Membrane Compartmentalisation and Endocytosis by Galectin-3 in Mammalian Cells Carola Benzing

A thesis in the fulfilment of the requirements for the degree of Doctor of Philosophy, written at the Centre for Vascular Research · School of Medical Sciences · Faculty of Medicine, submitted in August 2014

Originality Statement

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed...... Carola Benzing

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‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International.

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

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Abstract

Galectin-3 is a carbohydrate binding protein that is widely expressed and can be found in tissues and cells of the immune system. At the cell surface, galectin-3 interacts with various cell surface glycoproteins through its carbohydrate recognition domain (CRD) and exhibits multivalent binding properties by assembling into pentamers through its N-terminal domain. Galectin-3 molecules have been implicated in the formation of molecular lattices at plasma membranes and in endocytosis. The aim of this study was to examine the molecular arrangement of galectin-3 on the surface of fibroblasts and Hela cells as well resting and activated T cells, and investigate whether and how galectin-3 is internalized in T cells to generate signalling endosomes.

To map the molecular organisation of galectin-3 on the cell surface, direct stochastic optical reconstruction microscopy (dSTORM) and quantitative analysis of galectin-3 and galectin-3 ligands was established. It was found that galectin-3 clustering depended on glycosphingolipids in the plasma membrane and that galectin- 3-dependent clustering of known galectin-3 binding partners was sensitive to branched N-acetylglucosamine saccharides that were absent in β-1,6-N- acetylglycosaminyltransferase V (Mgat5)-deficient mouse embryonic fibroblasts (Mgat5-/- MEF). These data supports the concept that galectin-3 compartmentalises the plasma membrane. Next, it was demonstrated with confocal microscopy and flow cytometry that the binding of galectin-3 to the plasma membrane of T cells occurred in a carbohydrate-dependent fashion and led to internalisation. Further it was shown that uptake of galectin-3 was facilitated by different endocytic mechanisms suggesting that galectin-3 participates in various endocytic routes in T cells. Finally, in activated T cells, data is presented to show that galectin-3- positive vesicles were positioned at or near the immunological synapse and co-localised with proteins involved in signalling processes, suggesting that galectin-3 functions in the regulation of T cell signalling.

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In conclusion, the data presented in this PhD thesis suggest that galectin-3 binding to the cell surface creates distinct membrane domains in a glycosphingolipid- and branched N-glycosylation-dependent manner. In T cells, galectin-3 domains lead to the formation of galectin-3 vesicles that may function as signaling endosomes.

Acknowledgements

First and foremost I want to thank my parents for giving me the opportunity to complete a higher education, and for giving me the freedom of choice. I’m also thanking my sister Claudia for being my sister and knowing that she is around whenever I need her. By the way, Claudia, I did not have to clean any test tubes or feed the lab mice!

I want to thank Kat Gaus for providing me with the opportunity, the equipment, the outstanding resources, and her experience in science to complete my research in her group.

A huge thank you goes to Jeremie Rossy for his scientific advice throughout my graduate research career. I appreciate his knowledge, expertise and high scientific standard. I’m wishing him all the best as a new group leader and throughout his career.

I thank Sophie Pageon for her thorough proofreading, all her advice and help regarding formatting. She is just amazing with Microsoft Word!

I also thank Joanna for a brief but very helpful introduction to InkScape.

Publications

Benzing C., et al. (in preparation) Galectin-3 defines an endocytic pathway in T cells

Lakshminarayan R., Wunder, C., Becken U., Howes M.T., Benzing C., Arumungam S., Sales S., Ariotti N., Chambon V., Lamaze C., Loew D., Shevchenko A., Gaus K., Parton G.R., Johannes L. (2014). Galectin-3 drives Glycosphingolipid-dependent Biogenesis of Clathrin-independent Carriers. Nature Cell Biology 16, 595-606

Benzing, C., Rossy, J., Gaus, K. (2013). Do Signalling Endosomes play a role in T cell activation? FEBS Journal 280 (21), 5164-5176

Rossy, J., Williamson, D.J., Benzing C., Gaus K. (2012). The integration of signaling and the spatial organization of the T cell synapse. Frontiers in Immunology 3, 352

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Table of Contents

Originality Statement ...... ii Copyright Statement ...... iv Authenticity Statement ...... vi Abstract ...... viii Acknowledgements ...... x Publications ...... xii Table of Contents ...... xiiv List of Figures ...... xviii List of Tables ...... xx Abbreviations...... xxii Chapter 1 ...... 1 1.1 Lectins and galectins ...... 1 1.1.1 Expression, distribution and extracellular binding of galectin-3 ...... 4 1.2 Protein Glycosylation ...... 6 1.3 The organisation of the plasma membrane ...... 10 1.3.1 Plasma membrane domains ...... 10 1.3.2 Molecular lattices ...... 11 1.3.3 The main lipids of the plasma membrane ...... 14 1.3.4 Behaviour and function of glycosphingolipids ...... 15 1.3.5 Interactions of GSLs with galectins ...... 17 1.3.6 Plasma membrane domains and lipid rafts ...... 18 1.3.7. The role of lectins in the organisation of the plasma membrane ...... 21 1.4 Endocytosis ...... 24 1.4.1 Membrane Curvature ...... 27 1.4.2 Galectins in endocytosis ...... 29 1.4.3 N–glycosylation in endocytosis ...... 30 1.4.4 Retrograde Transport in Protein Trafficking ...... 30 1.5 Immunomodulatory functions of galectins ...... 31 1.5.1 T-cell receptor signalling and formation of the immunological synapse ...... 32 1.5.2 Vesicular traffic at the immunological synapse ...... 34 1.6 Fluorescence microscopy ...... 36 xiv

1.6.1 TIRF Microscopy ...... 37 1.6.2 Resolution and diffraction in fluorescence microscopy...... 38 1.6.3 Super-resolution microscopy ...... 40 1.7 Aim ...... 42 Chapter 2 ...... 45 2.1 Tissue culture ...... 45 2.2 Sample preparation using T cells ...... 48 2.3 Galectin-3: Expression, Purification & Labelling ...... 49 2.4 Galectin-3 Assays ...... 52 2.5 Pharmacological Inhibitors ...... 53 2.6 Preparation of sterol-complexed cyclodextrin ...... 55 2.7 Immunofluorescence ...... 56 2.8 Quantification of galectin-3 uptake by Flow cytometry ...... 56 2.9 Confocal Fluorescence Microscopy ...... 57 2.10 Pearson’s Coefficient ...... 57 2.11 dSTORM Single Molecule Microscopy ...... 57 2.12 Statistical Analyses ...... 60 Chapter 3 ...... 61 3.1 Introduction ...... 61 3.2 Glycosphingolipid-dependent galectin-3 clustering ...... 64 3.3 N-glycan-dependent clustering of galectin-3 ...... 69 3.4 Galectin-3 nanodomains in T cells ...... 71 3.5 Galectin-3 dependent surface expression of β1-integrin ...... 72 3.6 Galectin-3 dependent clustering of CD44 ...... 75 3.7 Discussion ...... 77 Chapter 4 ...... 83 4.1 Introduction ...... 83 4.2 Binding and uptake of galectin-3 ...... 85 4.3 Mechanisms of galectin-3 endocytosis ...... 89 4.4 Subcellular distribution of galectin-3 ...... 94 4.5 Discussion ...... 98 Chapter 5 ...... 103 5.1 Introduction ...... 103 5.2 Endogenous galectin-3 in TCR clustering ...... 105 xv

5.3 Exogenous galectin-3 in TCR clustering ...... 108 5.4 Galectin-3 at the immunological synapse ...... 110 5.5 Discussion ...... 112 Chapter 6 ...... 115 6.1 Conclusion ...... 115 6.2 Future outlook ...... 121 References ...... 124

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List of Figures

Figure 1.1. Schematic representation of structural subgroups within the galectin family…………………………………………………………………………………………………………………… 2 Figure 1.2. Glycosylated cell surface molecules interact with different extracellular components…………………………………………………………………………………….. 6 Figure 1.3. Generation of glycoproteins: processing of proteins and attachment of oligosaccharides in the ER and Golgi………………………………………………………………… 7 Figure 1.4. The major types of N-linked oligosaccharides…………………………………….. 9 Figure 1.5. Galectin-3 cross-links glycoproteins and –lipids at the cell surface…….. 18 Figure 1.6. Model of galectin-3 co-clustering and CLIC endocytosis……………………… 22 Figure 1.7. Mechanisms to generate membrane curvature………………………………….. 28 Figure 1.8. Structure of the immunological synapse and the TCR signalling assembly……………………………………………………………………………………………………………… 34 Figure 1.9. Total internal reflection fluorescence microscopy diagram………………… 38 Figure 1.10. The point spread function (PSF)……………………………………………………….. 39 Figure 3.1. dSTORM imaging and cluster analysis of Gal3-Alexa647 in control and PPMP-treated HeLa cells………………………………………………………………………………………. 67 Figure 3.2. Analysis for the effects of photoblinking…………………………………………….. 68 Figure 3.3 Clustering of galectin-3 in the presence or absence of Mgat5……………... 70 Figure 3.4. Cluster analysis of Gal3-Alexa647 nanodomains in Jurkat E6.1 T cells… 72 Figure 3.5. Integrin-β1 clusters on the plasma membrane of T cells…………………… 74 Figure 3.6. Effects of altered N-glycosylation on the clustering of CD44 in the presence of galectin-3………………………………………………………………………………………….. 76 Figure 4.1. Galectin-3 binding and uptake in T cells is carbohydrate-dependent….. 86 Figure 4.2. Confocal SIM image of galectin-3 vesicles in Jurkat T lymphocytes…….. 87 Figure 4.3. Confocal images and flow cytometry analysis of galectin-3 binding and endocytosis in energy-depleted Jurkat T lymphocytes…………………………………… 88 Figure 4.4. Time course of galectin-3 uptake by Jurkat cells…………………………………. 89 xviii

Figure 4.5. Effects of endocytic inhibitors on galectin-3 uptake…………………………… 90 Figure 4.6. Effects of lipids on the endocytosis of galectin-3………………………………… 93 Figure 4.7. Live cell imaging of galectin-3 binding and endocytosis in resting T cells……………………………………………………………………………………………………………………… 95 Figure 4.8. Analysis of the subcellular distribution of galectin-3…………………………… 97 Figure 5.1. Expression of intracellular galectin-3 in activated T cells……………………. 106 Figure 5.2. Clustering of CD3ζ-subunit in the presence or absence of intracellular galectin-3……………………………………………………………………………………………………………… 107 Figure 5.3. Clustering of phosphorylated CD3ζ in the absence or presence of extracellular galectin-3…………………………………………………………………………………………. 109 Figure 5.4. Immunological synapse with galectin-3 positive vesicles……………………. 110 Figure 5.5. Analysis of galectin-3 positive vesicles at the immunological synapse… 111 Figure 6.1. Model of galectin-3 driven membrane bending and subsequent endocytosis………………………………………………………………………………………………………….. 120 Figure 6.2. Different models of endosomal signalling………………………………………….. 122

