Structure-function relationships in the stem cell's mechanical world B: emergent anisotropy of the cytoskeleton correlates to volume and shape changing stress exposure

Abstract

In the preceding study (Part A), we showed that prescribed seeding conditions as well as seeding density can be used to subject multipotent stem cells (MSCs) to volume changing stresses and that changes in volume of the cell are associated with changes in shape, but not volume, of the cell nucleus. In the current study, we aim to control the mechanical milieu of live cells using these prescribed seeding conditions concomitant to delivery of shape changing stresses via fluid flow, while observing adaptation of the cytoskeleton, a major cellular transducer that modulates cell shape, stiffness and remodeling. We hypothesize that the spatiotemporal organization of tubulin and actin elements of the cytoskeleton changes in response to volume and shape changing stresses emulating those during development, prior to the first beating of the heart or twitching of muscle. Our approach was to quantify the change over baseline in spatiotemporal distribution of actin and tubulin in live C3H/10T1/2 model stem cells subjected to volume changing stresses induced by seeding at density as well as low magnitude, short duration, shape changing (shear) stresses induced by fluid flow (0.5 or 1.0 dyne/cm2 for 30/60/90 minutes). Upon exposure to fluid flow, both tubulin thickness (height) and concentration (fluorescence intensity) change significantly over baseline, as a function of proximity to neighboring cells (density) and the substrate (apical-basal height). Given our recently published studies showing amplification of stress gradients (flow velocity) with increasing distance to nearest neighbors and the substrate, i.e. with decreasing density and toward the apical side of the cell, tubulin adaptation appears to depend significantly on the magnitude of the stress to which the cell is exposed locally. In contrast, adaptation of actin to the changing mechanical milieu is more global, exhibiting less significant differences attributable to nearest neighbors or boundaries than differences attributable to magnitude of the stress to which the cell is exposed globally (0.5 versus 1.0 dyne/cm2). Furthermore, changes in the actin cytoskeletal distribution correlate positively with one pre-mesenchymal condensation marker (Msx2) and negatively with early markers of chondrogenesis (ColIIaI alone, indicative of pre-hypertrophic chondrogenesis) and osteogenesis (Runx2). Changes in the tubulin cytoskeletal distribution correlate positively with a marker of pericondensation (Sox9 alone), negatively with chondrogenesis (ColIIaI) and positively with adipogenesis (Ppar-gamma 2). Taken as a whole, exposure of MSCs to volume and shape changing stresses results in emergent anisotropy of cytoskeletal architecture (structure), which relate to emergent cell fate (function).

Figure 1. Tubulin and actin cytoskeleton

High resolution image stacks of the stem cell tubulin (left) and actin (right) cytoskeleton are acquired with a laser scanning confocal microscope and are reconstructed in three dimensions. These images are intentionally overexposed to clearly delineate the cytoskeletal boundaries.

Figure 2. 3D images of flow fields around stem cells, reconstructed using micro-particle image velocimetry data and live cell imaging

Red arrows indicate local flow velocity vectors, where microsphere displacements give vector magnitude and direction, around cells (green). The global flow direction is from the top left to the bottom right. Three dimensional confocal image stacks were analyzed to quantify the flow fields, spatiotemporally at subcelluar scale resolution, with respect to distance from the substrate and cell density. After [23]. Used with permission.

Figure 3. Seeding density determines the initial mechanical stress state and boundary condition of the cells

(a) Phase contrast image at 40×. Cells seeded at low density show little cell-cell contact and unoccupied substrate compared to those seeded at high density. Epifluorescent images at 100× of GFP-tagged (b) tubulin and (c) actin show that cells at low density exhibit larger cells with more defined cytoskeletal structures while cells at high density exhibit smaller cells with a less structured cytoskeleton. Note: Due to the less than 100% transfection efficiency of the fluorescent tag, we are able to identify individual cells, which would not be possible with perfect transduction or other labeling methods.

Figure 4. Schematic diagram of reference points for cell measurements

To measure mechanoadaptation quantitatively, normalized changes in intensities from a baseline control of fluorescent actin and tubulin are measured from the bisected regions along the direction of flow, in the front (flow-side) and back (non-flow-side) halves, and along the vertical profile of stem cells (apical-basal). The total amount of each fluorescent cytoskeletal element (tubulin, actin) as well its thickness is also measured.

Figure 5. Tubulin and actin concentration, normalized to cell volume, in LD and HD cells

Normalized tubulin concentration is significantly higher in cells seeded at LD compared to HD and tends to be lower (albeit no statistical significance shown) in LD compared to HD cells. Asterisk (*) indicates a significant difference (prob>|Z| was less than 0.05).

Figure 6. Adaptation of tubulin and actin to shape changing stress exposure

Changes from baseline of measured actin (right) and tubulin (left) over time at varying cell densities and shear stresses. (a) Changes in cell shape can be seen in the measurements of cytoskeletal thickness and total amount of cytoskeletal protein. (b) Emergent anisotropies in the direction of flow can be seen by comparing the front and back measurements of cytoskeletal protein. (c) Emergent anisotropies in the distance from the substrate can be seen by comparing the apical and basal measurements of cytoskeletal protein. Error bars show standard error; asterisks (*) indicate a significant (prob>|Z| was less than 0.05) difference from zero and brackets indicate a significant (prob>|Z| was less than 0.05) differences in means.