Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy

Abstract

SUMMARY 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, non-thermal motion. Here we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.

Hydrodynamic collective effects of active protein machines in solution and lipid bilayers

“…In addition, several studies have proposed specific explanations for the importance of nonthermal random motions in living cells, whose origin lies in the forces generated by the uncorrelated activity of protein machines. (26,(42)(43)(44) Such nonthermal fluctuations may also be a consequence of the universal hydrodynamic effects, described here, that arise from active conformational changes in molecular motors and other protein machines powered by ATP.…”

Intracellular and extracellular forces drive primary cilia movement

“…Cells are nonequilibrium systems with vigorous cytoskeletal dynamics. Aggregate random forces generated by stochastic motor activity have recently been documented to stir the cytoplasm (37) and enhance the transport of small proteins and organelles in the cytoplasm (38). The basal body is embedded in the actin cortex (Figs.…”

Disentangling Random Motion and Flow in a Complex Medium

“…Using the Stokes-Einstein equation for the diffusion coefficient of a sphere (30), ðD ¼ kT=ð6pRhÞÞ, gives an effective viscosity of h z 50 cP, or~50 times that of water. We note, however, that this approximation assumes thermally driven particle motion, and that the actual viscosity may be significantly higher given a higher effective temperature due to actively generated forces (6,31).…”