Trans-sialidase Stimulates Eat Me Response from Epithelial Cells

Epithelial cell invasion by the protozoan parasite Trypanosoma cruzi is enhanced by the presence of an enzyme expressed on its cell surface during the trypomastigote life cycle stage. The enzyme, trans-sialidase (TS), is a member of one of the largest gene families expressed by the parasite and the role of its activity in mediating epithelial cell entry has not hitherto been understood. Here we show that the T. cruzi TS generates an eat me signal which is capable of enabling epithelial cell entry. We have utilized purified, recombinant, active (TcTS) and inactive (TcTS2V0) TS coated onto beads to challenge an epithelial cell line. We find that TS activity acts upon G protein coupled receptors present at the epithelial cell synapse with the coated bead, thereby enhancing cell entry. By so doing, we provide evidence that TS proteins bind glycans, mediate the formation of distinct synaptic domains and promote macropinocytotic uptake of microparticles into a perinuclear compartment in a manner which may emulate entosis

Polarized secretion of Leukemia Inhibitory Factor

<p>Abstract</p> <p>Background</p> <p>The direction of cytokine secretion from polarized cells determines the cytokine's cellular targets. Leukemia inhibitory factor (LIF) belongs to the interleukin-6 (IL-6) family of cytokines and signals through LIFR/gp130. Three factors which may regulate the direction of LIF secretion were studied: the site of stimulation, signal peptides, and expression levels. Stimulation with IL-1β is known to promote IL-6 secretion from the stimulated membrane (apical or basolateral) in the human intestinal epithelial cell line Caco-2. Since LIF is related to IL-6, LIF secretion was also tested in Caco-2 following IL-1β stimulation. Signal peptides may influence the trafficking of LIF. Two isoforms of murine LIF, LIF-M and LIF-D, encode different signal peptides which have been associated with different locations of the mature protein in fibroblasts. To determine the effect of the signal peptides on LIF secretion, secretion levels were compared in Madin-Darby canine kidney (MDCK) clones which expressed murine LIF-M or LIF-D or human LIF under the control of an inducible promoter. Low and high levels of LIF expression were also compared since saturation of the apical or basolateral route would reveal specific transporters for LIF.</p> <p>Results</p> <p>When Caco-2 was grown on permeable supports, LIF was secreted constitutively with around 40% secreted into the apical chamber. Stimulation with IL-1β increased LIF production. After treating the apical surface with IL-1β, the percentage secreted apically remained similar to the untreated, whereas, when the cells were stimulated at the basolateral surface only 20% was secreted apically. In MDCK cells, an endogenous LIF-like protein was detected entirely in the apical compartment. The two mLIF isoforms showed no difference in their secretion patterns in MDCK. Interestingly, about 70% of murine and human LIF was secreted apically from MDCK over a 400-fold range of expression levels within clones and a 200,000-fold range across clones.</p> <p>Conclusion</p> <p>The site of stimulation affected the polarity of LIF secretion, while, signal peptides and expression levels did not. Exogenous LIF is transported in MDCK without readily saturated steps.</p

Two Distinct Integrin-Mediated Mechanisms Contribute to Apical Lumen Formation in Epithelial Cells

Background: Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of b1-integrins in epithelial polarization is well established but the significance of the accompanying a-subunits have not been analyzed in detail. Principal Findings: Here we demonstrate that two distinct integrin-dependent pathways regulate formation of apical lumens to ensure robust apical membrane biogenesis under different microenvironmental conditions; 1) a2b1- and a6b4integrins were required to establish a basal cue that depends on Rac1-activity and guides apico-basal cell polarization. 2) a3b1-integrins were implicated in positioning of mitotic spindles in cysts, a process that is essential for Cdc42-driven epithelial hollowing. Significance: Identification of the separate processes driven by particular integrin receptors clarifies the functional hierarchies between the different integrins co-expressed in epithelial cells and provides valuable insight into the complexity of cell-ECM interactions thereby guiding future studies addressing the molecular basis of epithelial morphogenesis durin

Negatively-stained polysomes on rough microsome vesicles viewed by electron microscopy: further evidence regarding the orientation of attached ribosomes

