Biophysics Program, Stanford University School of Medicine, Stanford, California; Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, Washington; Marine Biological Laboratory, Woods Hole, Massachusetts.
Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California; Marine Biological Laboratory, Woods Hole, Massachusetts.
Biophys J. 2023 Mar 7;122(5):767-783. doi: 10.1016/j.bpj.2023.01.040. Epub 2023 Feb 3.
The cytoplasm is a complex, crowded, actively driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40 nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of individual fission yeast (Schizosaccharomyces pombe) cellscells. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak nonergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, the actin and microtubule cytoskeletons, cell-cyle stage, and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not "well-mixed" but represents a highly heterogeneous environment in which subcellular components at the 40 nm size scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.
细胞质是一个复杂、拥挤、活跃的环境，其生物物理特性调节着关键的细胞过程，如细胞骨架动力学、相分离和干细胞命运。对于这些细胞质特性的变化知之甚少。在这里，我们使用基于遗传编码的多聚体 40nm 纳米颗粒（GEM）的粒子追踪纳米流变学来测量单个裂殖酵母（Schizosaccharomyces pombe）细胞细胞质内的扩散。我们发现，单个 GEM 颗粒的表观扩散系数在 400 倍范围内变化，而单个细胞之间的平均颗粒扩散系数差异则跨越了 10 倍的范围。为了确定这种异质性的起源，我们开发了一种 Doppelgänger 模拟方法，该方法使用 GEM 扩散的随机模拟来复制基于粒子的实验统计数据，使得每个实验轨迹和细胞都与它们的模拟对应物一一对应。这些模拟表明，扩散率的大的细胞内和细胞间差异不能用实验变异性来解释，但只能用假设细胞质粘度在细胞内和细胞间广泛变化的随机模型来重现。模拟结合细胞质粘度的细胞内和细胞间变化也预测了 GEM 扩散中的弱非遍历性，这与实验数据一致。为了探究这种变化的起源，我们发现 GEM 扩散率的方差在很大程度上与温度、肌动蛋白和微管细胞骨架、细胞周期阶段和空间位置等因素无关，但在高渗冲击下会放大。总的来说，我们的结果提供了一个惊人的例证，表明细胞质不是“均匀混合”的，而是代表一个高度异质的环境，在这个环境中，40nm 大小的亚细胞成分在单个细胞内以及在遗传上相同的群体中的不同细胞内经历着截然不同的有效粘度。这些发现对细胞和亚细胞水平上生物噪声的起源和调节具有重要意义。
