Diffusion and Binding of Mismatch Repair Protein, MSH2, in Breast Cancer Cells at Different Stages of Neoplastic TransformationReportar como inadecuado




Diffusion and Binding of Mismatch Repair Protein, MSH2, in Breast Cancer Cells at Different Stages of Neoplastic Transformation - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

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.



Autor: Justin Sigley, John Jarzen, Karin Scarpinato, Martin Guthold, Tracey Pu, Daniel Nelli, Josiah Low, Keith Bonin

Fuente: http://plos.srce.hr/



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