Genetic interactions influencing epithelial apicobasal polarization.
2011-11-15T01:14:06Z (GMT) by
<p>(A) shRNA-mediated depletion of centrosome components impairs polarization. Representative bright-field images are shown for results of untreated and control vector pLKO.1, shRNA-<i>AURKA</i>, shRNA-<i>BRCA1</i>, shRNA-<i>HMMR</i>, or shRNA-<i>TPX2</i> transduced cultures of MCF10A cells in rBM. Magnification is equivalent for all images and scale bars represent 20 µm. (B) Acini architecture was quantified from bright-field images of cultures treated as described above. For comparison between experiments, all values were normalized to untreated cultures within experiments and differences assessed statistically relative to pLKO.1. Shape factor values for single cells, or small clusters, are not plotted. The graph shows the results of at least four independent experiments. For all graphs, asterisks and circles indicate significant differences (two-sided <i>t</i> test <i>p</i><0.05 and <i>p</i><0.005, respectively) from controls (pLKO.1). (C) Representative bright-field images of acini from concurrent depletions (shRNA-mediated) as indicated. (D) <i>AURKA-HMMR</i> interact in the regulation of polarization: <i>HMMR</i> depletion rescues the abnormality seen in the shRNA-<i>AURKA</i> assay. Graph shows the results of three independent experiments. (E) Quantification of acini per well confirms the genetic interaction between <i>AURKA</i> and <i>HMMR</i>. Graph shows the results of duplicate experiments. (F) <i>TPX2</i> depletion is suppressive to abnormalities caused by shRNA-<i>BRCA1</i> and shRNA-<i>HMMR</i>. Graph shows the results of at least three independent experiments. (G) Prior to the shRNA assays, published data proposed the hypothesis of a signaling pathway from TPX2 to RHAMM regulating polarization; degradation of the microtubule-associated factor RHAMM, through BRCA1, was predicted as key to polarization. However, several observations from the single and concurrent depletion assays (depleted proteins are indicated in grey font) diverged from the expected results (divergent observations are italicized). RHAMM depletion impaired polarization in a manner that was rescued by concurrent depletion of AURKA or TPX2, but not BRCA1. On the other hand, concurrent depletion of BRCA1 and TPX2 revealed normal acini.</p>