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List of Tables

Table 3.1. Summary of galectin-3 in GSL-depleted HeLa cells………………………………. 68 Table 3.2. Summary of Mgat5-dependent clustering of galectin-3 at the plasma membrane of MEF……………………………………………………………………………………………… 71 Table 3.3. Summary of galectin-3-dependent β1-integrin clusters at the plasma membrane of T cells…………………………………………………………………………………………….. 75 Table 3.4. Summary of dMNJ-dependent galectin-3-induced CD44 clusters at the plasma membrane of MEF…………………………………………………………………………………… 77 Table 3.5. Summary of galectin-3 and galectin-3-induced clusters at cellular membranes of different mammalian cells (MEF, HeLa, T cells)…………………………….. 78 Table 5.1. Summary of TCR and pTCR clustering in the absence or presence of galectin-3……………………………………………………………………………………………………………… 113

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Abbreviations

APC Antigen presenting cell Asn Asparagine BSA Bovine serum albumin Cav1 Caveolin 1 CCD Charge-coupled device CCV Clathrin-coated vesicles CHO Chinese hamster ovary cells CIE Clathrin-independent endocytosis CLIC Clathrin-independent carrier CME Clathrin-mediated endocytosis CPZ Chlorpromazine CRD Carbohydrate recognition domain cSMAC Central supra-molecular activation cluster CtxB Cholera toxin, subunit B Cyto D Cytochalasin D d Distance dMNJ 1-Deoxymannojirimycin DMSO Dimethyl sulfoxide DNS Dynasore dSTORM Direct stochastic optical reconstruction microscopy DRM Detergent-resistant membranes DSOM Dynamic saturation optical microscopy DTT Dithiothreitol EGF Epidermal growth factor ER Endoplasmic reticulum FACS Fluorescence-activated cell sorting FAK Focal adhesion kinase FPALM Fluorescence photo-activated localisation microscopy xxii

FRAP Fluorescence recovery after photobleaching Fuc Fucose GalCer Galactosylceramide GalNAc N-acteylgalactosamine GEEC GPI-anchored protein enriched early endosomal compartment GEM GSL-enriched microdomain GlcCer glucosylceramide GLUT2 Glucose Transporter 2 Golgi Golgi complex GPI Glycosylphosphatidylinositol GSL Glycosphingolipid GSH Glutathione HUVEC Human vascular endothelial cells IL2R Interleukin 2 receptor IS Immunological synapse ITAM Immunoreceptor tyrosine-based activation motifs LAT Linker for activation of T-cells Lat A Latrunculin A ld Liquid-disordered lo Liquid-ordered MDCK Madin-Darby canine kidney MEA β-mercaptoethylamine MTOC Microtubule organizing centre mβCD Methyl-β-cyclodextrin Mgat5 N-acetylglucosaminyltransferase V MT Microtubules MTOC Microtubule organising centre N-linked Asparagine-linked NA Numerical aperture PALM Photo-activated localisation microscopy PBS Phosphate-buffered saline

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PFA Paraformaldehyde PLL Poly-L-lysine PDGF Platelet-derived growth factor PDI Protein disulphide isomerase pMHC Peptide-major histocompatibility complex PPMP DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol pSMAC Peripheral supra-molecular activation cluster RESOLFT Reversible saturable optical fluorescence transitions SA Sialic acid SEE Staphylococcal enterotoxin E SEM Standard error of the mean SIM Structured-illumination microscopy SL Sphingolipid SMLM Single-molecule localization microscopy STED Stimulated emission depletion STORM Stochastic optical reconstruction microscopy StxB Shiga toxin, subunit B SWM Standing-wave microscopy TCR T cell receptor TGFβ Transforming growth factor β TGN Trans-Golgi-network TIRF Total internal reflection fluorescence TRK Tropomyosin receptor kinase VEGF Vascular endothelial growth factor ZAP-70 ζ-chain TCR-associated protein kinase of 70kDa 7-KC 7-ketocholesterol λ Wavelength

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Amino acid 3-letter 1-letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Unspecified Aa - X

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Chapter 1

Introduction

1.1 Lectins and galectins

Carbohydrate-binding proteins and enzymes are found abundantly in all living cells and are involved in a multitude of biological functions. Carbohydrates constitute the bulk of the organic matter on earth and the generation of carbohydrates during the biochemical process of carbon dioxide fixation is fundamental to photo- and chemosynthesis. Sugars are the central energy source for the execution of mechanical work and chemical reactions, and they are also necessary for a multitude of essential cellular processes. Protein-saccharide interactions play important roles in biological recognition, cell adhesion, and cell signalling. The major and unique group of carbohydrate-binding proteins is formed by lectins that are highly specific for particular sugar moieties. The distribution of lectins in nature is wide and they can be found in many animals and plants1,2. Lectins serve various functions such as facilitating attachment and binding of bacteria and viruses, mediating the first-line defence against invading microorganisms in the innate immune system, and are involved in cellular recognition processes as well as in receptor-mediated endocytosis3. The cellular ligands for plant and animal lectins are carbohydrate chains of glycoproteins and glycolipids, and the binding of glycosylated cell surface molecules by lectins can lead to the formation of cross-linked complexes that are implicated in cellular responses. Herrmann Stillmark was a pioneer of lectin biology. In 1888 Stillmark isolated the toxic plant lectin ricin from castor beans (Ricinus communis) and described the agglutinating properties on erythrocytes of what he thought was pure ricin4, not knowing that he had also isolated a lectin called ricinus communis agglutinin, which

possesses much stronger agglutinating properties than ricin5. Nevertheless, the cornerstone for the study of lectins was laid and in the last century scientists revealed important functions for carbohydrate-binding proteins in cellular events.

The large group of lectins is further subdivided into different families; one of them is the family of galectins. Galectins have a high affinity for β-galactoside- containing glycoconjugates through conserved sequence elements present in the carbohydrate recognition domain, which is a common binding site of galectins for carbohydrates6. Studies using X-ray crystallography have revealed the sequence of participating amino acids and significant sequence similarities in the carbohydrate recognition domains intertwine galectins into one family7–9. So far 15 members of mammalian galectins have been identified (Figure 1.1), all of them containing a carbohydrate recognition domain (CRD) consisting of about 130 amino acids6. Based on their structure the galectin family is further subdivided into three groups6,10. Members of the proto-type group contain only one CRD (galectin-1, -2, -5, -7, -10, -11, -13, -14, -15), whereas members of the tandem repeat-group consist of two distinct but homologous CRDs (galectin-6, -8, -9, -12), and the only member of the vertebrate chimera-type group is galectin-3, comprising one C-terminal CRD that is connected to an N-terminal non-lectin domain that is rich in the amino acids proline and glycine6,11.

Figure 1.1. Schematic representation of structural subgroups within the galectin family. (A) Proto-type galectins contain one CRD that can dimerize; members of this group are galectins 1, 2, 5, 7, 10, 11, 13, 14, 15. (B) Galectin-3 is structurally unique and the only member of the chimera-type galectins comprising one CRD that is connected to a large non-lectin domain. Galectin-3 is monomeric but interactions between the N-terminal non-lectin domains lead to oligomerization. (C) Members of the tandem repeat-type are galectins 4, 6, 8, 9, 12. They contain two distinct CRDs connected by a short linker sequence and are constitutive dimers.

The ability of certain galectins to form dimers or oligomers under certain conditions (e.g. concentration or presence of ligands) enables bivalent or multivalent binding of carbohydrate ligands and this multimerisation appears to be crucial for their biological functions. The presence of galactose is essential for binding of galectins to their ligands and the most basic recognition unit for galectin-3 is lactose12. Studies on the carbohydrate-binding activity and specificity of galectins described N- acetyllactosamine (also known as galactose β1,4N-acetylglucosamine; Galβ1,4GlcNAc; GlcNAc; LacNAc) as the preferred ligand13,14, either as disaccharide units or as repeating units in a poly-N-acetyllactosamine chain6,11,15,16. Binding affinities of galectins are proportional to the content and branching of N-glycans available on the ligands11,15–19 and increase with the number of N-acetyllactosamine units per N-glycan and the number of N-glycans per protein18. Galectins exhibit more variability in binding to larger, naturally occurring and more complex oligosaccharides11. Some galectins contain an extended carbohydrate-binding site20,21, such as galectin-3 and -9, which can accommodate larger oligosaccharides and extended poly-N-acetyllactosamine chains. The crystal structures of those galectins that crystallize well have been reported including human galectin-1, -3, -7, -8, -9, bovine galectin-1, and the eel galectin congerin II among others8,22–29, and highly conserved residues between the CRDs of the different galectins have been detected. A comparison of galectin CRDs reveals eight amino acid residues that are involved in the binding to carbohydrates and are invariant. Additionally, another dozen residues seem to be highly conserved, contributing to the structure. The CRD of galectin subunits lacks an α-helix and is composed of antiparallel β-sheets that are arranged in a β-sandwich or β-barrel jellyroll fold30,31, this topology follows a typical folding pattern that all galectin CRDs exhibit.

Though most galectins have similar affinities for lactose or N-acetyllactosamine, their CRDs differ in their carbohydrate-binding specificities32 and contribute to selective binding of glycosylated ligands based on differences in N-glycan topology. These differences imply that each galectin may interact with a discrete spectrum of glycoconjugate receptors16,33–40, which in turn is linked to specific downstream effects. Not only have proteomic analyses of galectin binding proteins identified a variety of

ligands of different galectins that despite occasional overlap are largely distinct form each other33–37,41,42, but experimental approaches investigating galectin interactions with cells have also revealed differential binding and divergent effects of galectins. For example, galectin-1 and -3 both bind to the surface of T cells but recognize distinct sets of cell surface glycoprotein receptors, and although both galectins can bind CD45 at the surface of T cells, only binding of galectin-1 impacts on the distribution of CD45 at the cell surface35. Of four tested galectins regarding their effect on osteoblasts, only galectin-9 was capable of inducing proliferation in human osteoblasts whereas other galectins (galectin-1, -3, and -4) were not involved in any downstream effects43.

1.1.1 Expression, distribution and extracellular binding of galectin-3

Galectin-3 was identified for the first time at the cell surface of murine peritoneal macrophages in 198244. At that time it carried the names ‘Mac-2 antigen’, ‘L-29’ and ‘carbohydrate binding protein 35’, due to its molecular weight of 35 kDa. Soon after this initial discovery, extracellular and intracellular galectin-3 was detected in a variety of cell types and tissues45–49 and research on galectin-3 took its course.

The onset of galectin-3 expression during murine development occurs at the fourth day of gestation50; secretion of galectins during development and differentiation seems to be tightly controlled, and several other factors such as cytokines, membrane fusion events as well as cell activation can modulate galectin secretion from cells51–55. In adults, galectin-3 is expressed in a variety of different tissues and organs, including numerous cells and tissues of the immune system such as the thymus56, neutrophils57, eosinophils58, basophils and mast cells59,60, langerhans cells61, dendritic cells62, as well as monocytes63 and macrophages64 associated with different tissues. With regard to T lymphocytes, galectin-3 is expressed by activated T lymphocytes including CD4+ and CD8+ T cells, and its expression can be enhanced by certain cytokines such as IL-2, IL-4, and IL-765. Furthermore galectin-3 is expressed in a variety of tumours, where galectin-3 expression levels depend on tumour progression, invasiveness and metastatic potential66.

