Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

<div><p>The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.</p></div

Distinct Tpm isoforms differentially impact on the elastic modulus of the cell.

<p>Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from <i>n</i> = 3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01 compared to control cells. ***<i>P</i><0.001, ****<i>P</i><0.0001 compared to control.</p

Nanomechanical properties of mouse adipose tissue from Tpm3.1 transgenic mice.

<p>Elastic modulus values of epididymal fat isolated from the Tpm3.1-overexpressing TG and control (WT) mice. A total of 6 mice per group were tested, 10–50 indentation points per area (area was 30 μm²) was conducted and 5–10 areas per epididymal fat pad (both left and right) were employed. Data is visualised as a box plot showing the median with the interquartile range and all the data points. Data points represent the mean of all indentations conducted per animal. A nonparametric, Mann-Whitney test shows no statistical differences between WT and TG samples.</p

Tpm3.1 knockdown leads to a decrease in the cell’s elastic modulus.

<p>(A) Representative westerns of 10 μg of total cellular protein isolated from the Tpm3.1- overexpressing cells untreated (untreat) or exposed to lipofectamine (Lipofec), scramble siRNA (scRNA), Tpm2.1 siRNA or human Tpm3.1 siRNA probed with the γ/9d (mouse and human Tpm3.1) and GAPDH as loading control. (B) Quantification of the total levels of Tpm3.1 levels, <i>n</i> = 3. (C) The elastic modulus for each of the treated Tpm3.1-overexpressing cells was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. ≥ 25 cells for each treatment from <i>n</i> = 3 independent experiments. **<i>P</i><0.01, compared to control.</p

Overexpression of Tpm3.1 alters the F/G actin ratio.

<p>(A) 10 μg of total cellular protein from the B35 clones together with skeletal actin protein (0.025, 0.05, 0.1, 0.25 μg) was analysed by SDS-PAGE followed by western blotting and probe with an actin antibody and GAPDH as loading control. A representative blot is shown from <i>n</i> = 3 independent experiments. (B) Actin levels were determined by densitometry and expressed as ng of actin per μg of total cellular protein, <i>n</i> = 3. (C) Biochemical fractionation of the G and F-actin pools from the different Tpm-overexpressing B35 clones were performed followed by SDS-PAGE electrophoresis. Shown are representative western blots probed for actin. (D) The F-actin/G-actin ratio was determined by densitometric scan of the blots. Shown are the mean ± SEM, <i>n</i> = 5. **<i>P</i><0.01 compared to control.</p

Model proposing the potential impact of distinct actin filament populations on cell stiffness.

<p>We propose that Tpm can define different actin filament populations by dictating the recruitment of different actin binding proteins. Shown are crosslinked filaments associating with αactinin, tension bearing filaments with myosinII, bundles with fascin and branched actin filaments with Arp2/3 and Capping protein. The distinct organisation and functional properties of these Tpm-containing cortical actin filaments impact on cell stiffness.</p

Impact of the different Tpm isoforms on the organization of the actin cytoskeleton.

<p>(A) Representative immunofluorescence images of the Tpm-overexpressing B35 clones stained with 488-Atto phalloidin to visualise F-actin (green) and DAPI (blue) for the nucleus and the corresponding colored overlays defining F-actin in the cells. (B) A linear feature detection algorithm was employed to determine total F-actin length/per cell. Each clone was plated in 8 replicates, 6 random fields were imaged and a range of 320 to 590 cells were analysed from <i>n</i> = 3 independent experiments. Scale bar, 10 μm. ****<i>P</i><0.0001.</p

Total Tpm protein levels in the Tpm-overexpressing clones.

<p>The total Tpm protein expression levels were determined in each Tpm-overexpressing clone. The expression of each Tpm isoform, evaluated by western blotting, was determined with the use of Tpm recombinant proteins and expressed as μg of Tpm per μg of total cellular protein. The histogram depicts the sum of the most highly enriched Tpm isoforms (Tpm1.6, 1.7, 1.10, 1.11, 1.12, 2.1, 3.1, 4.2). <i>n</i> = 4 independent cell lysates, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001.</p

Effect of blebbistatin on the cell’s elastic properties.

<p>The elastic (Young) modulus for the Tpm1.12, Tpm4.2 and Tpm3.1-overexpressing clones was determined following DMSO or 50 μM of blebbistatin treatment for 30 mins. All data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 23–25 cells for each clone was measured from <i>n</i> = 3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01 compared to control cells.</p