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Internalization of exogenous Gal3 into enterocytes a Pulse-chase experiment of Gal3 uptake in enterocytes. Gal3 (green) was bound at 4 °C to enterocytes of DTT-treated jejunum. After washing, incubations were performed at 37 °C for the indicated periods of time. Cell surface-exposed Gal3 was removed with lactose wash. Note that no signal is detected when cells were not incubated at 37 °C (0 min). However, when shifted to 37 °C, distinct intracellular structures become visible as early as after 5 min of incubation (arrowheads). The fluorescence signal of internalized Gal3 is shown to the right in function of time (means ± SEM, n = 50 cells quantified per condition, one representative of three independent experiments). Nuclei in blue. Scale bars = 10 µm. b Apically internalized Gal3 accumulates at the basolateral membrane. Enterocytes were continuously incubated for 30 min at 37 °C with apically added Gal3 (green). Cell surface-exposed Gal3 was removed by lactose wash. Cells were labeled for the adherens junction marker E-cadherin, which is known to be localized to the basolateral membrane. A clear overlap between Gal3 and E-cadherin (RGB plot) indicates that Gal3 is indeed transcytosed in these enterocytes. Nuclei in blue. Scale bars = 10 µm. c Electron microscopy analysis of Gal3 uptake into enterocytes of the DTT-treated jejunum. After 15 min of incubation at 37 °C with 40 μg/mL of HRP-coupled Gal3, electron-dense DAB precipitates (arrowheads) are found in vacuolar structures close to the lateral membrane (Gal3-HRP/15 min micrograph) and mainly accumulate at the basal side after 30 min of incubation (Gal3-HRP/30 min micrograph). Some tubular crescent-shaped structures could be detected with typical CLIC morphology (Gal3-HRP/30 min/CLIC micrograph). For comparison, a cell that was incubated in the absence of Gal3-HRP is shown in the No HRP micrograph. Red-dashed insets represent endocytic structures. Scale bars = 500 nm for the bottom-right electron microscopy image and 1 µm for the others. d Endocytic structures containing electron-dense DAB precipitate were quantified in the indicated conditions. Percentage of DAB-positive endocytic structures (no HRP condition, n = 525 endocytic carriers; Gal3-HRP/15 min condition, n = 224 endocytic carriers; Gal3-HRP/30 min condition, n = 131 endocytic carriers were analyzed). e Two types of precipitates were detected, of which only the smaller one was specific for the Gal3-HRP condition. Quantification of the size of dark precipitates (means ± SEM; no HRP condition, n = 79 precipitates were measured within undefined structures; Gal3-HRP condition, n = 62 precipitates were measured within endocytic structures, and n = 49 precipitates in undefined structures). Ordinary one-way ANOVA, ∗∗∗∗P < 0.0001, ns non-significant.
Source publication
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 s...
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Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We start by introducing the cell biological aspects of these pathways, the concept of the sugar code, and provide an overview on the role of glycans in the targeting of lysosomal pr...
Citations
... Similar mechanisms could be operating for cellular lectins as Galectins. They are galactose-binding lectins involved in many physiological roles and related to protein transport [98][99][100]. Particularly galectin-3 can, upon oligomerization, bind glycosphingolipids and drive membrane bending and construction of tubular endocytic pits [101]. ...
Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways. Sphingolipids, a chemical diverse type of lipids with special physicochemical characteristics have been implicated in the selective transport of proteins. In this review, we will discuss current knowledge about how sphingolipids modulate protein trafficking through the endomembrane systems to guarantee that proteins reach their functional destination and the proposed underlying mechanisms.
... Source data are provided as a Source Data file. enterocytes ( Fig. S3d) 23 . Next, GII.4 association with gal-3 was assessed using fluorescence microscopy, gal-3 inhibition assays, and direct binding respectively (Figs. 3f, S3e, S3f, 3g, 3h, and 3i). ...
... The CLIC pathway is reported to be involved in uptake of abundant surface proteins such as CD44 and glycosylphosphatidylinositol-anchored proteins 25,26 , bacterial toxins (CTxB and Shiga) 24,48 , adeno-associated virus 49 and SARS-CoV-2 50 . Use of the CLIC pathway by GII.4 HuNoV in nontransformed HIEs is consistent with the CLIC pathway being operational in vivo in mouse enterocytes 23 and tubular invaginations observed in giant unilamellar vesicles treated with GII.4 VLPs 51 . Our study shows that gal-3 is recruited by GII.4 at the site of the GII.4-membrane interface and GII.4gal-3 interactions are necessary for infection, suggesting a putative role of gal-3 in endocytosis. ...
... Our study shows that gal-3 is recruited by GII.4 at the site of the GII.4-membrane interface and GII.4gal-3 interactions are necessary for infection, suggesting a putative role of gal-3 in endocytosis. Although intracellular gal-3 and extracellular gal-3 have a myriad of functions, extracellular gal-3 is a key player in the biogenesis of CLIC structures 22,23 . Gal-3 is also known for its crosslinking abilities, organizing membrane proteins/receptors and influencing their function and signaling 52 . ...
