Vimentin intermediate filament and cell mechanics

The mechanical properties of a cell determine many aspects of its behavior; these mechanics are largely determined by the cytoskeleton. While the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about  the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, studies are carried out with mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin^-/- mice. The VIFs contribute little to cortical stiffness but are critical in regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to approximately 10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.

Figure 1. Analysis of control (WT) and vimentin^-/- mouse embryonic fibroblast cells (mEFs). (A) Immunoblot analyses of cell lysates from WT and vimentin^-/- mEFs using antibodies to vimentin, actin and tubulin. Representative blots from 3 experiments. (B) Immunofluorescence using antibodies against vimentin in control (left; WT) and vimentin^-/- (right; Vim^-/-) mEFs. The cell boundary in vimentin^-/- mEF is represented by  the yellow line. Representative  images from 3 experiments. Scale 10 µm.

Figure 2. Optical tweezers measurement of intracellular mechanics. (A) Schematic of the optical tweezer experiment. 500-nm PEG coated inert particles are endocytosed into mEF cells, then trapped and manipulated by a spatially sinusoidal oscillating optical trap, which generates a force F at frequency ω. The frequency dependent complex spring constant is calculated by measuring the resultant displacement x of the bead in the trap oscillation, as F/x. (B) Typical displacements of the trapped bead and the optical trap oscillating at 1Hz.

Figure 3. Active microrheology with optical tweezers controlling 500 nm endocytosed beads in the cytoplasm of mEFs. (A) Frequency dependent cytoplasmic elastic moduli G’ (solid symbols) and loss moduli G” (open symbols) of the WT and Vim^-/- mEFs. The cytoplasm of the WT mEFs (triangles) is stiffer than that of the Vim^-/- mEFs (circles). (B) Cytoplasmic elastic moduli in the WT and Vim^-/- mEFs at 1 Hz. Error bars, s.e.m (*P<0.05).

Figure 4. Intracellular movement of endogenous vesicles inside WT and Vim^-/- mEFs. (A,B) 10 second trajectories of endogenous vesicles in the cytoplasm of (A) WT mEFs and (B) Vim^-/- mEFs. (C) Calculation of the mean square displacement of vesicles shows that vesicles move faster in the Vim^-/- mEFs as compared to the WT mEFs. (D) Illustration of random vesicle movement in networks with and without vimentin. In the wild type cells, vimentin network constrains the diffusive-like movement of organelles; in the vimentin^-/- cells, organelles move more freely.

Figure 5. Cell cortical material properties of WT and vimentin^-/- mEFs measured with optical magnetic twisting cytometry (OMTC). (A) Schematic of the OMTC measurement. A magnetic field introduces a torque that causes the 4.5-μm ferromagnetic bead to rotate and to deform the cell cortex to which it is bound. (B) Elastic, G' (closed symbol), and loss, G" (open symbol), moduli for WT (black triangles) and Vim^-/- (gray circles) mEFs cultured overnight on Collagen I coated rigid plastic dishes, as measured by OMTC. About 100 single cells are measured for each cell type.

Force Spectrum Microscopy (FSM): A new probe of stochastic properties of molecular motors in cells

Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like yet non-thermal motion. There is no existing technique to measure these random forces. Here we introduce Force-Spectrum Microscopy (FSM) to directly quantify the random forces and thereby probe motor activity. This assay combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. These fluctuating forces substantially enhance intracellular movement over a broad range of length scales. We use FSM to show that force fluctuations are three times larger in malignant cells than in their benign counterparts, consistent with the higher motility of cancer cells. In addition, using FSM, we show that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm. Thus, FSM probes important consequences of the average random forces in cells.

Figure 1. Movements of microinjected tracer particles in living cells.

Figure 2. Optical-tweezer active microrheology measurement shows that the cytoplasm is a weak elastic gel.

Figure 3. Ensemble aggregate intracellular force spectrum probed by FSM.

Figure 4. Intracellular mechanics, dynamics and forces in benign and malignant tumor cells.

Cell volume correlates with cell stiffness and affects gene expression patterning

Within a given microenvironment, how cells acquire and modulate shape stability can determine cell function and even influence stem cell fate. Here we identify an unexpected role of cell volume in these processes. As a cell spreads on a rigid substrate, the cell volume decreases as cell stiffness increases, and whether spreading on soft or rigid substrates, cell volume tracks cell spreading in an identical fashion. Moreover, changes in gene expression resulting from cell stiffening on rigid substrates could be replicated by altering cell volume by osmotic pressure. Taken together, these observations suggest that cell volume control through water release rather than through substrate rigidity underlies cell stiffness and downstream effects such as gene expression, and stem cell differentiation.

Figure 1. Morphology and volume of adherent cells change with increasing substrate stiffness.

Figure 2. Cell volume increases when the cell spread area is decreased by growing cells on micropatterned collagen 'islands' on glass.

Figure 3. Cell stiffness correlates with cell volume.

Figure 4. Differentiation of mouse mesenchymal stem cells (mMSC) is modulated by controlling cell volume.