Rough microsomes, derived from rough endoplasmic reticulum of rat liver, were studied by electron microscopy after negative staining, to seek further information about the orientation of ribosomal small and large subunits in bound polysomes. Rough microsomal vesicles were fixed with 2% formaldehyde, centrifuged onto electron-microscopic grid membranes, and were then negatively-stained with 2% phosphotungstic acid. In these preparations, viewed with the electron microscope, flattened rough microsomal vesicles with bound polysomes were sometimes discernible, and the individual ribosomes in the polysomes occasionally showed small and large subunits. The small subunits were uniformly oriented toward the inside of the polysomal curve. The large and small subunits appeared to be alongside one another on the membrane, consistent with the orientation that has been described by Unwin and his co-workers. The boundary between the small and large subunits occurred at approximately the same level in the ribosome where inter-ribosomal strands have been described previously in surface views of bound polysomes in positively-stained electron-microscopic tissue sections. This further confirms the identity of the strands as messenger RNA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47690/1/441_2004_Article_BF00343942.pd

Lowe Syndrome Protein OCRL1 Supports Maturation of Polarized Epithelial Cells

Mutations in the inositol polyphosphate 5-phosphatase OCRL1 cause Lowe Syndrome, leading to cataracts, mental retardation and renal failure. We noted that cell types affected in Lowe Syndrome are highly polarized, and therefore we studied OCRL1 in epithelial cells as they mature from isolated individual cells into polarized sheets and cysts with extensive communication between neighbouring cells. We show that a proportion of OCRL1 targets intercellular junctions at the early stages of their formation, co-localizing both with adherens junctional components and with tight junctional components. Correlating with this distribution, OCRL1 forms complexes with junctional components α-catenin and zonula occludens (ZO)-1/2/3. Depletion of OCRL1 in epithelial cells growing as a sheet inhibits maturation; cells remain flat, fail to polarize apical markers and also show reduced proliferation. The effect on shape is reverted by re-expressed OCRL1 and requires the 5′-phosphatase domain, indicating that down-regulation of 5-phosphorylated inositides is necessary for epithelial development. The effect of OCRL1 in epithelial maturation is seen more strongly in 3-dimensional cultures, where epithelial cells lacking OCRL1 not only fail to form a central lumen, but also do not have the correct intracellular distribution of ZO-1, suggesting that OCRL1 functions early in the maturation of intercellular junctions when cells grow as cysts. A role of OCRL1 in junctions of polarized cells may explain the pattern of organs affected in Lowe Syndrome

A Sub-Cellular Viscoelastic Model for Cell Population Mechanics

Understanding the biomechanical properties and the effect of biomechanical force on epithelial cells is key to understanding how epithelial cells form uniquely shaped structures in two or three-dimensional space. Nevertheless, with the limitations and challenges posed by biological experiments at this scale, it becomes advantageous to use mathematical and ‘in silico’ (computational) models as an alternate solution. This paper introduces a single-cell-based model representing the cross section of a typical tissue. Each cell in this model is an individual unit containing several sub-cellular elements, such as the elastic plasma membrane, enclosed viscoelastic elements that play the role of cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane is divided into segments where each segment (or point) incorporates the cell's interaction and communication with other cells and its environment. The model is capable of simulating how cells cooperate and contribute to the overall structure and function of a particular tissue; it mimics many aspects of cellular behavior such as cell growth, division, apoptosis and polarization. The model allows for investigation of the biomechanical properties of cells, cell-cell interactions, effect of environment on cellular clusters, and how individual cells work together and contribute to the structure and function of a particular tissue. To evaluate the current approach in modeling different topologies of growing tissues in distinct biochemical conditions of the surrounding media, we model several key cellular phenomena, namely monolayer cell culture, effects of adhesion intensity, growth of epithelial cell through interaction with extra-cellular matrix (ECM), effects of a gap in the ECM, tensegrity and tissue morphogenesis and formation of hollow epithelial acini. The proposed computational model enables one to isolate the effects of biomechanical properties of individual cells and the communication between cells and their microenvironment while simultaneously allowing for the formation of clusters or sheets of cells that act together as one complex tissue

Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

<p>Abstract</p> <p>Background</p> <p>Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled <it>in vitro </it>by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.</p> <p>Results</p> <p>Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase.</p> <p>Conclusion</p> <p>Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis.</p

Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol‐anchored proteins in biosynthetic and recycling routes in Madin‐Darby canine kidney cells

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal-to-apical transcytosis routes. The apical distribution of the t-SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical-destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI-APs in polarized MDCK cells