The localisation of galectin-3 has been mapped to two principle sites. Galectin-3 is present in the intracellular space where it can be found in the cytoplasm as well as the nucleus, suggesting a shuttling role for galectin-367. Furthermore, it is also translocated to the exterior and is found in the extracellular space and attached to the cell surface. Within the family of galectins, no evidence of a classical signal sequence for externalisation through the endoplasmic reticulum (ER) and Golgi complex (Golgi) has been found, and early studies have shown that galectin-3 secretion is insensitive to drugs such as brefeldin A and monensin that disrupt events in the classical secretory pathway involving vesicular trafficking through ER/Golgi compartments51,55. Presumably the presence of galectin-3 on the exterior of cells is a consequence of secretion via alternative, non-classical routes41. Cells differ widely in their propensity to secrete galectins, some secrete little to none, and others secrete almost half or all of their newly synthesised galectin41; secretion seems to be responsive to developmental events and differentiation41, and it can be stimulated in vitro by heat shock and calcium ionophores53,55.

At the cell surface galectin-3 interacts with various cell surface glycoproteins through the CRD and possession of an extended binding site to accommodate longer oligosaccharides such as polylactosaminoglycans, increases the spectrum of ligands6,21. The interaction of galectin-3 with a carbohydrate ligand through its CRD is accompanied by a conformational change13 and a rearrangement of the backbone loops near the binding site68. Mobility at or near the binding site may provide galectin- 3 with the ability to bind a variety of ligands terminating with galactose and other residues68. Although galectin-3 possesses only one CRD, it exhibits multivalent binding properties6,21,69: galectin-3 molecules assemble in a pentameric fashion in the presence of multivalent ligands, this oligomerization is mediated through the N-terminal domain of galectin-36,70.

The ability of galectins to form dimers or pentamers enables the formation of molecular lattices via interactions of galectins with different cell surface glycoproteins71 (Figure 1.2). The multivalency of lectins enables recognition of multiple binding partners, and allows them to play key roles in signal transduction in different biological processes72,73. Fluorescently labelled galectin-3 has been shown to form 5

stable oligomers when added to the surface of neutrophils and endothelial cells74. Lattice association of cell surface receptors restricts lateral diffusion as shown for the epidermal growth factor (EGF) receptor. The diffusion rate of the EGF receptor was reduced by the galectin lattice and prevented association of the EGF receptor with caveolin-175. Furthermore, galectin-3 cross-links N-glycans on EGF receptors and transforming growth factor β (TGFβ) receptors and delays their removal by constitutive endocytosis76. Glycan-based membrane domains generated by galectin-lattices have been proposed previously15 and galectins are involved in forming molecular lattices creating homotypic and heterotypic membrane domains15,37,77–80.

Figure 1.2. Glycosylated cell surface molecules interact with different extracellular components. Carbohydrates can mediate cell-cell interactions and the attachment of viruses, bacteria and toxins. Furthermore pentameric galectin-3 mediates cross-linking of N-glycan carrying cell surface proteins thus forming a molecular lattice.

1.2 Protein Glycosylation

Glycosylation is a common process occurring on cellular proteins and lipids. The very diverse and complex oligosaccharide chains found on cell surface proteins, extracellular proteins and membrane lipids are attached and modified by enzymes in the ER and the Golgi. Protein and lipid glycosylation is known to mediate many important cellular and biological functions, and mutations impairing proper glycosylation lead to severe diseases (Figure 1.2). The two dominant glycan modifications of proteins are additions of monosaccharides to serine or threonine 6 residues, known as O-glycosylation and the addition of oligosaccharides to asparagine residues known as N-glycosylation. In the process of N-glycosylation (Figure 1.3), an oligosaccharide precursor is transferred from the lipid donor dolichol to an asparagine (Asn, N) located on a nascent polypeptide in the ER81. After addition of this oligosaccharide precursor to the protein, processing by glycosidases and glycosyltransferases begins through the removal and addition of sugar residues leading to extension and diversification of glycans.

Figure 1.3. Generation of glycoproteins: processing of proteins and attachment of oligosaccharides in the ER and Golgi. Oligosaccharyltransferase transfers the precursor oligosaccharide Glc3Man9GlcNAc2 preassembled on the lipid donor dolichol to Asp-X-Ser/Thr motifs on glycoproteins in the ER. Newly generated glycoproteins then transit from the ER to the Golgi, traversing through the cis, medial, and trans cisternae before being transported to the cell surface. N-acetylglucosaminyltransferases, termed Mgat1, Mgat2, Mgat4, and Mgat5 according to their gene names, generate branched N-glycans that display a range of affinities for galectins. N-acetylglucosaminyltransferases are dependent on the availability of the common donor substrate UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc synthesis through the hexosamine pathway utilizes substrates (fructose-6P, glutamine, acetyl-CoA, and UTP) that are key metabolites in carbon, nitrogen, and energy homeostasis. Hence the extent of GlcNAc branching is dependent on branching enzyme kinetics, and metabolic flux through the hexosamine pathway to generate UDP-GlcNAc 82. (Figure from Dennis et al.83)

Oligosaccharide structural analysis of mature glycoproteins revealed three main categories of structures termed high-mannose-, hybrid-, and complex-type (Figure 1.4). All of them share a common core structure (Manα1-3(Manα1-6) Manβ1-4 GlcNAcβ1-4 GlcNAc-Asn) but differ considerably in the composition of the outer branches84. The structures of the complex-type and high-mannose-type oligosaccharides differ greatly, whereas hybrid structures share features with both high-mannose and complex-type oligosaccharides. The glycan moieties attached to glycoproteins are structurally very diverse. Variations in glycans occur in the number and length of oligosaccharide branches (or antennae) that are linked to glycan core structures, also alterations in substitutions at the periphery such as attachments of galactose residues, sialic acids and others create variety84,85. This variability attributes functional diversity to glysocylated molecules. A given glycan can have different roles at different developmental stages or in different tissues. Glycan-additions to proteins are essential mediators of uncountable biological processes such as protein folding, quality control, trafficking, and conferring resistance to proteases86–89, they are indispensable in processes such as fertilization, embryogenesis as well as proliferation of cells and organisation of cells into tissues90–98. Furthermore they have important functions in cell signalling99, protein interactions100, endocytosis101, control of receptor cell surface levels102, compartmentalisation into lipid rafts103, and immune regulation104. Glycosylation of molecules is also relevant in binding and recognition of pathogens to target cells (Figure 1.2), in the innate immune response, in inflammation and it also plays a role in cancer105–109. Several developmental abnormalities in human disorders, known as congenital disorders of glycosylation, underscore the importance of protein glycosylation and functionality of glycosylated proteins110,111. These disorders are caused by defects in the glycosylation machinery and can cause different symptoms ranging from slight mental retardation to multi-organ dysfunctions and can be associated with infantile lethality112,113.

Figure 1.4. The major types of N-linked oligosaccharides. The three main structures of oligosaccharides found on glycoproteins added at Asn-X-Ser/Thr sequences are the high- mannose-type, the complex-type, and the hybrid-type. The core structure common to all N- linked glycoproteins is a pentasaccharide containing Man3GlcNAc2Asn indicated by the boxed area. Glycosidic linkages are indicated next to bonds (β, β2, β4, α2, α3, α6).

High mannose-type oligosaccharides - besides comprising their own class of N- linked oligosaccharides - are also intermediates in the biosynthesis of hybrid- and complex-type oligosaccharides. Therefore, inhibitors that block processing of high- mannose-type oligosaccharides also block synthesis of hybrid- and complex-type oligosaccharides and can be used to assess the function of these types of N-linked oligosaccharides. Two distinct inhibitors of Golgi α-mannosidase I, 1- deoxymannojirimycin (dMNJ) and kifunensine, block the synthesis of hybrid- and complex-type oligosaccharides114,115. Thus, experiments using dMNJ have provided insights into N-glycan function; for example, dMNJ disrupted the cellular distribution of the α1-adrenergic receptor116 or the formation of capillary tubes in endothelial cells117. In contrast, swainsonine inhibits Golgi α-mannosidase II, acting at a point downstream from the key intermediate for synthesis of hybrid- but not complex-type oligosaccharides118. Further experiments showed that the ligand-dependent activation of the epidermal growth factor (EGF) receptor tyrosine kinase was inhibited by the GM3 ganglioside through carbohydrate interactions, and this effect was abrogated in cells treated with swainsonine119.

For most cell surface receptors and plasma membrane proteins in eukaryotic cells, about 70% of the Asn-X-Ser/Thr motifs are co-translationally modified via N-

glycoslylation in the ER. A protein contains on average 4.6 N-glycosylation sites but the number of N-glycosylations may be as high as 24 per molecule120. N-glycans are trimmed and modified upon their transit through the Golgi. The affinity of galectins increases with respect to the number of N-acetyllactosamine branches per N-glycan and their extension with polylactosamine11. In the medial Golgi, N- acetylglucosaminyltransferases I, II, IV, and V (termed Mgat1, Mgat2, Mgat4a/b, and Mgat5) initiate the addition of N-glycan branches in a sequential manner121. Branched N-glycans are further modified in the trans Golgi and variably elongated with polylactosamine (linear repeats of Galβ1,4GlcNAcβ1,3), sialic acid (SA), fucose (Fuc), and N-acteylgalactosamine (GalNAc). Therefore branching at the Asn-X-Ser/Thr site is variable, as is elongation, leading to the creation of heterogeneous structures on mature glycoproteins. The Mgat5 product contains tetra-antennary N-glycans and is preferentially elongated with poly-N-acetyllactosamine, producing N-glycans with higher affinities for galectin than less-branched structures. Thus Mgat5-/- cells were shown to exhibit reduced binding of galectin-3 to complex N-glycans on the cell surface71. Galectin-1 binding to CD43 and CD45 on the surface of T cells depends on specific oligosaccharide modifications on these glycoproteins35. Galectin-1 was shown to induce T cell death and can be positively or negatively regulated by CD45, depending on the glycosylation status of the cell and hence of CD45 itself; for instance, when CD45 N-linked glycans were sialylated, galectin-1 induced CD45 clustering and T cell death was prevented122,123. Thus, glycan structures presented at cell surfaces hold valuable information within their carbohydrate structures and modifications.

1.3 The organisation of the plasma membrane

1.3.1 Plasma membrane domains

Cell membranes display a tremendously complex array of lipids and proteins holding different cellular functions that need to be coordinated. In order to organise and integrate different molecules and functions, the plasma membrane needs to segregate and partition its constituents laterally into various subdomains and clusters

10 of macromolecules to be able to serve different functions effectively. Such domains include nanoscale domains such as lipid rafts enriched in cholesterol and sphingolipids (for further information on lipid rafts please refer to section 1.3.6, page 18); specialised membrane regions encompassing endocytic invaginations like clathrin- coated pits and caveolae; distinct membrane structures like cellular adhesions, immunological synapses and neural synapses; and molecular lattices formed by lectin- glycoprotein interactions. The formation of domains – i.e. structuring and compartmentalising the plasma membrane into functionalised regions – increases the local molecular concentrations of domain constituents and thus facilitates selective molecular interactions for the execution of specific cellular processes. Domain formation is tightly regulated by various components such as the actin cytoskeleton, scaffolding proteins (lectins, caveolin, clathrin), membrane lipids containing specific molecular structures (such as sphingolipids (SL) and glycosphingolipids (GSL)), as well as functional attachments found on lipids and proteins (such as membrane anchors and N-glycans).

