Karl S. Matlin

Integrins in epithelial cell polarity: Using antibodies to analyze adhesive function and morphogenesis

Epithelial cells polarize in response to cell-substratum and cell-cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin. Investigation of the roles of integrins in epithelial cell polarization has depended on the use of function-blocking antibodies both to determine ligand specificity of individual integrins and to disrupt and redirect normal morphogenesis. In this article we describe techniques for employing function-blocking anti-integrin antibodies in adhesion assays of the polarized Madin-Darby canine kidney (MDCK) cell line and to demonstrate the involvement of beta1 integrins in collagen-induced tubulocyst formation. These techniques can be easily expanded to other antibodies and epithelial cell lines to characterize specific functions of individual integrins in epithelial morphogenesis.

Autocrine Transforming Growth Factor-β1 Activation Mediated by Integrin αVβ3 Regulates Transcriptional Expression of Laminin-332 in Madin-Darby Canine Kidney Epithelial Cells

The expression of the extracellular matrix protein Laminin-332 is regulated transcriptionally by TGF-β1 as a function of cell confluence in MDCK epithelial cells. Latent TGF-β1 is secreted apically, sequestered from its receptors and activation machinery, dependent on integrin αVβ3, localized on the basolateral side of the epithelial barrier.

The Protein Kinase A Pathway Contributes to Hg2+-Induced Alterations in Phosphorylation and Subcellular Distribution of Occludin Associated with Increased Tight Junction Permeability of Salivary Epithelial Cell Monolayers

Hg2+ is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg2+ affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg2+ decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg2+-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg2+ treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg2+-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögrens syndrome, and possibly other disorders of fluid transport.

Activated PKCδ and PKCϵ Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl− Cotransporter NKCC1

The basolateral Na+-K+-2Cl− cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ϵ) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCϵ- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCϵ-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCϵ, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.

Laminin 511 partners with laminin 332 to mediate directional migration of Madin–Darby canine kidney epithelial cells

Directional migration of MDCK cells is regulated by the ratio of the deposited basement membrane proteins laminin-511 and laminin-332. Knockdown of laminin-511 or its receptor integrin α3 inhibits directional migration and destabilizes cell–cell contacts, disturbing the polarization machinery.

Medical Education Reform in Wuhan University, China: A Preliminary Report of an International Collaboration

Background : In 2008 Wuhan University Medical School in China proposed to reform its curriculum by adapting the curriculum of the University of Chicago Medical School. Description : An assessment of Wuhan University Medical Schools traditional curriculum conducted in 2009 informed the reform directions, which included course integration, use of clinical cases, improved relevance of basic sciences to clinical medicine, reduction of lecture time, increase in group and independent learning, and the use of formative assessments. Fifty student volunteers per year were chosen to participate in the reform, and the rest remained in the traditional curriculum. Evaluation : A student survey was conducted in 2011 to evaluate the reform by comparing the attitudes of those in the reform and standard curricula. Conclusions : The reform met the needs of the school, was generally well received, improved satisfaction in reform participants, and had a positive impact on students. Areas needing improvement were also identified.

αV-Integrins Are Required for Mechanotransduction in MDCK Epithelial Cells

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2β1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2β1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.

Mechanical Stimulation Increases Collagen Type I and Collagen Type III Gene Expression of Stem Cell: Collagen Sponge Constructs for Patellar Tendon Repair

Tendons (rotator cuff, Achilles and patellar tendons) are among the most commonly injured soft tissues [1]. Many repairs/reconstructions have been attempted using sutures, resorbable biomaterials, autografts, and allografts, but with varying success. A tissue engineered repair using mesenchymal stem cells (MSCs) is attractive [2–4] but often lacks initial stiffness and strength [5].Copyright

The strange case of the signal recognition particle

The discovery of the signal-recognition particle (SRP) and its receptor represented a huge step forwards in the study of protein translocation and secretion. Just as intriguing was the race to identify SRP, as two teams — one based in New York, the other in Heidelberg — took up the quest and scored complementary victories.

When Cell Biology Grew Up

On October 11, 1999 Günter Blobel of Rockefeller University in New York was awarded the Nobel Prize for Physiology or Medicine for his work on protein transport (1). The signal hypothesis, which formed the basis for his studies, was first described in a brief article by Blobel and Sabatini in 1971 (2). In it, they proposed that ribosomes bound to the surface of the endoplasmic reticulum are directed there by the amino-termini of nascent polypeptide chains. With continued synthesis, the polypeptides are extruded into the lumen and, upon completion, ribosomes and messenger RNA dissociate and return to a soluble cytoplasmic pool. Later, in 1975, Blobel and Dobberstein provided the first strong evidence for the hypothesis in back-to-back seminal papers published in the Journal of Cell Biology (3,4). Upon winning the Prize, press reports widely heralded Blobel’s discovery of protein addresses or zip codes, as signal sequences were called. The importance of Blobel’s work, however, extends far beyond signal sequences. His accomplishments, one can argue, represent the beginnings of molecular cell biology.