Final Report Summary - RHOGEFS IN EPITHELIA (The role of RhoGEFs in epithelial polarity using a three-dimensional model)
Manuel Gálvez-Santisteban, Alejo E Rodriguez-Fraticelli, Silvia Vergarajauregui, Ilenia Bernascone, Inmaculada Bañon and Fernando Martín-Belmonte
Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, C/Nicolás Cabrera 1, Madrid 28049, Spain
E-mail: fmartin@cbm.uam.es
See http://www.martinbelmontelab.net/fmb/Home.html for more details.
Project context and objectives
Epithelial organs are made of tubes and cavities lined by a monolayer of polarised cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. De novo epithelial lumen morphogenesis is a well-regulated process, which requires the orchestration of many different pathways in a very short period of time, especially internal membrane trafficking. Defects associated with these pathways lead to abnormalities in epithelial morphogenesis, and are common cause of major diseases like cancer (1).
1. A three-dimensional (3D)-functional-screening identifies a differential role for 15 candidate proteins in vertebrate epithelial morphogenesis.
During morphogenesis, gene expression is modified to accommodate the specific needs of individual cell types. However, how cell polarity is controlled at a transcriptional level during epithelial morphogenesis in vertebrates is poorly understood. We performed a two-step screening based on a transcriptomic analysis, followed by a functional screening with RNAi to identify new proteins required for lumen formation using MDCK 3D cell cultures. We identified 15 new candidate regulators of epithelial polarity and lumen formation, which are frequently downregulated in epithelial cancers, suggesting an importance in the maintenance of a differentiated epithelial phenotype in vivo (2). In addition, we characterised the mechanistic roles of Synaptotagmin-like protein 2a/exophilin (Slp2a), identified in the screening, and Slp4, another member of this family of Rab27-effectors. Slp2a localises to apical membranes and cell-cell junctions during lumen formation in a PtdIns4,5p2-dependent manner and is required to target Rab27-loaded vesicles to initiate the lumen. Slp4/granulophilin, which is also induced during morphogenesis and depends on Slp2a for proper function, is involved in the tethering of apical vesicles to form the lumen. The function and localisation of Slp4 depends on Rab27/Rab3 and the t-SNARE protein syntaxin-3. Thus showing two members of the same family of proteins functioning in the same cellular process at different levels.
Transcriptional and functional screenings have demonstrated to be powerful tools to identify novel factors in many biological processes. For instance, some functional screenings using RNAi techniques have been previously performed to identify regulators of epithelial morphogenesis. However, these studies were based in screenings targeting specific subset of genes, such as apical proteins or activators for Rho-family GTPases (3)(4), instead of a genome-wide screenings as the one performed by our laboratory. Therefore, we show here how a wider screening is a useful tool to identify specific regulators of epithelial morphogenesis that were never shown before related to this process. Interestingly, most of the potential novel regulators of lumen formation identified in the screening were for the most part uncharacterised proteins, and their functions and mechanism in epithelial morphogenesis are still to be elucidated. In addition, most of the genes identified to be upregulated during morphogenesis have been shown to be downregulated in many epithelial cancers, indicating they might be acting as tumour suppressors and thus providing potential clinical relevance.
2. ITSN2 is Ccd42 RhoGEF required for spindle orientation and epithelial morphogenesis.
The Rho guanosine tri-phosphatase (GTPase) Cdc42, which is a master regulator of cell polarity (5), regulates the formation of the central lumen in epithelial morphogenesis (6)(7). However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the 3D Madin-Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localises to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects (8).
A wealth of knowledge about the mechanisms that regulate the establishment of cell polarity has unravelled their important contributions to preventing the acquisition of tumorigenic characteristics (9). Given the crucial roles of Rho GTPases in the regulation of several pathways that are required for the proper function and proliferation of epithelial tissues, including the establishment of cell adhesion and the regulation of cell growth, they can be potential clinical prognostic factors. Future research will shed light on the molecular nature of their regulation to possibly influence their assembly through therapeutic interventions. Loss of the finely tuned functions of the small GTPases may also contribute to the early stages of tumorigenesis. The role of Rho GTPases, and other epithelial polarity proteins, in regulating the orientation of centrosomes and mitotic spindles, and thus asymmetric cell division, may affect the balance of proliferation and differentiation in epithelial tissues. Understanding the genetic and biochemical mechanisms that control self-renewal and asymmetric division and understanding the role of the stem cell niche in regulating the biological properties of both normal and cancer stem cells are key issues for the future, because preventing these abnormalities may limit the proportion of cells that fuel tumour growth in carcinomas.