1.3.2 Molecular lattices

The abundance, size and complexity of glycan structures present at the cell surface determine the constitution of a macromolecular structure termed glycocalyx124. The glycocalyx is a network of membrane-bound proteoglycans and glycoproteins; and plasma-membrane derived soluble molecules become integrated to participate in this network124. Structural and functional information in the glycocalyx is encoded in carbohydrate structures that are attached to molecules. In the glycocalyx of the ocular surface, galectin-3 was shown to function as a stabiliser at the epithelial barrier by binding and cross-linking distinct glycosylated membrane-associated mucins125,126.

Galectin-glycoprotein lattices support many functions in the regulation of cellular processes. To date, three major roles for lattices have been described. Firstly, in polarised cells, targeting of apical protein and formation of lipid rafts via galectin lattices aid in the organisation of apical and basolateral membrane domains. Secondly,

galectin lattices are involved in the regulation of signalling thresholds at the cell surface as shown by T cell antigen recognition and T cell apoptosis. Thirdly, the molecular lattices can determine receptor residency time at the cell surface by inhibiting their endocytosis. These three aspects are discussed in more detail below.

(1) The organisation of plasma membrane domains is particularly important in polarised cells and studies have demonstrated the contribution of N-glycans127 and carbohydrate-binding proteins128 in the regulation of apical targeting of proteins. Apically targeted vesicles in MDCK (Madin-Darby canine kidney) cells were associated with galectin-3 and depletion of galectin-3 from these cells by RNAi resulted in missorting of apical proteins129. Similarly, in the intestinal brush border, hydrolases that are expressed in polarised enterocytes were shown to bind to and depend on galectin-3. In galectin-3-null mice, these hydrolases, destined for the apical membrane, mislocalised to the basolateral membrane130. Other membrane abnormalities were observed in these mice130. These data indicate an important role for galectins in sorting of proteins and targeting of membranes in polarised cells.

(2) Regulation of signalling thresholds by galectin glycan lattices has been well documented, especially in T cells. The interaction of galectins with glycosylated cell surface proteins on T cells was suggested as a possible mechanism for influencing initiation of signalling and controlling signalling strength. The formation of a molecular lattice that negatively regulates T cell function has been shown by Chen et al. using Mgat5-null mice lacking specific N-glycan branches to which galectin-3 preferably binds131. The authors suggested that the galectin lattice not only retained CD45 in membrane microdomains but also prevented signalling molecules including the T cell receptor (TCR), co-receptor CD4 and kinases Lck and Zap70 from entering these domains. Furthermore, they proposed a general mechanism in which extracellular domains formed by the galectin-glycan lattice are counteracting cytoplasmic forces generated by the actin cytoskeleton and adaptor proteins132. During thymocyte development the process of positive and negative selection regulates thymocyte maturation. Using transgenic mouse-models, the Miceli group found that loss of galectin-1 expression promoted positive thymocyte selection and reduced negative selection suggesting that the galectin-1 lattice increases TCR signalling by stabilising 12 agonist-antigen complexes133. Galectin-1 deficient mice, as well as the previously mentioned Mgat5-deficient mice, showed an increased susceptibility for autoimmune disease and alterations in cytokine production71,134,135, supporting a role that galectin- lattices regulate antigen responses in T cells. Thus galectin lattices function in the regulation of signalling thresholds and are involved in T cell development.

(3) Cells receive information from their environment via cell surface receptors at the plasma membrane that mediate contact flexibly and swiftly. Regulation of cellular probing of the local environment is influenced by receptor residency time at the cell surface and often ligand binding triggers receptor endocytosis thus attenuating signalling. The galectin-lattice has been suggested to extend the residency time of cell surface receptors, thereby increasing the magnitude and/or the duration of signalling. Mgat5-modified N-glycans promote lattice formation, which results in the retention of EGF at the cell surface and promotes growth factor signalling. In contrast, in Mgat5- deficient cells, the cellular response to EGF is dampened due to reduced density of EGF receptors at the cell surface19. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that EGF receptor movement into endocytic vesicles was reduced in wild-type compared to Mgat5-deficient cells because EGF receptors were retained at the cell surface by the galectin lattice75. In experiments using Mgat4a- deficient mice, glucose transport was impaired and a rise in the intracellular localisation of glucose transporter 2 (GLUT2) receptors was observed. This suggested that increased endocytosis of the transporter was due to the absence of proper N- glycosylation causing improper lattice formation, which in turn failed to retain GLUT2 receptors at the cell surface136. In a study on angiogenesis in refractory tumours, it was shown that galectin-1 significantly prolonged cell-surface residency time of the vascular endothelial growth factor (VEGF) receptor 2, promoting angiogenesis and tumour progression137,138. Galectin-9 also participates in cell surface lattices. On CD4 Th2 cells but not Th1 cells, galectin-9 bound to the cell surface protein disulphide isomerase (PDI), leading to the retention of PDI at the cell surface139. Increased cell surface PDI regulated T cell migration and potentiated infection with HIV. Collectively, these data indicate critical tasks for galectin-glycan lattices in biological functions in order to maintain well-balanced cellular processes.

1.3.3 The main lipids of the plasma membrane

Cellular membranes are made of lipids and membrane-associated or -embedded proteins. In eukaryotic membranes, three main classes of lipids are found: sterols, glycerophospholipids, and sphingolipids. Cholesterol is the most abundant sterol in mammalian membranes and its levels are tightly controlled by a complex mechanism140. The tetracyclic sterol-ring structure of cholesterol is buried within non- polar phospholipid tails in the membrane and its hydroxyl group interacts with polar head groups of phospho- and sphingolipids. Thus cholesterol increases membrane packing and is a key regulator of membrane fluidity through interactions with fatty acid hydrocarbon chains of neighbouring phospholipids, stabilising the membrane at high temperatures and preventing membrane stiffening at low temperatures. Phospholipids contain a hydrophilic head group made of a negatively charged phosphate group and glycerol, and two hydrophobic saturated or unsaturated fatty acid hydrocarbon chains. The saturation state of the fatty acid chains also impacts on the fluidity of the lipid bilayer; saturated phospholipids have straight fatty acid chains and consequently pack into the bilayer more densely whereas bent unsaturated chains cannot be packed as densely and thus confer higher fluidity to the membrane. Ceramide-based sphingolipids have the tendency to self-aggregate in cellular membranes and form separate phases that are less fluid compared to bulk phospholipids. All lipids within the large family of sphingolipids (SLs) share ceramide as their common core structure, and glycosphingolipids - a sub-division of this family - comprise an important group. Glycosphingolipids can be found in cell membranes of all organisms ranging from bacteria to man. Nearly all glycolipids found in vertebrates are glycosphingolipids (GSLs) and they are represented in all vertebrate tissues without exception. So far, hundreds of unique GSL structures have been identified. The ceramide backbone of these lipids consists of sphingosine, a long-chain amino alcohol, linked to a fatty acid via an amide linkage. The major difficulties encountered by J.L.W. Thudichum in 1884 in the determination of the structure of sphingosine, which contains both amine and alcohol, are responsible for its name referring to the enigmatic Egyptian Sphinx141. Variations in ceramide structures regarding length, hydroxylation, and saturation of sphingosine as well as the fatty acid moieties, 14 contribute to the structural diversity of these lipids which in turn impacts on the presentation of attached glycans at the membrane surface; thus major structural and functional classifications have traditionally been made according to these glycans. During the first step of glycosylation, attachment of glucose or galactose yields two very basic GSLs, glucosylceramide (GlcCer), or galactosylceramide (GalCer), respectively. From these basic GSLs more complex structures are generated142,143. Different glycan extensions and modifications create a series of structures that form the basis for nomenclature and classification of GSLs into subfamilies that are expressed in tissue-specific patterns. Evidently, this diversity among GSLs reflects important differences in their functions.

1.3.4 Behaviour and function of glycosphingolipids

GSLs are primarily located in the outer leaflet of the plasma membrane, mediating cell-cell interactions and the regulation of protein activities within the plasma membrane. GSLs are not essential for cell homoeostasis but are implicated in cell regulatory processes and signal transduction. GSLs provide structural integrity to plasma membranes and play critical roles in modulating cellular signalling and gene expression142,144,145. Through these operations, GSLs alter various aspects of cell function, including apoptosis, proliferation, autophagy, endocytosis, transport, migration, senescence, and inflammation146–149. Essential functions of GSLs are also present in developmental processes; here GSLs are required in the mediation and modulation of intercellular coordination in multicellular organisms and the differentiation of cells143,150–153.

The composition of membrane lipids in plasma membranes of higher animals comprises from <5% to >20% GSLs. Their distribution within the plasma membrane is not uniform but clustered into GSL-enriched microdomains (GEMs) or lipid rafts, creating small lateral lipid microdomains of self-associating membrane molecules. The concept of lipid raft was introduced in 1988 to explain the different protein and lipid compositions in the apical and basolateral membranes of epithelial cells154. Later, this hypothesis was adapted to generally account for lipid-mediated compartmentalisation

of all cellular membranes that functions in many cellular processes such as Golgi- trafficking, cellular signalling, endocytosis and other membrane functions155. Data generated since the beginnings of the raft hypothesis suggest a role for GSLs in the dynamic organisation of plasma membranes as well as in the formation of signalling complexes. Further studies revealed specific biological roles for certain GSLs. There are over 400 different species of GSLs in mammalian cells and the current view assigns a key role to GSLs in mammalian plasma membranes.

In mammals the complexity of oligosaccharide groups varies greatly among GSLs ranging from simple two sugar molecules to complex oligosaccharide chains containing covalently bound sialic acid150. Accordingly, a multitude of different GSLs has been characterized such as globo-, (neo)lacto-, sulfo-, or phosphoglycosphingolipids, and GSLs containing neuraminic acid moieties, termed gangliosides156. Compared to the commonly found bulk membrane glycerophospholipids, the ceramide acyl chains of mammalian GSLs are longer and highly saturated resulting in different phase transition temperatures157 and provides them with the ability of forming gel-like structures that segregate laterally from other lipids with lower phase transition temperatures. Thus, the chemical and biophysical properties of distinct GSLs species may result in differential organisation of biological membranes. This is illustrated by studies examining the clustering behaviour of gangliosides GM1 and GM3. Gangliosides have been implicated in exerting multiple physiological functions in cellular membranes and GM1 and GM3 were shown to exhibit a differential sensitivity to cholesterol depletion or actin depolymerisation leading to a reduction in clustering of both of these gangliosides158,159. Post-translational modifications of membrane proteins with glycosylphosphatidylinositol (GPI) or palmytic acid groups mediate the partitioning of membrane proteins within membrane nanodomains160–162 and membrane associated proteins can interact directly with specific GSLs through their glycosylated headgroups163,164. A study focusing on the action of GM3 in EGF receptor regulation revealed that the lipid composition exerts regulatory effects on kinase domain activation and showed a direct impact of GSLs on signalling proteins. In this context, GM3 limited autophosphorylation of EGFR and its dimerization165. Other studies demonstrated that the ganglioside GM1 regulates the membrane localisation of the

16 platelet-derived growth factor (PDGF) receptor, displacing it from caveolae when GM1 levels at the cell surface are increased166. GM1 is furthermore assumed to regulate the function of the tropomyosin receptor kinase (TRK) receptor by promoting autocrine stimulation through its own ligand167,168. These and other discoveries identify GSLs as key players in plasma membrane compartmentalisation and signalling platform formation.

