Genetic interactions influencing epithelial apicobasal polarization.

2011-11-15T01:14:06Z (GMT) by Christopher A. Maxwell Javier Benitez Laia Gómez-Baldó Ana Osorio Núria Bonifaci Ricardo Fernández-Ramires Sylvain V. Costes Elisabet Guinó Helen Chen Gareth J. R. Evans Pooja Mohan Isabel Català Anna Petit Helena Aguilar Alberto Villanueva Alvaro Aytes Jordi Serra-Musach Gad Rennert Flavio Lejbkowicz Paolo Peterlongo Siranoush Manoukian Bernard Peissel Carla B. Ripamonti Bernardo Bonanni Alessandra Viel Anna Allavena Loris Bernard Paolo Radice Eitan Friedman Bella Kaufman Yael Laitman Maya Dubrovsky Roni Milgrom Anna Jakubowska Cezary Cybulski Bohdan Gorski Katarzyna Jaworska Katarzyna Durda Grzegorz Sukiennicki Jan Lubinski Yin Yao Shugart Susan M. Domchek Richard Letrero Barbara L. Weber Frans B. L. Hogervorst Matti A. Rookus J. Margriet Collee Peter Devilee Marjolijn J. Ligtenberg Rob B. van der Luijt Cora M. Aalfs Quinten Waisfisz Juul Wijnen Cornelis E. P. van Roozendaal Douglas F. Easton Susan Peock Margaret Cook Clare Oliver Debra Frost Patricia Harrington D. Gareth Evans Fiona Lalloo Rosalind Eeles Louise Izatt Carol Chu Diana Eccles Fiona Douglas Carole Brewer Heli Nevanlinna Tuomas Heikkinen Fergus J. Couch Noralane M. Lindor Xianshu Wang Andrew K. Godwin Maria A. Caligo Grazia Lombardi Niklas Loman Per Karlsson Hans Ehrencrona Anna von Wachenfeldt Rosa Bjork Barkardottir Ute Hamann Muhammad U. Rashid Adriana Lasa Trinidad Caldés Raquel Andrés Michael Schmitt Volker Assmann Kristen Stevens Kenneth Offit João Curado Hagen Tilgner Roderic Guigó Gemma Aiza Joan Brunet Joan Castellsagué Griselda Martrat Ander Urruticoechea Ignacio Blanco Laima Tihomirova David E. Goldgar Saundra Buys Esther M. John Alexander Miron Melissa Southey Mary B. Daly Rita K. Schmutzler Barbara Wappenschmidt Alfons Meindl Norbert Arnold Helmut Deissler Raymonda Varon-Mateeva Christian Sutter Dieter Niederacher Evgeny Imyamitov Olga M. Sinilnikova Dominique Stoppa-Lyonne Sylvie Mazoyer Carole Verny-Pierre Laurent Castera Antoine de Pauw Yves-Jean Bignon Nancy Uhrhammer Jean-Philippe Peyrat Philippe Vennin Sandra Fert Ferrer Marie-Agnès Collonge-Rame Isabelle Mortemousque Amanda B. Spurdle Jonathan Beesley Xiaoqing Chen Sue Healey Mary Helen Barcellos-Hoff Marc Vidal Stephen B. Gruber Conxi Lázaro Gabriel Capellá Lesley McGuffog Katherine L. Nathanson Antonis C. Antoniou Georgia Chenevix-Trench Markus C. Fleisch Víctor Moreno Miguel Angel Pujana
<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>