Lecinski Sarah, Shepherd Jack W, Bunting Kate, Dresser Lara, Quinn Steven D, MacDonald Chris, Leake Mark C
Department of Physics, University of York, York YO10 5DD, UK.
Department of Biology, University of York, York YO10 5DD, UK.
Interface Focus. 2022 Oct 14;12(6):20220042. doi: 10.1098/rsfs.2022.0042. eCollection 2022 Dec 6.
In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize and then apply these insights to live cells of budding yeast . It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.
在真核生物中，细胞内的物理化学性质，如大分子拥挤和细胞质粘弹性，会影响代谢活动、分子扩散和蛋白质折叠等关键过程。然而，绘制活细胞中的拥挤和粘弹性情况仍然具有挑战性。一种方法是使用被动流变学，即追踪细胞内内化的外源荧光颗粒的扩散，并从导出的均方位移关系推断物理化学性质。最近，crGE2.3荧光共振能量转移生物传感器被开发出来用于量化细胞内的拥挤情况，不过尚不清楚这种读数如何依赖于粘弹性和拥挤剂的分子量。在这里，我们提供相关的多维数据，以利用超分辨荧光显微镜和系综时间分辨光谱来探索分子拥挤剂的扩散和分子拥挤特征。我们首先进行表征，然后将这些见解应用于出芽酵母的活细胞。据我们所知，这是首次尝试这样做。我们证明这些方法在活细胞中以及内源性表达传感器的情况下都是可用的。最后，我们提出一种将荧光珠作为粘弹性标记内化到活酵母细胞质中的方法，并讨论这种方法的局限性，包括细胞功能的损伤。
