Marcel Vergés

Membrane traffic in polarized epithelial cells

Epithelial cells contain apical and basolateral surfaces with distinct compositions. Sorting of certain proteins to the basolateral surface involves the epithelial-specific mu 1b clathrin adaptor subunit. Recent results have shown that targeting to the basolateral surface utilizes the exocyst, whereas traffic to the apical surface uses syntaxin 3. Endocytosis at the apical surface is regulated by ARF6. Transcytosis of IgA is regulated by the p62Yes tyrosine kinase.

The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor

Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions. After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface. Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan. The polymeric immunoglobulin receptor (pIgR) transcytoses polymeric IgA (pIgA) from the basolateral to the apical surface of epithelial cells and hepatocytes. However, the molecular machinery that controls polarized sorting of pIgR–pIgA and other receptors is only partially understood. The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains two sub-complexes. One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding. The other is the Vps35p–Vps29p–Vps26p subcomplex, which provides cargo specificity. The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway. Here, we show a function for the mammalian Vps35–Vps29–Vps26 retromer subcomplex in promoting pIgR–pIgA transcytosis.

A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor.

Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes–EGFR–ERK cascade, decreased pIgA–pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA–pIgR transcytosis. Our results reveal a novel Yes–EGFR–ERK–FIP5 signalling network for regulation of pIgA–pIgR transcytosis.

A Missense Mutation in the Sodium Channel β2 Subunit Reveals SCN2B as a New Candidate Gene for Brugada Syndrome

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%–25% of BrS patients carry genetic defects that cause loss‐of‐function of the voltage‐gated cardiac sodium channel. Thus, 70%–75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa) from cells coexpressing Nav1.5 and wild‐type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Nav1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Nav1.5 cell surface expression. The effect of the mutant β2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.

Retromer and sorting nexins in development.

Trafficking and signaling processes involve common molecular components. The machinery that controls intracellular trafficking is vital in ensuring that signaling mechanisms take place correctly. An illustrative example of this relationship is the sustained signaling of endocytosed membrane receptors, such as receptor Tyr kinases and G-protein coupled receptors, after ligand-induced activation. An intriguing role in controlling the fate of these and other receptors at the endosome has been attributed to members of the sorting nexin protein family. The best characterized sorting nexins are subunits of a multimeric complex, termed retromer. It was first found in yeast that retromer mediates endosome-to-Golgi retrieval of receptors after they have delivered soluble hydrolase precursors into the vacuole, the organelle equivalent to the mammalian lysosome. Work in cultured mammalian cells later demonstrated that retromer performs an analogous function in higher eukaryotes. Data from genetically modified mice, and from a simpler organism such as the nematode Caenorhabtidis elegans, has revealed that retromer performs an essential role during embryogenesis. This review will discuss implications of recent work on this subject.

Subcellular localisation of retromer in post-endocytic pathways of polarised Madin–Darby canine kidney cells

Retromer is required for endosome‐to‐Golgi retrieval of the cation‐independent mannose 6‐phosphate receptor (CI‐MPR), allowing delivery of hydrolases into lysosomes. It is constituted by a conserved heterotrimer formed by vacuolar protein sorting (Vps) gene products Vps26, Vps35 and Vps29, which is in charge of cargo selection, and a dimer of phosphoinositide‐binding sorting nexins (SNXs), which has a structural role. Retromer has been implicated in sorting of additional cargo. Thus, retromer also promotes polymeric immunoglobulin A (pIgA) transcytosis by the pIgA receptor (pIgR) in polarised cells, and considerable evidence implicates retromer in controlling epithelial cell polarity. However, the precise localisation of retromer along the endocytic pathway of polarised cells has not been studied in detail.

