Chapman Cole D, Gorczyca Stephanie, Robertson-Anderson Rae M
Department of Physics, University of California San Diego, La Jolla, California.
Department of Physics, University of San Diego, San Diego, California.
Biophys J. 2015 Mar 10;108(5):1220-8. doi: 10.1016/j.bpj.2015.02.002.
Despite the ubiquity of molecular crowding in living cells, the effects of crowding on the dynamics of genome-sized DNA are poorly understood. Here, we track single, fluorescent-labeled large DNA molecules (11, 115 kbp) diffusing in dextran solutions that mimic intracellular crowding conditions (0-40%), and determine the effects of crowding on both DNA mobility and conformation. Both DNAs exhibit ergodic Brownian motion and comparable mobility reduction in all conditions; however, crowder size (10 vs. 500 kDa) plays a critical role in the underlying diffusive mechanisms and dependence on crowder concentration. Surprisingly, in 10-kDa dextran, crowder influence saturates at ∼20% with an ∼5× drop in DNA diffusion, in stark contrast to exponentially retarded mobility, coupled to weak anomalous subdiffusion, with increasing concentration of 500-kDa dextran. Both DNAs elongate into lower-entropy states (compared to random coil conformations) when crowded, with elongation states that are gamma distributed and fluctuate in time. However, the broadness of the distribution of states and the time-dependence and length scale of elongation length fluctuations depend on both DNA and crowder size with concentration having surprisingly little impact. Results collectively show that mobility reduction and coil elongation of large crowded DNAs are due to a complex interplay between entropic effects and crowder mobility. Although elongation and initial mobility retardation are driven by depletion interactions, subdiffusive dynamics, and the drastic exponential slowing of DNA, up to ∼300×, arise from the reduced mobility of larger crowders. Our results elucidate the highly important and widely debated effects of cellular crowding on genome-sized DNA.
尽管分子拥挤现象在活细胞中普遍存在，但人们对拥挤对基因组大小的DNA动力学的影响却知之甚少。在这里，我们追踪单个荧光标记的大DNA分子（11,115千碱基对）在模拟细胞内拥挤条件（0-40%）的葡聚糖溶液中的扩散情况，并确定拥挤对DNA迁移率和构象的影响。两种DNA在所有条件下均表现出遍历性布朗运动和相当的迁移率降低；然而，拥挤剂大小（10 kDa与500 kDa）在潜在的扩散机制以及对拥挤剂浓度的依赖性方面起着关键作用。令人惊讶的是，在10 kDa的葡聚糖中，拥挤剂的影响在约20%时达到饱和，DNA扩散下降约5倍，这与随着500 kDa葡聚糖浓度增加而呈指数级减慢的迁移率以及弱反常亚扩散形成鲜明对比。当受到拥挤时，两种DNA都会伸长为低熵状态（与无规卷曲构象相比），其伸长状态呈伽马分布且随时间波动。然而，状态分布的宽度以及伸长长度波动的时间依赖性和长度尺度取决于DNA和拥挤剂的大小，而浓度的影响出人意料地小。结果共同表明，大的拥挤DNA的迁移率降低和卷曲伸长是由于熵效应和拥挤剂迁移率之间的复杂相互作用。虽然伸长和初始迁移率减慢是由耗尽相互作用驱动的，但亚扩散动力学以及DNA高达约300倍的急剧指数减慢是由较大拥挤剂迁移率降低引起的。我们的结果阐明了细胞拥挤对基因组大小的DNA的高度重要且备受争议的影响。
