageJ to mark trajectories of single cells. (B) Histogram of whole-cell-trajectory lengths manually measured from time-stacked images of hour-long bright-field movies (from 1 to 2 h post-induction) without and with 10% strain. Unstrained n = 80; Strained n = 101. Dotted lines show experimental data while solid lines show Gaussian fits. Inset shows the mean values. Error bars represent standard error. ^**p = 2E-13. (C) Scatter plot of nuclear area fluctuations vs. nuclear trajectory length (both measured over 30 min duration time-lapse images of H2B-GFP labeled OPC nuclei). Gray dots represent combined data from unstrained [n = 25 (1 h), n = 20 (24 h), n = 11 (48 h)] and strained [n = 15 (1 h), n = 7 (24 h), n = 9 (48 h)] OPCs. Extreme data points with nuclear trajectory length or nuclear fluctuations amplitude higher than mean + 2^*S.D. were considered outliers and removed before linear fitting (Figure S8). Solid black line shows linear fit (Pearson's r = 0.63). (D) Average normalized nuclear-trajectory length plotted as a function of time post-induction. Data has been normalized to average nuclear-trajectory length in unstrained or strained OPCs at 1-h post-induction respectively. Solid black arrow lines drawn manually to highlight the decreasing trend with 10% strain. ^**p = 0.06.

Figure 4

Effect of mechanical strain on OPC cytoskeleton. (A) Total fluorescence intensity of F-actin and (B) tubulin normalized by actin and tubulin volume, respectively, at 1 and 24 h post-induction of differentiation, for strained and unstrained OPCs. F-actin levels increase to similar extent for strained and unstrained cells with differentiation time progression from 1 to 24 h; F-actin expression is reduced in response to strain, by 6 and 15% at 1 and 24 h post induction, respectively. Tubulin levels increases by 70% in strained cells at 24 h time point. (C) Examples of F-actin (red) and microtubule (green) cytoskeleton in unstrained (top) and strained (bottom) cells at 24 h time point; Hoechst nuclear staining in blue. Scale bar 5 μm. N = at least 20 cells per condition. Error bars are SEM (standard error of the mean); ^*p < 0.05, ^***p < 0.001.

Figure 5

Effect of strain on cellular and nuclear dynamics of differentiating OPCs. OPCs induced simultaneously by chemical cues and mechanical strain exhibit significantly lower cell-migration and nuclear-fluctuations (at 24 h post-induction) than cells induced by chemical cues alone.