References:
1. Rodriguez-Fraticelli, A. E., Galvez-Santisteban, M., Martin-Belmonte, F. Divide and polarize: recent advances in the molecular mechanism regulating epithelial tubulogenesis. Curr Opin Cell Biol 23, 638 - 46 (2011);
2. Gálvez-Santisteban, M. R.-F. Alejo E.; David M. Bryant, Silvia Vergarajauregui, Anirban Datta, Natalie Spivak, Kitty Young, Christiaan L. Slim, Paul R. Brakeman, Mitsunori Fukuda, Keith E. Mostov, and Martín-Belmonte, F. Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells. Nat Cell Biol in revision (2011);
3. Quyn, A.J. et al. Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue. Cell Stem Cell 6, 175 - 81 (2010);
4. Torkko, J. M., Manninen, A., Schuck, S., Simons, K. Depletion of apical transport proteins perturbs epithelial cyst formation and ciliogenesis. J Cell Sci 121, 1193 - 203 (2008);
5. Etienne-Manneville, S. Cdc42-the centre of polarity. J Cell Sci 117, 1291 - 300 (2004);
6. Martin-Belmonte, F. et al. PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell 128, 383 - 97 (2007);
7. Martin-Belmonte, F. et al. Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis. Curr Biol 18, 507 - 13 (2008);
8. Rodriguez-Fraticelli, A. E. et al. The Cdc42 GEF Intersectin 2 controls mitotic spindle orientation to form the lumen during epithelial morphogenesis. J Cell Biol 189, 725-38 (2010);
9. Martin-Belmonte, F., Perez-Moreno, M. Epithelial cell polarity, stem cells and cancer. Nature reviews. Cancer 12, 23-38 (2011).
Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, C/Nicolás Cabrera 1, Madrid 28049, Spain
E-mail: fmartin@cbm.uam.es
See http://www.martinbelmontelab.net/fmb/Home.html for more details.
Project context and objectives
Epithelial organs are made of tubes and cavities lined by a monolayer of polarised cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. De novo epithelial lumen morphogenesis is a well-regulated process, which requires the orchestration of many different pathways in a very short period of time, especially internal membrane trafficking. Defects associated with these pathways lead to abnormalities in epithelial morphogenesis, and are common cause of major diseases like cancer (1).
1. A three-dimensional (3D)-functional-screening identifies a differential role for 15 candidate proteins in vertebrate epithelial morphogenesis.
During morphogenesis, gene expression is modified to accommodate the specific needs of individual cell types. However, how cell polarity is controlled at a transcriptional level during epithelial morphogenesis in vertebrates is poorly understood. We performed a two-step screening based on a transcriptomic analysis, followed by a functional screening with RNAi to identify new proteins required for lumen formation using MDCK 3D cell cultures. We identified 15 new candidate regulators of epithelial polarity and lumen formation, which are frequently downregulated in epithelial cancers, suggesting an importance in the maintenance of a differentiated epithelial phenotype in vivo (2). In addition, we characterised the mechanistic roles of Synaptotagmin-like protein 2a/exophilin (Slp2a), identified in the screening, and Slp4, another member of this family of Rab27-effectors. Slp2a localises to apical membranes and cell-cell junctions during lumen formation in a PtdIns4,5p2-dependent manner and is required to target Rab27-loaded vesicles to initiate the lumen. Slp4/granulophilin, which is also induced during morphogenesis and depends on Slp2a for proper function, is involved in the tethering of apical vesicles to form the lumen. The function and localisation of Slp4 depends on Rab27/Rab3 and the t-SNARE protein syntaxin-3. Thus showing two members of the same family of proteins functioning in the same cellular process at different levels.