Reorganization of the endocytic compartment in regenerating liver

Antibodies raised to two membrane proteins present in rat liver endosomal fractions were used to study changes occurring in the endocytic compartment of hepatocytes during liver regeneration. Antibodies to the 42-kDa subunit (RHL-1) of the asialoglycoprotein receptor showed, by Western blotting of liver microsomes and endosomes, that there was a reduced expression of the receptor in liver 24 h following a partial hepatectomy. Immunocytochemical staining of thin sections of regenerating livers using these antibodies indicated that there was an intracellular relocation of endocytic structures in hepatocytes. The two main endocytic regions immunocytochemically stained in normal liver--one located beneath the sinusoidal plasma membrane and the other abutting the bile canaliculus--were replaced, in regenerating liver, by staining more closely associated with a region underlying the baso-lateral plasma membrane. A 140-kDa pI 4.3 calmodulin-binding protein located in endocytic and plasma membranes was also demonstrated, using a radio-iodinated calmodulin-binding assay, to be present at reduced levels in endosomes isolated from regenerating livers. Antibodies to this calmodulin-binding protein stained the hepatocytes cytoplasm in a punctate manner. However, in regenerating liver, the staining was located in regions underlying the baso-lateral and apical plasma membrane of hepatocytes. Together, the results demonstrate that a reorganization of the endocytic compartment has occurred in hepatocytes 24 h following hepatectomy, with two endosomal proteins becoming relocated to a region below the baso-lateral-apical surface regions of hepatocytes.

Trafficking and localization to the plasma membrane of Nav1.5 promoted by the β2 subunit is defective due to a β2 mutation associated with Brugada syndrome

Cardiac channelopathies arise by mutations in genes encoding ion channel subunits. One example is Brugada Syndrome (BrS), which causes arrhythmias and sudden death. BrS is often associated with mutations in SCN5A, encoding Nav1.5, the α subunit of the major cardiac voltage‐gated sodium channel. This channel forms a protein complex including one or two associated β subunits as well as other proteins.

Retromer in Polarized Protein Transport

Retromer is an evolutionary conserved protein complex required for endosome-to-Golgi retrieval of receptors for lysosomal hydrolases. It is constituted by a heterotrimer encoded by the vacuolar protein sorting (VPS) gene products Vps26, Vps35, and Vps29, which selects cargo, and a dimer of phosphoinositide-binding sorting nexins, which deforms the membrane. Recent progress in the mechanism of retromer assembly and functioning has strengthened the link between sorting at the endosome and cytoskeleton dynamics. Retromer is implicated in endosomal sorting of many cargos and plays an essential role in plant and animal development. Although it is best known for endosome sorting to the trans-Golgi network, it also intervenes in recycling to the plasma membrane. In polarized cells, such as epithelial cells and neurons, retromer may also be utilized for transcytosis and long-range transport. Considerable evidence implicates retromer in establishment and maintenance of cell polarity. That includes sorting of the apical polarity module Crumbs; regulation of retromer function by the basolateral polarity module Scribble; and retromer-dependent recycling of various cargoes to a certain surface domain, thus controlling polarized location and cell homeostasis. Importantly, altered retromer function has been linked to neurodegeneration, such as in Alzheimers or Parkinsons disease. This review will underline how alterations in retromer localization and function may affect polarized protein transport and polarity establishment, thereby causing developmental defects and disease.

A mechanism for NaV1.5 downregulation and sodium current decrease in heart failure

The pore-forming α-subunit of the cardiac voltage-gated sodium channel, NaV1.5, is responsible for the initial upstroke of the cardiac action potential. NaV1.5 cell surface expression and function are modulated by its interaction with regulatory proteins and by posttranslational modifications, such as phosphorylation, arginine methylation or ubiquitination 1. Genetic mutations in the SCN5A gene, which encodes NaV1.5, have been associated with a variety of inherited cardiac arrhythmias, including long QT syndrome type 3, Brugada syndrome, atrial fibrillation, and congenital sick sinus syndrome. In addition, abnormal NaV1.5 plasma membrane expression or sodium current (INa) density have also been observed in acquired cardiac disorders, such as heart failure (HF), although the molecular mechanisms that trigger these alterations are not well understood. This article is protected by copyright. All rights reserved.