12 research outputs found
Diffusion and Binding of Mismatch Repair Protein, MSH2, in Breast Cancer Cells at Different Stages of Neoplastic Transformation
<div><p>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 (MutSĪ± and MutSĪ²) and we assume MSH2 is in the complex MutSĪ±, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutSĪ±-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14ā24 Ī¼m<sup>2</sup>/s for EGFP and 3ā7 Ī¼m<sup>2</sup>/s for MutSĪ±-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutSĪ± produces an <i>effective</i> diffusion coefficient that, coupled with the free EGFP diffusion measurements, can be used to extract a pure diffusion coefficient and a pseudo-equilibrium constant <i>K</i>*. The MutSĪ± nuclear <i>K</i>* increased sixfold in the first stage of cancer and then decreased in the more advanced stages. The ratio of nuclear to cytoplasmic <i>K</i>*for MutSĪ± 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.</p></div
Statistical significance of results for the <i>EGFP proteins</i>.
<p>Statistical significance of results for the <i>EGFP proteins</i>.</p
FRAP fluorescence vs. time.
<p>Representative FRAP curve showing the recovery of fluorescence with time after the photobleach, which ended at t = 0. Red points/line are the experimental data, and the blue points/line are the best fit to the data using Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170414#pone.0170414.e002" target="_blank">2</a>). These data are from a noncancerous cell in the cytoplasm. Inset: Same data and fit shown for the first 10 seconds to better show the details during recovery. For the full curve, note that only the 1200 points starting at t = 0 and after were corrected for photobleaching in order to perform the FRAP fit. Thus the first 300 points before the bleach at t = 0 exhibit photobleaching.</p
Diffusion data.
<p>Diffusion data separated by protein, cell type and cellular region: (A) EGFP and (B) MutSĪ±-EGFP. Each x-axis is ordered according to the neoplastic progression of the cancer cellānoncancerous on the left and metastatic on the right. For each cell type cytoplasmic and nuclear values are plotted next to each other (cytoplasmāsolid; nucleusāhatched). Error bars are +/- SEM.</p
Diffusion and binding data.
<p>(A) Line plot of EGFP diffusion vs. cancer cell type (neoplastic transformation). (B) Line plot of the pseudo-equilibrium constant <i>K</i>* for MutSĪ±-EGFP as a function of cancer cell type (neoplastic transformation) for both cell regions. Each x-axis is ordered according to the neoplastic progression of the cancer cellānoncancerous on the left and metastatic on the right. For each cell type cytoplasmic and nuclear values are plotted on the same graph (cytoplasmāblue; nucleusāred). Error bars are +/- SEM.</p
Log<sub>10</sub> scale plot of the cancer response activity (CRA) vs. cancer cell type (stage of neoplastic transformation).
<p>Log<sub>10</sub> scale plot of the cancer response activity (CRA) vs. cancer cell type (stage of neoplastic transformation).</p
Free diffusion coefficient estimates assigned to the MutSĪ±-EGFP complex and values for the pseudo-equilibrium constants in the different cell types and regions.
<p>Free diffusion coefficient estimates assigned to the MutSĪ±-EGFP complex and values for the pseudo-equilibrium constants in the different cell types and regions.</p
The human mammary epithelial cell types used here.
<p>The human mammary epithelial cell types used here.</p
EGFP diffusion measurements in water-glycerol mixtures.
<p>EGFP diffusion measurements in water-glycerol mixtures.</p
Summary of <i>MutSĪ±</i>Āresults and their significance.
<p>Summary of <i>MutSĪ±</i>Āresults and their significance.</p