Transcriptional and functional screenings have demonstrated to be powerful tools to identify novel factors in many biological processes. For instance, some functional screenings using RNAi techniques have been previously performed to identify regulators of epithelial morphogenesis. However, these studies were based in screenings targeting specific subset of genes, such as apical proteins or activators for Rho-family GTPases (3)(4), instead of a genome-wide screenings as the one performed by our laboratory. Therefore, we show here how a wider screening is a useful tool to identify specific regulators of epithelial morphogenesis that were never shown before related to this process. Interestingly, most of the potential novel regulators of lumen formation identified in the screening were for the most part uncharacterised proteins, and their functions and mechanism in epithelial morphogenesis are still to be elucidated. In addition, most of the genes identified to be upregulated during morphogenesis have been shown to be downregulated in many epithelial cancers, indicating they might be acting as tumour suppressors and thus providing potential clinical relevance.
2. ITSN2 is Ccd42 RhoGEF required for spindle orientation and epithelial morphogenesis.
The Rho guanosine tri-phosphatase (GTPase) Cdc42, which is a master regulator of cell polarity (5), regulates the formation of the central lumen in epithelial morphogenesis (6)(7). However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the 3D Madin-Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localises to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects (8).
A wealth of knowledge about the mechanisms that regulate the establishment of cell polarity has unravelled their important contributions to preventing the acquisition of tumorigenic characteristics (9). Given the crucial roles of Rho GTPases in the regulation of several pathways that are required for the proper function and proliferation of epithelial tissues, including the establishment of cell adhesion and the regulation of cell growth, they can be potential clinical prognostic factors. Future research will shed light on the molecular nature of their regulation to possibly influence their assembly through therapeutic interventions. Loss of the finely tuned functions of the small GTPases may also contribute to the early stages of tumorigenesis. The role of Rho GTPases, and other epithelial polarity proteins, in regulating the orientation of centrosomes and mitotic spindles, and thus asymmetric cell division, may affect the balance of proliferation and differentiation in epithelial tissues. Understanding the genetic and biochemical mechanisms that control self-renewal and asymmetric division and understanding the role of the stem cell niche in regulating the biological properties of both normal and cancer stem cells are key issues for the future, because preventing these abnormalities may limit the proportion of cells that fuel tumour growth in carcinomas.
References:
1. Rodriguez-Fraticelli, A. E., Galvez-Santisteban, M., Martin-Belmonte, F. Divide and polarize: recent advances in the molecular mechanism regulating epithelial tubulogenesis. Curr Opin Cell Biol 23, 638 - 46 (2011);
2. Gálvez-Santisteban, M. R.-F. Alejo E.; David M. Bryant, Silvia Vergarajauregui, Anirban Datta, Natalie Spivak, Kitty Young, Christiaan L. Slim, Paul R. Brakeman, Mitsunori Fukuda, Keith E. Mostov, and Martín-Belmonte, F. Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells. Nat Cell Biol in revision (2011);
3. Quyn, A.J. et al. Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue. Cell Stem Cell 6, 175 - 81 (2010);
4. Torkko, J. M., Manninen, A., Schuck, S., Simons, K. Depletion of apical transport proteins perturbs epithelial cyst formation and ciliogenesis. J Cell Sci 121, 1193 - 203 (2008);
5. Etienne-Manneville, S. Cdc42-the centre of polarity. J Cell Sci 117, 1291 - 300 (2004);
6. Martin-Belmonte, F. et al. PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell 128, 383 - 97 (2007);
7. Martin-Belmonte, F. et al. Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis. Curr Biol 18, 507 - 13 (2008);
8. Rodriguez-Fraticelli, A. E. et al. The Cdc42 GEF Intersectin 2 controls mitotic spindle orientation to form the lumen during epithelial morphogenesis. J Cell Biol 189, 725-38 (2010);
9. Martin-Belmonte, F., Perez-Moreno, M. Epithelial cell polarity, stem cells and cancer. Nature reviews. Cancer 12, 23-38 (2011).
