Rashid Rafi, Chee Stella Min Ling, Raghunath Michael, Wohland Thorsten
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.
Phys Biol. 2015 Apr 30;12(3):034001. doi: 10.1088/1478-3975/12/3/034001.
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.
大分子拥挤效应（MMC）已被用于各种体外实验系统，以模拟体内生理状态。这是因为细胞内拥挤的细胞质含有许多溶解在水性介质中的不同类型溶质。细胞外微环境中的MMC参与维持干细胞的未分化状态（生态位），并在干细胞迁移到生态位外的新位置后帮助其分化。在化学诱导的脂肪生成过程中，生理相关分数体积占有率（FVO）下的MMC显著增强人骨髓间充质干细胞的成脂分化。MMC产生这种增强作用的机制尚不完全清楚。在细胞外胶原蛋白沉积的背景下，我们最近报道了优化FVO同时最小化本体粘度的重要性。两个相反的特性将决定生化反应的净速率：本体粘度的负面影响和排阻体积的正面影响，后者由FVO表示。在本研究中，我们更仔细地研究了粘度对反应速率的影响。我们使用荧光法测量肌动蛋白聚合速率，并使用荧光相关光谱法（FCS）测量各种探针在含有生理浓度拥挤剂聚蔗糖的溶液中的扩散。与它对胶原蛋白的作用类似，聚蔗糖尽管增加了本体粘度，但仍提高了肌动蛋白聚合速率。我们的FCS测量揭示了异常扩散的一个相对较小的成分。此外，我们的测量确实表明，在拥挤环境中微观粘度变得重要。我们通过在甘油中进行肌动蛋白聚合试验，排除了本体粘度是速率增强的原因。聚蔗糖和甘油的这些相反作用使我们得出结论，在拥挤的微环境中，微观粘度在反应分子的长度尺度上变得重要。排阻体积效应（由拥挤引起）增加了肌动蛋白的有效浓度，从而提高了反应速率，而微观粘度没有增加到足以降低反应速率的程度。这项研究揭示了关于MMC机制的更精细细节。