1.3.5 Interactions of GSLs with galectins

Although the formation of lattice structures was initially proposed to occur mainly through cross-linking of glycoproteins, accumulating evidence suggests that binding of galectins to glycolipids also takes place and hence galectin engagement of GSLs may contribute to lattice formation (Figure 1.5). GSLs belong to the most diverse and abundant cell surface glycolipids in the outer leaflet of plasma membranes of animals and have been demonstrated to interact with a variety of different molecules. Interactions among GSLs through carbohydrate moieties have been reported169 and GSL interactions with cell surface integrins170, galectins171–178 and other carbohydrate binding proteins173,179,180 have also been demonstrated. Owing to their interactions with lectins, GSLs have been implicated in cellular recognition processes as well as modulation of signal transduction by influencing cell surface receptors181,182. The importance of GSLs in plasma membrane domain formation and their necessity in immune cell regulation was established in mice deficient in the biosynthesis of lacto/neolacto-series GSLs. These mice exhibited increased levels of B cell activation as well as hyperproliferation178. In this study the authors furthermore hypothesised a function for galectins and certain GSLs in plasma membrane domain formation. Also, gangliosides engage in galectin-dependent cell-cell adhesion, growth control and signal transduction174,183,184. Using enzyme-linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR), the carbohydrate binding specificity of the N-terminal CRD of galectin-8 to GSLs carrying 3-O-sulfated 3Galactose/Sialylα2 residues was shown175. Furthermore this study demonstrated that galectin-8 associated with GSLs in Chinese hamster ovary (CHO) cells. Similarly, galectin-4 bound to GSLs carrying 3-Ο-sulfated

galactose residues and colocalised with specific GSLs (SB1a and GM1) on the cell surface of human colon adenocarcinoma cells176. In other studies, galectins-1, -4 and -9 were shown to bind GSLs172,185,186. The binding of galectin-3 to GSLs has been established by others11 and interactions with mammalian GSLs have been analysed using X-ray crystal structures of the galectin-3 CRD bound to GSLs171. Using super- resolution microscopy GSLs were shown to induce clustering of galectin-3 in this thesis and a published report187, furthermore membrane bending induced by galectin-3 has been demonstrated187. Collectively, these observations imply a function for galectins in GSL-dependent plasma membrane binding and suggest a contribution of GSLs together with glycoproteins in the formation of molecular lattices.

Figure 1.5. Model of galectin-3 cross-linking glycoproteins and –lipids at the cell surface. After synthesis on cytoplasmic ribosomes, monomeric galectin-3 is secreted by a non-classical pathway and is thought to bind to the cell surface in an autocrine or paracrine fashion. Monomeric galectin-3 is bound by N-glycans of plasma membrane glycoproteins and GSLs. Different GSLs can be involved in galectin binding. Galectin-3 can form pentamers through its N-terminal domain cross-linking glycosylated cell surface molecules thus forming membrane domains.

1.3.6 Plasma membrane domains and lipid rafts

Currently, membrane rafts are viewed as highly dynamic nanoscale assemblies of 10 -200 nm in size, enriched in sterols and sphingolipids, containing associated plasma membrane proteins, including GPI-anchored proteins188. Cholesterol is a critical component for lipid rafts providing a highly condensed structure of the lipid bilayer, and depletion of cholesterol reduces the abundance of lipid rafts and dissociation of 18 associated raft proteins. Lipid raft disruption can be achieved using different compounds that sequester, deplete or complex cholesterol. Treatments using saponin, digitonin, filipin, nystatin, or methyl-β-cyclodextrin (mβCD) are commonly applied in order to study the disruption and impact of plasma membrane domains in cellular events189–191. Oxysterols such as 7-ketocholesterol (7-KC), which is chemically derived from cholesterol, decrease membrane order by disrupting the tight packing of saturated acyl chains of membrane lipids192,193. The chemical composition of hydrocarbon chains of SLs aids in the formation of raft domains and contributes to the phase separation of membrane lipids via their saturation state. Here, cholesterol- dependent lateral segregation occurs due to the preference of the planar rigid sterol ring to interact with straighter hydrocarbon chains of saturated lipids194, leading to a physical segregation in the plane of the membrane and creating thicker liquid-ordered phases that co-exist with thinner liquid-disordered phases195. SLs display longer and saturated hydrocarbon chains, which favour the interaction with cholesterol and also lead to ordered phases with increased thickness. Phase-separation was first observed in lipid bilayers in model-membrane systems196,197. The co-existence of liquid-ordered

(lo) and liquid-disordered (ld) domains in hydrated bilayers underscores the raft hypothesis in living cells. The raft concept has long been controversial – due to the small size and dynamic nature of these domains it has been difficult to prove the existence of lipid rafts in cells with most experimental approaches. However, improved methodology using biochemical crosslinking as well as advances in microscopy including lipid order sensitive dyes such as Laurdan (6-dodecanoyl-2- dimethylaminonaphtalene) and fluorescence resonance energy transfer measurements have contributed to evidence that supports the lipid raft hypothesis198– 204. Laurdan, a fluorescent lipid probe whose emission wavelength depends on the lipid environment in which the Laurdan molecule resides203,205, can be used to measure membrane order in living cells. The tight packing of SLs and cholesterol in membrane rafts confers resistance to detergent solubilisation with non-ionic detergents206, hence lipid rafts are biochemically characterised as ‘detergent-resistant membranes’ (DRM). The association of proteins with lipid rafts has mainly been assessed by isolation of DRM fractions. However, this association reflects the preference of proteins for DRMs

but does not prove their association prior to the experimental manipulation207. Thus, the use of raft markers such as the ganglioside GM1, CtxB, or GPI-anchored proteins or fluorescent membrane probes such as Laurdan have greatly improved the investigation of membrane rafts208–214.

Lipid rafts are considered to play an important role in signal transduction by forming concentrating platforms for individual receptors. The residency in lipid rafts creates an environment for protein interactions and modification of the phosphorylation state. A connection between lipid rafts and spatiotemporal regulations of cell signalling is well established and lipid rafts have been shown to act as signalling platforms for many signalling pathways215–222. For instance, rafts have been identified as signalling platforms at the immunological synapse (IS) in T cells after the discovery that the T cell receptor (TCR) promotes partitioning of central T cell signalling proteins such as CD3ζ, LAT (linker for activation of T cells), ZAP-70 (ζ-chain TCR-associated protein kinase of 70kDa) and phosphoinositide 3-kinase into DRMs223– 225. Lck, which is the first signalling protein involved in the signalling cascade after the TCR, was found to be greatly enriched in DRM fractions compared to detergent-soluble fractions in Jurkat T cells226. In a recent study investigating the migration capabilities of galectin-3-/- dendritic cells (DC), the authors detected impaired cell migration and attenuated contact hypersensitivity responses in galectin-3-/- mice227. The observation of defective signalling in galectin-3-/- cells upon chemokine receptor activation and an enrichment of galectin-3 in lipid rafts in stimulated DCs and macrophages227, implied an involvement of galectin-3 in the signalling processes leading to DC migration. A study investigating the effects of galectin-9 on osteoblast proliferation and signalling mechanisms demonstrated that galectin-9 induced clustering of lipid rafts in the plasma membrane and subsequent phosphorylation of the c-Src/ERK pathway. In contrast, when galectin-9 induced lipid rafts were disrupted by mβCD, phosphorylation of c-Src and proliferation of osteoblasts were reduced43. These findings suggest that activation of c-Src is a direct effect of galectin-9 leading to the idea that galectin-9 triggers c-Src signalling via lipid rafts. The presence of lectins, galectins and galectin- 3228 in lipid rafts has previously been reported43,227,229–234 and a participation of galectin-3 in signalling processes initiated in lipid rafts seems likely.

1.3.7. The role of lectins in the organisation of the plasma membrane

Lipid rafts may fuse, possibly in response to signalling events, into larger, more stable signal transduction platforms235, and the process of merging and coalescing can be facilitated through specific lipid-lipid, protein-lipid, and protein-protein interactions188,207,236. One possibility is that coalescence of rafts can be promoted through clustering of raft-components and thus the process of merging may be induced by lectins and galectins. Selective binding of galectins to cell surface receptors and segregation of proteins and lipids into special domains can potentially lead to the merging of smaller pre-clustered domains. Galectins can also contribute to the formation of membrane domains by specifically sorting proteins and lipids. It is currently assumed that the increased thickness and lipid order of lipid rafts surrounded by a disordered lipid environment could promote membrane bending237,238. Galectins are susceptible to further support this lipid-induced membrane bending via membrane binding and self-oligomerization187. In a recent study that includes data presented in this thesis, galectin-3-induced clustering, membrane bending and endocytosis were investigated, and a model was proposed in which monomeric galectin-3 is recruited to the plasma membrane by binding to glycosylated cargo proteins such as CD44 and β1- integrin. Membrane bound galectin-3 then forms oligomers and interacts with GSLs. Subsequently co-clustering of GSLs and cargo proteins generates mechanical strain on the plasma membrane, leading to the formation of endocytic invaginations187. Thus, galectin-3 induces clustering and participates in the generation of local specialized membrane regions that form entry portals for endocytosis (Figure 1.6). In polarised epithelial cells, directed transport of distinct proteins and cargoes to their apical or basolateral membranes was demonstrated, and it is commonly accepted that lipid rafts play a role in this directed trafficking237. Here N- and O-glycans are thought to act as sorting signals for apical trafficking, and galectins including galectin-3 were shown to participate in intracellular sorting, targeting and transport of cargo destined for the apical membrane129,130,239,240. As rafts exist also on internal membranes, they could play an important role in the formation of apical or basolateral transport carriers and domain-induced budding could provide the driving force for cargo formation involving 21

outward bending of raft clusters237, here galectins could be key players in generating specialized domains. Membrane domains supported by galectins are also involved in cell migration. A study by Hsu et al. reports that galectin-3 is associated with membrane ruffles and specifically with lipid rafts in migrating DCs227. Here galectin-3 deficiency resulted in structural differences in ruffles, suggesting that galectin-3 is necessary for the formation of more complex ruffle structures and for the regulation of cell migration by coordinating the local architecture of the cell membrane227.

Figure 1.6. Model of galectin-3 co-clustering and CLIC endocytosis. Monomeric galectin-3 is synthesised on cytoplasmic ribosomes and secreted by a non-classical pathway. Through its affinity to carbohydrates, monomeric galectin-3 is recruited to the plasma membrane where it binds to glycosylated cell surface receptors, such as CD44 and β1-integrin. N-glycan-bound galectin-3 oligomerises through its N-terminal domain and gains GSL binding capacity. Co- clustering of GSLs and cell surface receptors by pentameric galectin-3 generates mechanical stress and induces endocytic membrane invaginations leading to clathrin-independent endocytosis187.

