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

The formin INF2 regulates basolateral-to-apical transcytosis and lumen formation in association with Cdc42 and MAL2

Transcytosis is a widespread pathway for apical targeting in epithelial cells. MAL2, an essential protein of the machinery for apical transcytosis, functions by shuttling in vesicular carriers between the apical zone and the cell periphery. We have identified INF2, an atypical formin with actin polymerization and depolymerization activities, which is a binding partner of MAL2. MAL2-positive vesicular carriers associate with short actin filaments during transcytosis in a process requiring INF2. INF2 binds Cdc42 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are necessary for apical transcytosis and the formation of lateral lumens in hepatoma HepG2 cells. INF2 and MAL2 are also essential for the formation of the central lumen in organotypic cultures of epithelial MDCK cells. Our results reveal a functional mechanism whereby Cdc42, INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation. © 2010 Elsevier Inc.This work was supported by grants (BFU2006-01925, BFU2009-07886, and CONSOLIDER COAT CSD2009-00016) to M.A.A. from the Ministerio de Ciencia e Innovación (MICINN), Spain. R.M. is the holder of a contract from the Ramón y Cajal Program of the MICINN. The authors declare no competing financial interests

Genome wide analysis of gene expression changes in skin from patients with type 2 diabetes

Non-healing chronic ulcers are a serious complication of diabetes and are a major healthcare problem. While a host of treatments have been explored to heal or prevent these ulcers from forming, these treatments have not been found to be consistently effective in clinical trials. An understanding of the changes in gene expression in the skin of diabetic patients may provide insight into the processes and mechanisms that precede the formation of non-healing ulcers. In this study, we investigated genome wide changes in gene expression in skin between patients with type 2 diabetes and non-diabetic patients using next generation sequencing. We compared the gene expression in skin samples taken from 27 patients (13 with type 2 diabetes and 14 non-diabetic). This information may be useful in identifying the causal factors and potential therapeutic targets for the prevention and treatment of diabetic related diseases

Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell-cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell-cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesi

Myosin II isoforms play distinct roles in adherens junction biogenesis

International audienceAdherens junction (AJ) assembly under force is essential for many biological processes like epithelial monolayer bending, collective cell migration, cell extrusion and wound healing. The acto-myosin cytoskeleton acts as a major force-generator during the de novo formation and remodeling of AJ. Here, we investigated the role of non-muscle myosin II isoforms (NMIIA and NMIIB) in epithelial junction assembly. NMIIA and NMIIB differentially regulate biogenesis of AJ through association with distinct actin networks. Analysis of junction dynamics, actin organization, and mechanical forces of control and knockdown cells for myosins revealed that NMIIA provides the mechanical tugging force necessary for cell-cell junction reinforcement and maintenance. NMIIB is involved in E-cadherin clustering, maintenance of a branched actin layer connecting E-cadherin complexes and perijunctional actin fibres leading to the building-up of anisotropic stress. These data reveal unanticipated complementary functions of NMIIA and NMIIB in the biogenesis and integrity of AJ

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

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