Globally, most cases of gastroenteritis are caused by pandemic GII.4 human norovirus (HuNoV) strains with no approved therapies or vaccines available. The cellular pathways that these strains exploit for cell entry and internalization are unknown. Here, using nontransformed human jejunal enteroids (HIEs) that recapitulate the physiology of the gastrointestinal tract, we show that infectious GII.4 virions and virus-like particles are endocytosed using a unique combination of endosomal acidification-dependent clathrin-independent carriers (CLIC), acid sphingomyelinase (ASM)-mediated lysosomal exocytosis, and membrane wound repair pathways. We found that besides the known interaction of the viral capsid Protruding (P) domain with host glycans, the Shell (S) domain interacts with both galectin-3 (gal-3) and apoptosis-linked gene 2-interacting protein X (ALIX), to orchestrate GII.4 cell entry. Recognition of the viral and cellular determinants regulating HuNoV entry provides insight into the infection process of a non-enveloped virus highlighting unique pathways and targets for developing effective therapeutics.
... Glycolipids can also serve as glycan ligands for galectins 1,2 . Through distinct oligomeric quaternary organization, galectins -in particular galectin-3 -when bound to cell surface glycoconjugates, can regulate endocytosis, receptor activation, lattice formation and the secretion of glycoproteins 145,[154][155][156][157][158] . ...
The galectin family consists of carbohydrate (glycan) binding proteins that are expressed by a wide variety of cells and bind to galactose-containing glycans. Galectins can be located in the nucleus or the cytoplasm, or can be secreted into the extracellular space. They can modulate innate and adaptive immune cells by binding to glycans on the surface of immune cells or intracellularly via carbohydrate-dependent or carbohydrate-independent interactions. Galectins expressed by immune cells can also participate in host responses to infection by directly binding to microorganisms or by modulating antimicrobial functions such as autophagy. Here we explore the diverse ways in which galectins have been shown to impact immunity and discuss the opportunities and challenges in the field. Galectins can modulate immune cells by binding to glycosylated proteins and lipids on the cell surface, or intracellularly via carbohydrate-dependent or carbohydrate-independent interactions. This Review explores the diverse ways in which galectins affect immunity and discusses the challenges in the field.
... Many cargoes whose endocytosis follows the predictions of the GL-Lect hypothesis, such as CD44, β1 integrin (ITGB1) and CD166, are linked to cell migration or cell polarity (Delacour et al., 2005;Lakshminarayan et al., 2014;Lim et al., 2017;Mishra et al., 2010;reviewed in Shafaq-Zadah et al., 2020). Recently, the validity of the GL-Lect hypothesis has been tested in vivo (Ivashenka et al., 2021). The authors reported that the extracellular lectin Gal3 is a novel binding partner of lactotransferrin. ...
... The authors reported that the extracellular lectin Gal3 is a novel binding partner of lactotransferrin. The Gal3lactotransferrin complex is transported in a glycosphingolipiddependent manner from the apical membrane to the basolateral membrane in enterocytes of the mouse intestine (Ivashenka et al., 2021). One of the common features of CIE processes is that they are inhibited upon perturbation of actin or raft-type membrane nanodomains (Boucrot et al., 2015;Day et al., 2015;Lakshminarayan et al., 2014). ...
Endocytosis is indispensable for multiple cellular processes, including signalling, cell adhesion, migration, as well as the turnover of plasma membrane lipids and proteins. The dynamic interplay and regulation of different endocytic entry routes requires multiple cytoskeletal elements, especially motor proteins that bind to membranes and transport vesicles along the actin and microtubule cytoskeletons. Dynein and kinesin motor proteins transport vesicles along microtubules, whereas myosins drive vesicles along actin filaments. Here, we present a brief overview of multiple endocytic pathways and our current understanding of the involvement of these motor proteins in the regulation of the different cellular entry routes. We particularly focus on structural and mechanistic details of the retrograde motor proteins dynein and myosin VI (also known as MYO6), along with their adaptors, which have important roles in the early events of endocytosis. We conclude by highlighting the key challenges in elucidating the involvement of motor proteins in endocytosis and intracellular membrane trafficking.
... Also, different galectins can contribute to the formation of distinct CLIC subpopulations [58]. Of note, this galectin-3edriven CLIC endocytosis works in vivo in transcytosis [60]. ...