Lipid rafts have also been shown to play a role in viral infection. Budding viruses acquire their membrane from the host cell. Consequently, viral membranes are enriched in raft lipids241 and contain proteins associated with rafts242, indicating that membrane rafts may also exist in the membrane of viruses. More importantly, viruses depend not only on raft lipids for entry into their host cells but also use lectins or galectins as receptors234,243–246, suggesting that lectin- or galectin-supported microdomains may play a central role in viral infection. Lectins are also key organisers 22 of plasma membrane microdomains in the brush border. Galectin-4 is found to associate with the exoplasmic leaflet of the plasma membrane in the intestinal brush border. Here it cross-links glycoproteins and –lipids, associates with lipid rafts and aids in the creation of lipid rafts through the clustering of abundantly present glycolipids into membrane domains230,247. Release of galectin-4 from plasma membranes in the brush border removes essential digestive proteins such as aminopeptidase N and alkaline phosphatase from lipid rafts, indicating that the brush border relies on galectin-4 as a plasma membrane organiser and raft stabiliser229, probably in order to assure the association and concentration of important enzymes within membrane domains. Similarly, intelectin, a mammalian Ca2+-dependent lectin expressed in the small intestine, was found to associate with lipid rafts in the brush border231. Galectin- 4 was organised in stable glycolipid-based raft domains suggesting a similar function for this lectin in membrane organisation and stabilisation, preventing loss of digestive enzymes248. Thus galectin-4 and intelectin have been found to be key components coordinating plasma membrane microdomains. Further, it has been implied that other members of the galectin family229 or other lectins248 that are expressed in the gastrointestinal tract, contribute to the organisation of the plasma membrane. Formation of membrane domains by galectins does not necessarily require the presence of cholesterol, as cholesterol depletion by mβCD does not affect the stability of lipid rafts in microvilli of the brush border. Instead, the formation of these domains relies solely on the presence of GSLs, and possibly galectin-4249. This finding supports the possibility that lectins can contribute to the formation and stability of various distinct populations of membrane domains. A similar heterogeneity of membrane domains has been observed for two microvillar proteins in MDCK cells - prominin and placental alkaline phosphatase (PLAP). Both proteins were shown to reside in separate subsets of membrane rafts and the co-existence of different cholesterol-based lipid rafts was suggested by the authors250.

Membrane domains supported by lectins do not only play a role in shaping membrane architecture, but also contribute to the regulation of cell signalling, as mentioned above. The scaffolding function of galectins promotes recruitment of proteins and lipids to these plasma membrane domains, so that the dynamics and

functionality of signalling molecules can be modulated. Functional lattice formation and its effects have already been reported: galectin-3 binds to and oligomerises at the surface of neutrophils and endothelial cells74, plasma membrane bound galectin in turn regulates diffusion rates of cell surface receptors such as integrins and EGF receptors in the presence of Mgat5 generated N-glycans75,251. The lattice can inhibit or promote signalling events, as shown by different studies: in T cells, the lattice formed between Mgat5 generated N-glycans and galectin partitions the CD45 receptor inside rafts while the TCR-CD4-Lck-Zap70 complex is retained outside rafts, impeding TCR activation131. In this context, lattice functionality is underscored by the fact that mice lacking Mgat5 exhibit hypersensitive T cells due to the absence of lattice regulation71. On the other hand, lattice formation has been shown to facilitate and foster signalling: in T cells galectin-1 binds to certain glycosylated cell surface receptors, including CD7, CD43 and CD45, and these glycoproteins can regulate susceptibility to apoptosis of T cells, induced by galectin-135. In osteoblasts, galectin-9-induced clustering of lipid rafts leads to c-Src phosphorylation followed by proliferative activity43. Considering the ability of galectins to compartmentalise the plasma membrane and form functional membrane domains, it appears likely that galectins may be capable of forming endocytic domains.

1.4 Endocytosis

The traditional view of endocytosis as a way to merely internalise nutrients and extracellular molecules that became associated with the plasma membrane is not sufficient to encompass the sheer complexity of this molecular mechanism. The plasma membrane fences off the cellular interior and is a large surface through which cellular communication with the surrounding environment occurs. Together with the plasma membrane, endocytic events are major organisers between the intra- and extracellular space, and must be tightly regulated in order to enable cells to properly respond to their environment. Cellular signalling and signal transduction are influenced by the availability of cell surface receptors, whose presence at the membrane is itself regulated by endocytosis and recycling. Thus endocytosis plays an important role and

24 influences a variety of cellular processes including fluid and nutrient uptake, regulation of signalling, cell motility, antigen presentation, lipid homeostasis, and plasma membrane remodelling, just to name a few252–258. There are various ways in which cells mediate the internalisation of cargo from the plasma membrane. For instance, in phagocytic events, there is a progressive formation of invagination around the cargo but this mechanism is restricted to specialised mammalian cells; and during macropinocytosis internalisation occurs from highly ruffled plasma membrane regions. In order to sustain cellular homeostasis, eukaryotic cells maintain several distinct, constitutive pathways of endocytosis. These are clathrin-mediated endocytosis (CME)259, endocytosis via caveolae256, and several non-caveolar clathrin-independent endocytic (CIE)256 routes.

The first identified and best-known endocytic process is CME; here the cargo is recruited into developing clathrin-coated pits that subsequently turn into clathrin- coated vesicles (CCV). Caveolae represent a particular type of microdomain, forming smooth cholesterol-sensitive flask-shaped invaginations at the plasma membrane260– 262. Regulators, cargoes and dynamics of CME as well as caveolar pathways have been identified and are reasonably well understood. In comparison, much less is known about the mechanisms of non-caveolar CIE; nevertheless several types of CIE are known to date, which have been characterised based on the participation of certain proteins and lipids, the sensitivity to pharmacological inhibition, and the internalisation of particular cargoes256,263. Although CME accounts for a plethora of endocytic processes, and had long been assumed to be the main pathway for receptor downregulation259, several studies have shown that many cell surface receptors are in fact internalised through clathrin-independent pathways256,264. In the CLIC/GEEC pathway (clathrin-independent carrier/GPI-enriched early endosomal compartment), specific domains are first formed in the plasma membrane before they become CLICs that acquire Rab5 and EEA1 molecules and mature into GEECs that consequently merge with early endosomes265. Parameters defining the CLIC pathway include dynamin-independence266, enrichment in GPI-anchored proteins266, reliance on Arf1 activity266,267 as well as Cdc42 regulation263, and sensitivity to cholesterol depletion261,268. Cdc42 is a small G protein that induces cytoskeletal changes and

promotes the actin polymerization required for vesicle internalisation265,268. At the plasma membrane, Cdc42 binds preferentially to sphingomyelin- and cholesterol- enriched membranes 269,270, establishing a link between lipid rafts and endocytosis. A variety of small G proteins contribute to endocytic events such as the Arf, Rab and Rho families. Their regulation is tightly coupled to their affiliation with the plasma membrane and they are cycling through membrane-bound and cytosolic stages depending on their association with GTP or GDP. Distinct pathways in endocytosis seem to be reliant on specific subsets of small G proteins, which are involved in cytoskeletal regulation in a complex manner. They activate a variety of effectors that impact on cytoskeletal rearrangements and plasma membrane events that are necessary for endocytosis. Arf proteins are found predominantly at the plasma membrane and take part in the regulation of CIE. They participate in plasma membrane and cytoskeletal functions, regulating actin polymerization and the local 271,272 production of PtdIns(4,5)P2 at the plasma membrane . Arf1 and Arf6 have been shown to generate membrane curvature through insertion of an amphipathic helix into the proximal lipid layer273.

A prerequisite for endocytosis is the formation of a budding structure with or without clathrin coating that eventually dissociates from the plasma membrane. A diversity of adaptor and accessory proteins is required to achieve vesicle formation. Dynamin is a large GTPase, which is critical for the final scission from the membrane. Dynamin forms a helical polymer around the neck of the budding vesicle and mediates fission from the plasma membrane in order to release the vesicle to the intracellular space274,275. Dynamin functions in many processes of endocytosis and has been shown to be indispensable for CME as dominant-negative dynamin constructs inhibit CME276. Dynamin is also involved in several clathrin-independent endocytic processes. During caveolin-dependent endocytosis caveolin-1 and dynamin form a complex in vivo and dynamin can be found at the neck of caveolae, suggesting a function for dynamin in the fission of caveolar uptake277–279. In flotillin-associated endocytosis, both dependence and independence of dynamin has been reported280,281.

Lipids also have crucial functions in the modulation of endocytic events and membrane-associated signalling. Evidence for raft-mediated routes of endocytosis has 26 established a function for lipid rafts as well as caveolae in the internalisation of raft- associated proteins204. The interleukin 2 receptor (IL2R) is a lymphocyte growth factor receptor that is synthesised and secreted in response to T lymphocytic activation and is essential for proliferation. The IL2R is constitutively associated with lipid rafts and while its internalisation process depends on dynamin, it is independent of clathrin and caveolins282, potentially indicating a distinct raft-mediated pathway for IL2R endocytosis. Several extracellular ligands are known to bind to GSLs and to be endocytosed; these include antibodies, bacterial toxins, viruses, and lectins283. It has previously been shown that some endocytosed molecules that bind to GSLs are transported to the ER from where they can enter the cytosol284.

1.4.1 Membrane Curvature

Another underlying feature of plasma membrane areas engaged in endocytic events is the necessity to create a membrane curvature in order to initiate the budding process (Figure 1.7). In recent years, a large variety of cellular proteins have been found to generate membrane curvature and increasing evidence shows that the composition and organisation of membrane lipids plays a role in this process. Epsin, which is known to have a function in CME, induces membrane curvature by inserting an amphipathic helical structure into the proximal membrane monolayer, resulting in splayed phospholipid moieties285. Proteins within the BAR domain superfamily have been shown to be linked with endocytic events and fulfil functions in sensing and generating membrane curvature and possibly effector recruitment to curved membrane sites286. BAR protein dimers are linked in a particular angle, creating a highly curved interface reminiscent of a boomerang shape. BAR domain proteins can sense curved membranes and bind membranes that fit their intrinsic molecular curvature via electrostatic interactions 286. Due to their ability to oligomerise and bind to the extracellular moiety of cell surface proteins and lipids, lectins can also generate membrane curvature. Our recent study shows that galectin-3 induced GSL-dependent membrane bending as a first step of CLIC biogenesis to drive clathrin-independent uptake structures187. GSLs were required for galectin-3 induced membrane curvature.

GSLs have been implicated in endocytic processes through modulation of membrane curvature owing to their biophysical properties. Studies investigating the internalisation of Shiga toxin revealed that binding of the toxin subunit B to the ganglioside Gb3 induces formation of a local ordered phase287, which in turn facilitates membrane curvature288 and aids in the formation of membrane tubules164. The GSL lactosylceramide affected the clustering of β1-integrin in GSL-enriched microdomains and stimulated its endocytosis289. Our data revealed that β1-integrin was endocytosed via galectin-3 in MEF cells and the uptake of CD44 depended on the presence of galectin-3, GSLs and N-glycans187.

