Developmental regulation of apical endocytosis controls epithelial patterning in vertebrate tubular organs

© 2015 Macmillan Publishers Limited. Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.MINECO (BFU2011-22622) and CONSOLIDER (CSD2009-00016); Fundación Obra Social `La Caixa' PhD fellowship. G.A. was supported by the Amarouto Program for senior researchers from the Comunidad Autónoma de Madrid.Peer Reviewe

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe Smoothelin-like 2 (SMTNL2) function, a member of the smooth-muscle related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during the development of multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of Coronin-1B. While Coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to Coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular corte

Identificación y estudio de nuevos mecanismos moleculares implicados en la morfogénesis epitelial: mecanotransducción, orientación del huso mitótico y endocitosis

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 11-07-2014Epithelial cells represent the founding stone at the dawn of metazoan evolution. Epithelial cell polarity is apparent in the organization of different membrane domains, which carry out essential functions in the animal, such as nutrient uptake and excretion. Epithelial tissues are frequently convoluted and wrapped inside the animal body, in the form of tubes, providing protection from the environment and augmenting the exchange surface for different physiological functions. Epithelial organ morphogenesis is the process by which epithelial cells proliferate and organize the three-dimensional architecture of the final organ while generating and maintaining the polarized phenotype. Using the 3D-MDCK cell culture system and zebrafish gut morphogenesis as in vitro and in vivo models of epithelial morphogenesis we have investigated three mechanisms that regulate epithelial morphogenesis in vertebrates. Firstly, through use of adhesive micropatterns, we have characterized that spatial confinement of cell adhesion provides essential mechanophysical cues for the acquisition of 3D epithelial polarity. Second, through the analysis of the small GTPase Cdc42, a master regulator of cell polarity, we have found that mitotic spindle formation and orientation are required for the maintenance of planar symmetric cell divisions, which is necessary for single lumen formation. Finally, we characterized a specific gene set induced in vivo and in vitro during epithelial morphogenesis, and we contributed to elucidate that one of these genes is responsible for the fine regulation of endocytosis during development to control the process of epithelial morphogenesis and differentiation. In conclusion, we have analyzed three mechanisms involved in the process of epithelial morphogenesis in vertebrates. Furthermore, these mechanisms are common to most epithelial glands, and therefore provide essential knowledge on how polarity and proliferation are controlled during development, and point to new approaches to unravel the process of polarity loss and dysplasia at the origin of carcinomagenesi

Methods for analysis of apical lumen trafficking using micropatterned 3D systems

Epithelial organs are made of interconnected branched networks of tubules, with a central lumen lined by a monolayer of epithelial cells. Certain epithelial cell lines can be converted into organotypic cultures by the addition of extracellular matrix components. When cultured in these conditions, epithelial cells reorient the axis of polarity, reorganize the membrane surfaces, and transport apical proteins to form the lumen in a process that recapitulates essential aspects of de novo apical membrane formation during epithelial organ morphogenesis. Micropatterns are a simple technique that allows cell culture in a controlled adhesive environment with extremely high precision, close to the nanometer scale. We have recently developed a method to culture MDCK cysts on micropatterns of different sizes and composition. Using this method we found that changes in micropattern shape and size can be used to modify cell contractility to understand its contribution to apical membrane formation. When imaging cysts on micropatterns the main advantage is that apical-directed vesicle trafficking is visualized in the x- y plane, which presents higher resolution on confocal microscopes. Thus, the use of micropatterns is an efficient setup to analyze polarized secretion with unprecedented higher resolution in both time and space. © 2013 Elsevier Inc.Human Frontiers Science Program (HFSP-CDA 00011/2009); MICINN (BFU2011-22622), CONSOLIDER (CSD2009-00016); CSIC, Fundación Ramón ArecesPeer Reviewe

