Sigley Justin, Jarzen John, Scarpinato Karin, Guthold Martin, Pu Tracey, Nelli Daniel, Low Josiah, Bonin Keith
Department of Physics, Wake Forest University, Winston-Salem, North Carolina, United States of America.
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
PLoS One. 2017 Jan 26;12(1):e0170414. doi: 10.1371/journal.pone.0170414. eCollection 2017.
The interior of cells is a highly complex medium, containing numerous organelles, a matrix of different fibers and a viscous, aqueous fluid of proteins and small molecules. The interior of cells is also a highly dynamic medium, in which many components move, either by active transport or passive diffusion. The mobility and localization of proteins inside cells can provide important insights into protein function and also general cellular properties, such as viscosity. Neoplastic transformation affects numerous cellular properties, and our goal was to investigate the diffusional and binding behavior of the important mismatch repair (MMR) protein MSH2 in live human cells at various stages of neoplastic transformation. Toward this end, noncancerous, immortal, tumorigenic, and metastatic mammary epithelial cells were transfected with EGFP and EGFP-tagged MSH2. MSH2 forms two MMR proteins (MutSα and MutSβ) and we assume MSH2 is in the complex MutSα, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutSα-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14-24 μm2/s for EGFP and 3-7 μm2/s for MutSα-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutSα produces an effective diffusion coefficient that, coupled with the free EGFP diffusion measurements, can be used to extract a pure diffusion coefficient and a pseudo-equilibrium constant K*. The MutSα nuclear K* increased sixfold in the first stage of cancer and then decreased in the more advanced stages. The ratio of nuclear to cytoplasmic K*for MutSα increased almost two orders of magnitude in going from noncancerous to immortal cells, suggesting that this quantity may be a sensitive metric for recognizing the onset of cancer.
细胞内部是一种高度复杂的介质，包含众多细胞器、不同纤维组成的基质以及由蛋白质和小分子构成的粘性水性流体。细胞内部也是一种高度动态的介质，其中许多成分通过主动运输或被动扩散而移动。蛋白质在细胞内的移动性和定位能够为蛋白质功能以及诸如粘度等一般细胞特性提供重要见解。肿瘤转化会影响众多细胞特性，我们的目标是研究重要的错配修复（MMR）蛋白MSH2在肿瘤转化不同阶段的活人类细胞中的扩散和结合行为。为此，用增强绿色荧光蛋白（EGFP）和EGFP标记的MSH2转染非癌性、永生化、致瘤性和转移性乳腺上皮细胞。MSH2形成两种MMR蛋白（MutSα和MutSβ），我们假设MSH2存在于MutSα复合物中，不过无论哪种情况我们的结果都相似。与结合核DNA的MutS复合物不同，EGFP可自由扩散。使用光漂白后荧光恢复技术在每种细胞类型的细胞质和细胞核中测定EGFP和MutSα - EGFP的扩散系数。EGFP的扩散系数为14 - 24μm²/s，MutSα - EGFP的扩散系数为3 - 7μm²/s。从非癌性细胞到永生化细胞，EGFP扩散增加，表明粘度降低，在后续阶段变化较小。MutSα产生一个有效扩散系数，结合自由EGFP扩散测量结果，可用于提取纯扩散系数和伪平衡常数K*。MutSα的核K在癌症第一阶段增加了六倍，然后在更晚期阶段下降。从非癌性细胞到永生化细胞，MutSα的核与细胞质K之比增加了近两个数量级，这表明该量可能是识别癌症发生的一个敏感指标。