Asymmetric lipid compositions can also contribute to membrane bending. Plasma membranes exhibit an asymmetry regarding different phospholipids in the inner and outer leaflets. These differences are generated by P4-ATPases or lipid flippases that catalyse the translocation of phospholipids from the exoplasmic to the cytosolic leaflet and have crucial functions in vesicular transport pathways290,291. It is believed that the unidirectional transport of phospholipids is involved in bending the membrane. The enrichment of specific phospholipids may also control the recruitment and function of proteins that are part of the vesicle budding machinery to these sites292. At the cytosolic side of the plasma membrane, the actin cytoskeleton provides pushing and/or pulling forces to help generate and maintain the shape of budding vesicles and to promote the process of fission by dynamin293,294.

Figure 2.7. Mechanisms to generate membrane curvature. (A, B) Scaffolding proteins: (A) Oligomerisation of certain proteins bound to membranes forms a curved structure that causes membrane bending. (B) A rigid protein containing an intrinsic curvature binds to a curved membrane to stabilise it or bends the membrane to induce curvature. (C) Helix insertion: several proteins bind to membranes by insertion of an amphipathic helix. Helix insertions may act as a wedge curving the membrane curvature by pushing lipids apart. (D) Lipid-induced curvature: Asymmetric membrane structure is generated as certain phospholipids become enriched in one leaflet. 28

1.4.2 Galectins in endocytosis

Although endocytosis of galectin-3 was observed in several cell types, its endocytic pathways and entry mechanisms are still poorly understood. The process of binding and endocytosis of galectin-3 does not appear to follow the same mechanisms in all cell types. Earlier studies in breast carcinoma cells indicate a rapid internalisation of galectin-3 upon addition to these cells295–297. In this cell type, galectin-3 internalisation was inhibited using filipin but not chlorpromazine297. Filipin is a sterol- binding agent that is used to disrupt caveolae and caveolae-like structures including lipid rafts whereas chlorpromazine is a cationic amphiphilic inhibiting clathrin- dependent endocytosis. Inhibition of endocytosis by filipin and not chlorpromazine suggests that internalisation of galectin-3 occurs via cholesterol-rich domains and excludes uptake by CME in breast carcinoma cells. On the other hand, in M1 macrophage-like cells, galectin-3 was internalised by CME as demonstrated by inhibition with chlorpromazine298. The dependence of galectin-3 uptake on carbohydrates for binding and internalisation was established with the mutant CHO cell line Lec1 lacking LacNAc moieties in N-glycans of extracellular glycoproteins on the plasma membrane16. Also, in human vascular endothelial cells (HUVEC), galectin-3 could associate with and become internalised into these cells in a carbohydrate- dependent manner299. Results of galectin-3 uptake in macrophage model cell lines varied; in M2 cells galectin-3 uptake was carbohydrate-dependent but carbohydrate- independent in M1 macrophage-like cells298. In polarised MDCK cells, exogenous galectin-3 was rapidly internalised by clathrin-independent endocytosis300, enriched in tubular and vesicular Rab11-positive recycling endosomes301 and recycled back to the cell surface300. Galectin-4 in polarised enterocyte-like cells travelled through early endosomes, following the apical endocytic-recycling pathway and was required for the apical trafficking of glycoproteins302. Internalisation of galectin-1 in T cells occurred in a carbohydrate-dependent fashion and galectin-1 localised to the Golgi within 1 hour of internalisation. Galectin-1 followed two distinct pathways involving clathrin-coated vesicles and raft-dependent endocytosis303. In HUVECs, galectin-3 was shown to be transported to early and recycling endosomes and partitioned into two routes upon uptake – galectin-3 was either targeted to late endosomes and lysosomes or recycled 29

back to the plasma membrane299. Thus galectins in general and galectin-3 in particular appear to follow different modes of uptake and no unified mechanism regarding intracellular transport could so far be identified.

1.4.3 N–glycosylation in endocytosis

As mentioned above, lectins and galectins bind cell surface receptors and transporters with affinities that depend on the structure and number of N-glycans, regulating protein trafficking by either tethering proteins to one another thus largely immobilising them, or by sorting and guiding them into domains, hereby creating endocytic structures. Several biological functions for N-glycans have been determined, and N-glycans seem to play a role in endocytic processes. Examination of binding kinetics and uptake studies of glycoproteins and -peptides in rat hepatocytes showed that endocytosis depends on the structure of attached oligosaccharides101. Here, proteins with certain oligosaccharide structures were not endocytosed. Furthermore, N-glycans can act as determinants in the folding of proteins and the stability in secretory and endocytic membrane trafficking, and glycosylation-deficiency leads to accelerated turnover of cell surface proteins304. Endocytic studies revealed that galectin-3 binds to glycosylated cargoes and GSLs at the cell surface thus inducing membrane curvature and the generation of CLICs. This led to the proposed model that recruitment of galectin-3 to the plasma membrane involves binding to glycosylated cargo molecules and involves GSL to facilitate endocytosis of galectin-3 and associated molecules187. By binding to GSLs and forming oligomers, galectin-3 initiated membrane bending and formation of invaginations. This was demonstrated using a mutant version of the cargo protein CD44 lacking N-glycans that was not internalised by galectin, indicating a function for N-glycans in the mediation of endocytosis187.

1.4.4 Retrograde Transport in Protein Trafficking

The trans-Golgi network (TGN) holds a central role as a traffic hub and is a site of protein sorting305,306. The role of the TGN as organiser and distributor of proteins

30 received from the ER is not limited to forward transport. The TGN also receives proteins by retrograde transport, most notably from endosomes307. In retrograde sorting, endocytic cargo exits from endosomes and is transported to the TGN, Golgi membranes and occasionally to the ER308,309. The retrograde transport route is also exploited by invading pathogens upon cell entry. Cycling of cargoes through the retrograde route may have different purposes: it can be part of a crucial regulatory circuit required for protein function and an integral part in the cycle of the functional Wnt proteins310, or it can serve to promote protein transport to the ER as suggested for antigenic proteins in dendritic cells to aid antigen cross-presentation311. It can also be a mechanism to stock up on cargo proteins ready for secretion, as seen in matrix metalloproteases that are maintained at the TGN in order to be released when cleavage of extracellular matrix components is required during angiogenesis or tissue remodelling312. Cycling between endosomes and the TGN has been described for mammalian proteins such as the SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment receptors) including VAMP4 and GS15, the transmembrane cell adhesion protein P-selectin, the glucose transporter GLUT4 that is recycled and repackaged into vesicles, and the EGF signalling receptor for translocation purposes313. Many toxins such as cholera, shiga, botulinum and tetanus toxin have been shown to undergo retrograde transport314–319. Also, plant toxins which happen to be lectins, such as ricin320 have been shown to travel through the Golgi to the ER after endocytosis, and other lectins have been demonstrated to be retrogradely trafficked321. In addition, galectin-1 is translocated to the Golgi after its endocytosis from the plasma membrane303, indicating a possible pathway for cell surface-bound carbohydrate binding proteins in retrograde trafficking after their internalisation.

1.5 Immunomodulatory functions of galectins

Galectins are found in various cells and tissues of the immune system and galectin expression is modulated during the activation and differentiation of immune cells and may change depending on physiological conditions322. Several galectins are known to critically function in cellular apoptosis, in particular in the regulation of

immune cell apoptosis. Galectin-1 has been implicated in several studies to be involved in inhibiting cell growth, and regulating cell cycle and cell death323–325, and has been shown to participate in the T cell selection process as an inducer of apoptosis in developing thymocytes323–325. Resistance to galectin-1-induced cell death was conferred by CD45-positive T cells lacking branched O-glycan structures due to a deficiency in the core-2-β-1,6-N-acetylglucosaminyltransferase122. Galectin-2 also acts in apoptosis of cells326 and galectin-3 can either promote or protect cells from apoptosis327–329. Similarly to galectin-1, murine galectin-9 plays a role in mouse embryonic development and induces apoptosis in thymocytes330. As previously described, the galectin lattice functions in setting a threshold for T cell activation and protects from autoimmunity71. Previous studies have suggested a role for galectin-3 in regulating the immune response and inflammation. Galectin-3 exhibits pro- inflammatory effects331–333, is involved in phagocytic events334, modulates T cell activation71, can recognise invading pathogens and may function in innate immunity335, and is suggested to participate in the pathogenesis of autoimmune diseases, as shown in Crohn’s disease and systemic lupus erythematosus336,337. Collectively, these discoveries suggest crucial roles for galectins in immunomodulatory functions.

1.5.1 T-cell receptor signalling and formation of the immunological synapse

Signalling is initiated at the plasma membrane when the T cell receptor (TCR) recognises and binds antigenic peptide displayed by the major histocompatibility complex (pMHC) on antigen presenting cells (APCs). The formation of a specialised cell- cell interface between T cells and APCs with a distinct patterning of cellular receptors and cytoskeletal organisation is referred to as the immunological synapse. Engagement of TCRs results in the formation of TCR microclusters, which form at the periphery of the immunological synapse and are transported toward its centre338–340. These dynamic microclusters contain TCRs, kinases and adaptor proteins that serve to transduce signals for T cell activation from the plasma membrane to the intracellular space (Figure 1.8). The reorganisation of TCR microclusters within the plasma

32 membrane immediately after activation is reasonably well described. Lateral organisation can be imaged with a high signal-to-noise ratio with total internal reflection fluorescence (TIRF) microscopy when T cells are activated with antibody- coated glass surfaces or with supported lipid bilayers in which activating proteins are embedded (antibodies or pMHCs and adhesion proteins such as ICAM1 and B7). After initial activation or ~2 minutes, rearrangements occur within the plasma membrane to generate a mature synapse; TCR microclusters move to the centre of the activation site to form the central supra-molecular activation cluster (cSMAC). The cSMAC is considered as a site for endocytosis of signalling complexes and plays a role in terminating TCR signalling. A peripheral zone termed peripheral supra-molecular activation cluster (pSMAC) surrounds the cSMAC and consists of adhesion molecules such as LFA-1 and ICAM-1341,342.

The TCR comprises the antigen-binding TCRα and TCRβ chains that are associated with the signal transducing CD3 chains that consist of γε, δε and ζζ dimers. Ligation of the TCR induces phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAM) of CD3 chains that are constitutively associated with the TCR. This phosphorylation is mediated by the tyrosine kinase Lck and facilitates recruitment of the kinase ZAP-70 that binds to phosphorylated ITAMs. Subsequently ZAP-70 phosphorylates the adapter protein linker for activation of T cells (Lat)343 and the cytoplasmic adapter SLP-76344. Lat is a transmembrane protein and has multiple tyrosine phosphorylation sites on its intracellular domain. It therefore acts as a scaffold that links TCR triggering to several downstream signalling proteins and pathways that ultimately result in gene expression and cytokine secretion. Phosphorylated Lat and SLP-76 assemble at the plasma membrane via GADS345, forming a signalling complex that recruits multiple downstream components of T cell activation, including signal transduction components and mediators of cytoskeletal rearrangement.

