[1] Large, inert flow tracers are injected into live cells and become lodged inside the cytoskeletal mesh, wherein it is oscillated by repercussions from active motor proteins.
Tracking the fluctuations of tracer particles using optical microscopy can isolate the contribution of active random forces to intracellular molecular transport from that of Brownian motion.
FSM was developed by Ming Guo and David A. Weitz to probe stochastic intracellular forces generated by motor proteins.
[1] Far from a liquid void, the cytoplasm contains a complex meshwork of actin and myosin conferring structural support to the cell, as well as harbouring vesicles and mitochondria among other organelles.
[2] Recent research on the macromolecular crowding inside the cytoplasm raises concerns whether diffusive-like motion of large molecules have been mistakenly attributed to Brownian forces.
[3][4] Guo et al. developed an assay to distinguish whether particle motion inside cells are driven by thermal diffusion or by repercussions from active motor proteins like non-muscle myosin II shaking the cellular cytoskeleton.
FSM relies on injecting tracer particles coated with polyethylene glycol (PEG) larger than the cytoskeletal mesh size (>50 nm),[5] settling in between an internetwork of actin filaments and myosin motor proteins.
Thus, by recording the displacement of tracer oscillations, FSM can gauge and derive the magnitude of forces exerted by active motor proteins.
By using an optical tweezer to apply a prescribed force to a tracer particle, FSM can measure the resulting displacement in order to estimate the elastic spring constant.
[7][8] Directed oscillation of tracer particles using optical tweezers resulted in displacement that was nearly synchronized with applied force, suggesting that the cytoplasm is materially closer to an elastic solid.
[1] This is in stark contrast to previous hypothesis that the cytoplasm is a viscoelastic fluid in which large molecules can freely diffuse.
[9] In ATP-depleted cells, in which non-muscle myosin II are inactivated, FSM experiments reveal that tracer particles cease to oscillate as if the cytoplasm had solidified.
[11] Thus, ATP-hydrolysis by motor proteins appear to be critical to sustain cytoplasmic fluidity, which is crucial to vesicle transport and diffusive motion in the cytoskeleton.
In a 3D matrix, MDA-MB-231 metastatic breast cancer cells had comparatively more solid cytoplasm than counterparts cultured on 2D plates.