Mechanical control of epithelial lumen formation

Epithelial cells differentiate and polarize to build complete epithelial organs during development. The study of epithelial morphogenesis is instrumental to the understanding of disease processes where epithelial polarity is disrupted. Recently, we demonstrated that matrix-induced cell confinement controls the acquisition of three-dimensional epithelial polarity, by modulating the initiation of the apical membrane to form a central lumen (J Cell Biol 2012; 198:1011-1026). Cell confinement can be achieved by use of micropatterned culture chips that allow precise micrometric-scale control of the cell adhesion surface and its composition. Using micropattern chips, we demonstrated that polarizing epithelial cells require high confinement conditions to properly position the centrosome and the trafficking machinery toward the cell-cell contacts and to initiate lumen morphogenesis. Low confinement induces LKB1 and RhoA-mediated cell contractility, which inhibits this mechanism for lumen formation. Deactivation of Myosin-II-mediated contractility rescued normal lumen initiation in low confinement conditions. Our results indicate that a mechanotransduction pathway coordinates nuclear and centrosome positioning to initiate epithelial morphogenesis. Here we discuss the potential candidates that control this process, specifically the polarized activation of Rho and Rab-family GTPases, and also a group of recently characterized nuclear transcription factors. © Landes Bioscience.Human Frontiers Science Program (HFSP-CDA 00011/2009); MICINN (BFU2011-22622) and CONSOLIDER (CSD2009-00016); CSIC; Fundación Ramón ArecesPeer Reviewe

Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis

Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA-Rho kinase-myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear-centrosomal orientation and lumen initiation during 3D epithelial morphogenesis. © 2012 Rodríguez-Fraticelli et al.Human Frontiers Science Program; Marie Curie, Ministerio de Ciencia e Innovación; CONSOLIDERPeer Reviewe

Divide and polarize: recent advances in the molecular mechanism regulating epithelial tubulogenesis

Epithelial organs are generated from groups of non-polarized cells by a combination of processes that induce the acquisition of cell polarity, lumen formation, and the subsequent steps required for tubulogenesis. The subcellular mechanisms associated to these processes are still poorly understood. The extracellular environment provides a cue for the initial polarization, while cytoskeletal rearrangements build up the three-dimensional architecture that supports the central lumen. The proper orientation of cell division in the epithelium has been found to be required for the normal formation of the central lumen in epithelial morphogenesis. Moreover, recent data in cellular models and in vivo have shed light into the underlying mechanisms that connect the spindle orientation machinery with cell polarity. In addition, recent work has clarified the core molecular components of the vesicle trafficking machinery in epithelial morphogenesis, including Rab-GTPases and the Exocyst, as well as an increasing list of microtubule-binding and actin-binding proteins and motors, most of which are conserved from yeast to humans. In this review we will focus on the discussion of novel findings that have unveiled important clues for the mechanisms that regulate epithelial tubulogenesis.Work supported by grants from Human Frontiers Science Program (HFSP-CDA 00011/2009), Marie Curie (IRG-209382), MICINN (BFU2008-01916) and CONSOLIDER (CSD2009-00016) to FM-B. AER-F is recipient of a JAE fellowship, from CSIC, and MG is recipient of a FPI fellowship, from MICINN. An institutional grant from Fundación Ramón Areces to CBMSO is also acknowledged.Peer reviewe

Methods and a device for the formation of three-dimensional multicellular assemblies

[EN] The present invention relates to devices and associated methods for forming three- dimensional multicellular assemblies in vitro. Specifically, the present invention relates to devices comprising at least one three-dimensional multicellular assembly immobilised on a two-dimensional adhesive pattern, wherein said three-dimensional multicellular assembly has an organised structure with a normalised polarity and methods for the formation of three-dimensional multicellular assemblies having an organised structure.Peer reviewedCYTOO (Francia), Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagminlike proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27- loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell.Human Frontiers Science Program (HFSP-CDA 00011/2009); Marie Curie (IRG-209382); MICINN (BFU2008-01916, BFU2011-22622); CONSOLIDER (CSD2009-00016) ; The March of Dimes Basil O’Connor Starter Research Award; Fundacion Ramon ArecesPeer Reviewe