Figure 1.8. Structure of the immunological synapse and the TCR signalling assembly. The immunological synapse is generated upon engagement of TCR with peptide-MHC. TCR ligation and engagement of co-receptors, such as CD4 and CD28, recruit key signalling proteins to the T cell-APC contact area. The tyrosine kinase Lck phosphorylates the ITAM domains of the CD3 chains. Phosphorylated ITAM domains recruit ZAP-70, which in turn phosphorylates Lat and SLP-76. Phosphorylated Lat and SLP-76 both assemble at the plasma membrane via GADS and form a signalling complex that recruits downstream components of T-cell activation. The contact site is stabilised by interactions of LFA-1 and ICAM-1. Activation of PLC-γ by ITK 2+ generates inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), resulting in Ca flux within the cell and in downstream activation of kinases and effectors that influence gene transcription. The Lat-SLP-76 complex also functions as a platform for molecules involved in regulating actin organisation, such as VAV-1, RAC, Cdc42, WASP, and ARP2/3. Intracellular pools of Lck and Lat can be delivered to the immunological synapse by vesicles and endosomes.

1.5.2 Vesicular traffic at the immunological synapse

At the immunological synapse, the orchestrated action of proteins involved in vesicle trafficking aids in the localisation and maintenance of key signalling proteins within and around the synapse. T cell organelles polarise together with microtubules towards the APC, and antigen stimulation triggers intracellular rearrangements and recruitment of molecules and vesicles that are determinants in cytoskeletal changes such as the movement of clathrin-containing multi-vesicular bodies toward the immunological synapse in order to promote localised actin polymerisation346.

Coordinated movement of vesicles is necessary for TCR signal propagation and is guided by actin filaments and microtubules. This is illustrated by the role played by end-binding protein 1 (EB1) in T cell signalling. EB1 belongs to the group of end-binding proteins that are able to bind to microtubules through a specialised domain. In activated T cells, EB1 also bound to the ITAM domains of CD3ε and CD3ζ347. The association of EB1 with CD3 and microtubules suggests that EB1 facilitated the movement of CD3-enriched vesicles via dynein/dynactin motors in and out of the plane of the immunological synapse. EB1 knockdown altered TCR dynamics at the immunological synapse, prevented signal propagation to Lat, and inhibited PLCγ1 activation. Consistent with the current understanding that the microtubular network is highly dynamic and an essential feature of vesicle trafficking at the immunological synapse, signal propagation from the activation site to the Lat/PLCγ1 signalling cassette could not occur when EB1-dependent binding to microtubules (MT) was disrupted347.

Another molecule associated with intracellular membrane compartments and cytoskeletal components is IFT20, an intraflagellar transport protein essential for ciliary assembly, which is expressed in human lymphoid and myeloid cells lacking a primary cilium. IFT20 associates with the microtubule organising centre (MTOC), Golgi and post-Golgi compartments and binds to the TCR subunits CD3ζ and CD3ε upon T cell activation. Recycling endosomes failed to polarise towards the immune synapse in IFT20 knock-down T cells. As a consequence, TCR-CD3 clustering, TCR signalling and TCR recycling back to the cell surface after stimulation were impaired348. The myelin and lymphocyte protein (MAL) is an integral membrane protein implicated in polarised intracellular trafficking349. Sequestration of Lck into endosomal vesicles and recycling to plasma membrane was disrupted in MAL-depleted cells. Time-lapse video- microscopy showed that Lck and MAL travelled in the same vesicles destined for the cell surface350. These are some of the examples in which vesicle trafficking regulates TCR signalling and T cell activation.

Many immune cells produce galectin-3 and galectin-3 is expressed by CD4+ and CD8+ T cells after activation351. Secreted galectin-3 may affect surrounding cells by an autocrine or paracrine mechanism triggering cellular responses and may be 35

internalised and exert intracellular functions6. In activated CD4+ T cells endogenous galectin-3 was recruited to the immunological synapse after TCR engagement and was found to localise primarily to the pSMAC352. The authors found galectin-3 to act as a suppressor in early activation events and to exert a destabilising function regarding the formation of the synapse. This suggests a negative regulatory function for endogenously expressed galectin-3 in T cell activation. Similarly, as described above, externally bound galectin-3 forms a lattice at the plasma membrane of T cells, which negatively regulates T cell signalling and thus increases the signalling threshold in the presence of Mgat5-modified N-glycans71. In conclusion, these data collectively highlight the importance of vesicular traffic at the immunological synapse, and furthermore suggest a possible function for internalised galectin-3 in the regulation of TCR signalling.

1.6 Fluorescence microscopy

Cell biology relies extensively on fluorescence light microscopy and this optical technique has provided the scientific community with valuable insights into molecular structures and cellular processes. However, fluorescence microscopy is limited by the diffraction of light to an optical resolution of ~250 nm laterally and ~600 nm axially353. The diffractive limitation greatly impairs the retrieval of structural information in the sub-diffraction range, preventing access to information on the single-molecule level. Enhancing the resolution beyond the diffraction limit, so called super-resolution fluorescence microscopy provides great possibilities to explore and unravel the complexity of nature. Super-resolution microscopy is an invaluable tool for the study of biological and biochemical processes in single cells as well as multicellular organisms using a non- or minimally-invasive technique resolving ultra-structural details. Recent advances in single-molecule localisation microscopy (SMLM) have achieved resolutions in the sub-nanometre range354.

1.6.1 TIRF Microscopy

Total internal reflexion fluorescence (TIRF) microscopy is founded on an optical phenomenon in which a propagating wave, such as light, impinges on an interface of two different media. Here, two crucial factors must be met: (1) the propagating wave (e.g. laser light) must strike the medium boundary at an angle larger than a particular critical angle and (2) the refractive index of the medium on the opposite side of the boundary must be lower. In the presence of these two factors the light wave cannot pass through and is entirely reflected355. When the incident light is totally internally reflected at the interface, an evanescent wave is created and decays exponentially from the interface, thus penetrating the sample medium only to a depth of approximately 100 nm. This evanescent wave can selectively excite fluorophores that are located at or near the interface, e.g. the medium-coverslip interface. Hence TIRF enables a selective visualisation of regions such as the plasma membrane (Figure 1.9). The idea of combining TIRF with imaging was first described in 1956 by E.J. Ambrose and later improved by D. Axelrod356,357. TIRF microscopy is very sensitive and provides a superb signal-to-noise ratio. Since its first application, it has been used to unravel many biological processes358. TIRF microscopy has been a brilliant tool in combination with fluorescently tagged molecules for the observation of real time proceedings at and close to the plasma membrane. Researchers observed in fixed and live cells processes such as membrane dynamics, vesicular trafficking and cytoskeletal remodelling359–364.

Figure 1.9. Total internal reflection fluorescence microscopy diagram. (A) The sample is adhered to a high-precision coverglass, specifically manufactured for applications in TIRF microscopy. The excitation beam travels through the objective and impinges on the glass- water interface. Here, the evanescent wave is generated at this interface expanding into the sample; the range and shallow penetration depth of this evanescent wave is shown in red. The signal or emission beam generated by the sample is traveling through the objective to the detector (not shown). (B) The diffraction and reflection of light in EPI (epifluorescence), HILO (highly inclined laminated optical sheet) and TIRF (total internal reflection fluorescence). In EPI light is minimally diffracted, in HILO the diffracted light exits at a shallow angle, in TIRF the light is totally internally reflected, generating an exponentially decaying evanescent wave in the medium with the lower refractive index (liquid medium).

1.6.2 Resolution and diffraction in fluorescence microscopy

In conventional light microscopy, the resolution limit is ~250 nm in х and y direction and <600 nm in z direction. This limit defines the spread of a single point of light that is diffracted through the microscope as the emitted light travels away from its source and encounters changes in refractive indices, for example within the sample and optics of the microscope. This diffraction causes the spread of a single point into a three-dimensional intensity distribution that is called the point-spread function (PSF) (Figure 1.10). Objects that are smaller than the PSF appear to be the same size as the PSF, and objects that are closer together than the width of the PSF are not distinguishable as separate objects. The PSF appears with a fixed size and a shape that is represented by an airy disk pattern. This spatial broadening effect is caused by two

38 things: (1) the emission wavelength (λ) of the fluorophore and (2) the numerical aperture (NA) of the objective. Using these parameters, the resolution power of a microscope is expressed according to Ernst Abbe’s ‘Theory of Image Formation’ from the year 1873353.

Using Rayleigh’s criterion, the resolution limit or minimum resolvable distance (d) is a function of the applied wavelength of light and the numerical aperture of the instrument and can be calculated according to the equation below.

d 1.22

If the resolution limit is overcome by at least a factor of two, the method is considered a super-resolution technique.

Figure 1.10. The point spread function (PSF). (A) The PSF represents a three-dimensional structure. Its axial orientation or z dimension is longer compared to its lateral orientation or x-, y-dimensions. (B) The intensity distribution of a single-point source as it is recorded by the detector according to the PSF of the microscope. (C) Overlapping PSFs of multiple point sources emitting at the same time received by the microscope’s detector. Due to the overlap of each PSF the individual sources become indistinguishable from each other creating a bright blurred spot.

1.6.3 Super-resolution microscopy

To resolve subcellular structures that are below the limitations of fluorescence microscopy different conceptual strategies have been employed that enable scientists to overcome the limitation of the diffraction barrier. The implication of these strategies allows the analysis of biological structures at the level of super-resolution. In super-resolution microscopy, excitation is either temporally or spatially modulated and different methods haven been introduced. These include standing-wave microscopy (SWM)365, structured-illumination microscopy (SIM)366, and dynamic saturation optical microscopy (DSOM)367. Far-field microscopy techniques achieving super resolution to a precision of 20-30 nm laterally and 50-60 nm axially have been developed. These include stimulated emission depletion (STED)368–371, stochastic optical reconstruction microscopy (STORM)372–374, photo-activated localisation microscopy (PALM)375, and fluorescence photo-activated localisation microscopy (FPALM)376.

In structured illumination microscopy (SIM), a grid is used to illuminate the sample with patterned light, leading to an increase in resolution by measuring the fringes from the interference of the illumination pattern and the sample. The excitation grid is rotated, generating a series of images with different fringes. A high- resolution 3D image can be reconstructed with an improved resolution by a factor of 2 to about ~100 nm. An extension of SIM exploits a non-linear phenomenon arising from saturation of excited molecular states of fluorophores and thus has been termed saturated structured illumination microscopy (SSIM)366. SSIM can potentially generate a lateral resolution of 50 nm. In stimulated emission depletion (STED)369 a patterned illumination can be used to spatially modulate the fluorescence within a diffraction- limited region. In STED two lasers are applied: a focused laser beam is used for excitation of fluorophores and overlaps with a second ‘STED’ laser beam featuring strong intensities at the focal periphery and a focal centre without any intensity resulting in a doughnut shaped emission of laser light. This setup depletes the fluorescence from fluorophores exposed to the STED beam by transferring them back to their ground state resulting in the detection of fluorescence only within the centre of the doughnut377. Focusing the visible light in a subdiffraction-sized spot results in

40 higher resolution and yields a precision of about 20 – 50 nm. