Light – microscopic analysis of the physical properties of cytoplasm in living cells

Abstract

We have used a variety of optical techniques to explore the intracellular constraints on diffusion, cytoplasmic compartmentalization and biomechanics of living tissue culture cells. Fluorescence ratio imaging measurements of solvent viscosity in single cells indicate that the solvent viscosity of cytoplasm does not differ detectably from bulk water. Nevertheless, data obtained by fluorescence recovery after photobleaching (FRAP) of inert tracer molecules suggest that macromolecular crowding and molecular sieving retard the long-range translational diffusion of protein-sized molecules from 4 to 50-fold relative to their diffusion in dilute aqueous solution.Transient binding interactions with intracellular components may additionally retard the diffusion of proteins. Recent FRAP studies of fluorescent analogs of calmodulin suggest that as little as 5% of this protein is freely diffusing, even in unstimulated cells where intracellular [Ca2+] is below the threshold for activation of calmodulin binding to calcium-dependent targetsin vitro. Preliminary data show that binding is mitigated or abolished by charge reversal mutations in the central helix of calmodulin or by a point mutation in calcium binding loop 4 that greatly reduces the affinity of calmodulin for Ca2+.