Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization.
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.
NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore. NUS Centre for BioImaging Sciences, Faculty of Science, National University of Singapore, Singapore.
Rafi
R
Rashid
Stella Min Ling
SM
Chee
Michael
M
Raghunath
Thorsten
T
Wohland
D000199:Actins / Q000737:chemistry*; D004058:Diffusion; D005362:Ficoll; D005470:Fluorometry; D008954:Models, Biological; D058105:Polymerization; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014783:Viscosity
D000199:Actins; D012996:Solutions; D005362:Ficoll