Endocytosis mediates the uptake of extracellular proteins, micronutrients and transmembrane cell surface proteins. Importantly, many viruses, toxins and bacteria hijack endocytosis to infect cells. The canonical pathway is clathrin-mediated endocytosis (CME) and is active in all eukaryotic cells to support critical house-keeping functions. Unconventional mechanisms of endocytosis exit in parallel of CME, to internalize specific cargoes and support various cellular functions. These clathrin-independent endocytic (CIE) routes use three distinct mechanisms: acute signaling-induced membrane remodeling drives macropinocytosis, activity-dependent bulk endocytosis (ADBE), massive endocytosis (MEND) and EGFR non-clathrin endocytosis (EGFR-NCE). Cargo capture and local membrane deformation by cytosolic proteins is used by fast endophilin-mediated endocytosis (FEME), IL-2Rβ endocytosis and ultrafast endocytosis at synapses. Finally, the formation of endocytic pits by clustering of extracellular lipids or cargoes according to the Glycolipid-Lectin (GL-Lect) hypothesis mediates the uptake of SV40 virus, Shiga and cholera toxins, and galectin-clustered receptors by the CLIC/GEEC and the endophilin-A3-mediated CIE.
... Curvature generation is also used to drive the endocytosis of endogenous proteins via the GL-Lect mechanism (Ref. [42][43][44], reviewed in [10]). ...
Many bacteria secrete toxic protein complexes that modify and disrupt essential processes in the infected cell that can lead to cell death. To conduct their action, these toxins often need to cross the cell membrane and reach a specific substrate inside the cell. The investigation of these protein complexes is essential not only for understanding their biological functions but also for the rational design of targeted drug delivery vehicles that must navigate across the cell membrane to deliver their therapeutic payload. Despite the immense advances in experimental techniques, the investigations of the toxin entry mechanism have remained challenging. Computer simulations are robust complementary tools that allow for the exploration of biological processes in exceptional detail. In this review, we first highlight the strength of computational methods, with a special focus on all-atom molecular dynamics, coarse-grained, and mesoscopic models, for exploring different stages of the toxin protein entry mechanism. We then summarize recent developments that are significantly advancing our understanding, notably of the glycolipid–lectin (GL-Lect) endocytosis of bacterial Shiga and cholera toxins. The methods discussed here are also applicable to the design of membrane-penetrating nanoparticles and the study of the phenomenon of protein phase separation at the surface of the membrane. Finally, we discuss other likely routes for future development.
As biologics used in the clinic outpace the number of new small molecule drugs, an important challenge for their efficacy and widespread use has emerged, namely tissue penetrance. Macromolecular drugs - bulky, high-molecular weight, hydrophilic agents - exhibit low permeability across biological barriers. Epithelial and endothelial layers, for example within the gastrointestinal tract or at the blood-brain barrier, present the most significant obstacle to drug transport. Within epithelium, two subcellular structures are responsible for limiting absorption: cell membranes and intercellular tight junctions. Previously considered impenetrable to macromolecular drugs, tight junctions control paracellular flux and dictate drug transport between cells. Recent work, however, has shown tight junctions to be dynamic, anisotropic structures that can be targeted for delivery. This review aims to summarize new approaches for targeting tight junctions, both directly and indirectly, and to highlight how manipulation of tight junction interactions may help usher in a new era of precision drug delivery.
Targeted drug delivery in cancer typically focuses on maximising the endocytosis of drugs into the diseased cells. However, there has been less focus on exploiting the differences in the endocytosis pathways of cancer cells versus non-cancer cells. An understanding of the endocytosis pathways in both cancer and non-cancer cells allows for the design of nanoparticles to deliver drugs to cancer cells whilst restricting healthy cells from taking up anticancer drugs, thus efficiently killing the cancer cells. Herein we compare the differences in the endocytosis pathways of cancer and healthy cells. Second, we highlight the importance of the physicochemical properties of nanoparticles (size, shape, stiffness, and surface chemistry) on cellular uptake and how they can be adjusted to selectively target the dominated endocytosis pathway of cancer cells over healthy cells and to deliver anticancer drug to the target cells. The review generates new thought in the design of cancer-selective nanoparticles based on the endocytosis pathways.
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. Up to date, the premises of the GL-Lect hypothesis have been based on model membrane and cell culture experiments. It has therefore become urgent to extend its exploration to complex organisms. In the current protocol, we describe methods to study the endocytosis and transcytosis of a key driver of the GL-Lect mechanism, the cellular galectin-3, and of one of its cargoes, lactotransferrin, in enterocytes of the intact jejunum of mice. In a step-by-step manner, we present the generation of fluorescent endocytic ligands, tissue preparation for cellular uptake measurements, binding and internalization assays, tissue fixation and preparation for sectioning, light and electron microscopical observations, and quantification of data by image processing. Pitfalls are discussed to optimize the chances of success with the described methods.
Background information:
Like most other cell surface proteins, α5 β1 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 α5 β1 integrin that was purified from a natural source, i.e., rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were non-glycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of β1 integrin. The extensive glycan information that results from our study was projected onto a map of α5 β1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5 β1 integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis.
Conclusion:
We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5 β1 integrin activity and endocytic trafficking.
Significance:
Glycosylation of α5 β1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5 β1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of 'native' glycoproteins. This article is protected by copyright. All rights reserved.
