Figure_4.tif (998.7 kB)

Genetic interactions influencing epithelial apicobasal polarization.

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posted on 2011-11-15, 01:14 authored by Christopher A. Maxwell, Javier Benítez, 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 Lubiński, 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

(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-AURKA, shRNA-BRCA1, shRNA-HMMR, or shRNA-TPX2 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 t test p<0.05 and p<0.005, respectively) from controls (pLKO.1). (C) Representative bright-field images of acini from concurrent depletions (shRNA-mediated) as indicated. (D) AURKA-HMMR interact in the regulation of polarization: HMMR depletion rescues the abnormality seen in the shRNA-AURKA assay. Graph shows the results of three independent experiments. (E) Quantification of acini per well confirms the genetic interaction between AURKA and HMMR. Graph shows the results of duplicate experiments. (F) TPX2 depletion is suppressive to abnormalities caused by shRNA-BRCA1 and shRNA-HMMR. 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.