Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization

Abstract

Macromolecular crowding (MMC) has been used in various in vitro experimental systems to mimic in vivo physiology. This is because the crowded cytoplasm of cells contains many different types of solutes dissolved in an aqueous medium. MMC in the extracellular microenvironment is involved in maintaining stem cells in their undifferentiated state (niche) as well as in aiding their differentiation after they have travelled to new locations outside the niche. MMC at physiologically relevant fractional volume occupancies (FVOs) significantly enhances the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells during chemically induced adipogenesis. The mechanism by which MMC produces this enhancement is not entirely known. In the context of extracellular collagen deposition, we have recently reported the importance of optimizing the FVO while minimizing the bulk viscosity. Two opposing properties will determine the net rate of a biochemical reaction: the negative effect of bulk viscosity and the positive effect of the excluded volume, the latter being expressed by the FVO. In this study we have looked more closely at the effect of viscosity on reaction rates. We have used fluorimetry to measure the rate of actin polymerization and fluorescence correlation spectroscopy (FCS) to measure diffusion of various probes in solutions containing the crowder Ficoll at physiological concentrations. Similar to its effect on collagen, Ficoll enhanced the actin polymerization rate despite increasing the bulk viscosity. Our FCS measurements reveal a relatively minor component of anomalous diffusion. In addition, our measurements do suggest that microviscosity becomes relevant in a crowded environment. We ruled out bulk viscosity as a cause of the rate enhancement by performing the actin polymerization assay in glycerol. These opposite effects of Ficoll and glycerol led us to conclude that microviscosity becomes relevant at the length scale of the reacting molecules within a crowded microenvironment. The excluded volume effect (arising from crowding) increases the effective concentration of actin, which increases the reaction rate, while the microviscosity does not increase sufficiently to lower the reaction rate. This study reveals finer details about the mechanism of MMC.

The effect of crowder charge in a model polymer–colloid system for macromolecular crowding: Polymer structure and dynamics

“…11,37 Other studies have also reported diffusion coefficients of proteins that suggest a difference between micro-viscosity and bulk viscosity. 38,39 Rashid et al 13 have reported that the micro-viscosity experienced by a fluorescent probe molecule in Ficoll70 is up to 8 times smaller than the bulk viscosity in the limit of crowding, roughly consistent with our findings.…”

“…[6][7][8][9] In addition to these, solution heterogeneity and micro-viscosity has also been identified to play a big role in macromolecular crowding. [10][11][12][13] As a result, depending on the environment, macromolecules can either compact into smaller localized regions (as happens with DNA in the presence of added polymer and salt solutions 14 ) or adopt more complex conformations. Thus, a careful unraveling of the effect of intermolecular interactions on macromolecular conformations and dynamics in crowded environments has been recognized to be important.…”

Reorganization of Lipid Diffusion by Myelin Basic Protein as Revealed by STED Nanoscopy

“…Differences in lipid compositions may also explain why the lipid analogs diffuse much more slowly in membrane sheets of oligodendrocytes as compared with the MBP-enriched domains of the PtK2 cells. The oligodendrocyte-specific enrichment in long-chain, saturated lipids (>C 24 ) ( 6 , 8 ) increases not only the membrane order or packing but also the probability of inner-leaflet coupling, which in turn results in increased friction, viscosity, and lower lipid mobility ( 53 ). It would be interesting to study the diffusion of fluorescent lipid analogs with long-chain lipids (>C 24 ), but unfortunately, so far we have only been able to efficiently incorporate fluorescent lipid analogs with relatively short-chain fatty acids (<C 20 ) into cellular membranes.…”

Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems

“…[15][16][17][18] However, others have found anomalous subdiffusion where MSD = Kt α with K being the transport coefficient and α being in the range of 0.4-0.9. 4,[19][20][21] These studies have also shown evidence of macromolecular compaction, swelling, and elongation depending on type and size of tracer macromolecule and crowder. 4,5,22 These crowdinginduced conformational changes in turn impact the resulting transport properties.…”