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Diffusion and binding of mismatch repair protein, MSH2, in breast cancer cells at different stages of neoplastic transformation

Bonin, Keith

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title
Diffusion and binding of mismatch repair protein, MSH2, in breast cancer cells at different stages of neoplastic transformation
author
Sigley, Justin
author
Jarzen, John
author
Scarpinato, Karin
author
Guthold, Martin
author
Pu, Tracey
author
Nelli, Daniel
author
Low, Josiah
author
Bonin, Keith
abstract
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 (MutS--alpha and MutS--alpha) and we assume MSH2 is in the complex MutS--alpha, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutS--alpha-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14-24 m2/s for EGFP and 3-7 m2/s for MutS--alpha-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutS--alpha 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 MutS--alpha 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 MutS--alpha 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.
description
Data Availability: The diffusion measurements for individual cells that went into determining the averaged results summarized in Table 3, and the t-test results of Tables 4 B&C are given in a publicly accessible server at Wake Forest University (http://hdl.handle.net/10339/62002). The EGFP fluorescence vs. time data that is fit with a model to determine the cell's diffusion values given in the paper are also accessible at the same url. The fluorescence vs. time data that is fit with a model to determine the EGFP diffusion values in the three glycerol control mixtures are also accessible at the same url. Finally, the sets of measured diffusion values in different control runs that were used to determine the averages in Table 2 are also to be found at the same url.
subject
neoplastic
cancer
diffusion
FRAP
EGFP
MSH2
viscosity
date
2016-08-01T19:50:26Z (accessioned)
2016-08-01T19:50:26Z (available)
2016-08-01 (issued)
identifier
http://hdl.handle.net/10339/62002 (uri)
type
Dataset

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