Exploration into Galectin-3 Driven Endocytosis and Lattices.

Essentially all plasma membrane proteins are glycosylated, and their activity is regulated by tuning their cell surface dynamics. This is achieved by glycan-binding proteins of the galectin family that either retain glycoproteins within lattices or drive their endocytic uptake via the clathrin-independent glycolipid-lectin (GL-Lect) mechanism. Here, we have used immunofluorescence-based assays to analyze how lattice and GL-Lect mechanisms affect the internalization of the cell adhesion and migration glycoprotein αβ integrin.

SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.

Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasingly dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism.

Endocytic Roles of Glycans on Proteins and Lipids.

Most cell surface proteins are decorated by glycans, and the plasma membrane is rich in glycosylated lipids. The mechanisms by which the enormous complexity of these glycan structures on proteins and lipids is exploited to control glycoprotein activity by setting their cell surface residence time and the ways by which they are taken up into cells are still under active investigation. Here, two mechanisms are presented, termed galectin lattices and glycolipid-lectin (GL-Lect)-driven endocytosis, which are among the most prominent to establish a link between glycan information and endocytosis.

Galectin-3 Binding to αβ Integrin in Pore Suspended Biomembranes.

Galectin-3 (Gal3) is a β-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein αβ integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and αβ were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA.

Solubilization and Purification of αβ Integrin from Rat Liver for Reconstitution into Nanodiscs.

Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling.

Transcytosis of Galectin-3 in Mouse Intestine.

The GlycoLipid-Lectin (GL-Lect) hypothesis provides a conceptual framework to explain how endocytic pits are built in processes of clathrin-independent endocytosis. According to this hypothesis, oligomeric cellular or pathogenic lectins interact with glycosylated plasma membrane lipids in a way such as to drive the formation of tubular endocytic pits that then detach to generate clathrin-independent endocytic carriers for the cellular uptake of cellular or pathogenic products. This process operates in a complementary manner to the conventional clathrin pathway for biological function linked to cell polarity.

Site-specific N-glycan profiles of α β integrin from rat liver.

Background Information: Like most other cell surface proteins, α β integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined.

Results: Here, we have established the first comprehensive site-specific glycan map of α β integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition.

Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism.

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to the immune synapse (IS).

Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes.

Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family.

Quantitative Methods to Study Endocytosis and Retrograde Transport of Cargo Proteins.

Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot.

Clathrin-independent endocytosis, retrograde trafficking, and cell polarity.

Several mechanisms allow for cargo internalization into cells within membrane-bound endocytic carriers. How these internalization processes couple to specific pathways of intracellular distribution remains poorly explored. Here, we review uptake reactions that are independent of the conventional clathrin machinery.

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo.

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined.

Glycolipids and Lectins in Endocytic Uptake Processes.

A host of endocytic processes has been described at the plasma membrane of eukaryotic cells. Their categorization has most commonly referenced cytosolic machinery, of which the clathrin coat has occupied a preponderant position. In what concerns intra-membrane constituents, the focus of interest has been on phosphatidylinositol lipids and their capacity to orchestrate endocytic events on the cytosolic leaflet of the membrane.

Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex.

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/β) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome.

Persistent cell migration and adhesion rely on retrograde transport of β(1) integrin.

Integrins have key functions in cell adhesion and migration. How integrins are dynamically relocalized to the leading edge in highly polarized migratory cells has remained unexplored. Here, we demonstrate that β1 integrin (known as PAT-3 in Caenorhabditis elegans), but not β3, is transported from the plasma membrane to the trans-Golgi network, to be resecreted in a polarized manner.

Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells.

E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown.

SNAP-tagging the retrograde route.

We have developed a chemical biology strategy to identify proteins that follow the retrograde transport route from the plasma membrane to the Golgi apparatus, via endosomes. The general principle is the following: plasma membrane proteins are covalently tagged with a first probe. Only the ones that are then transported to trans-Golgi/TGN membranes are covalently bound to a capture reagent that has been engineered into this compartment.

AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine.

Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C.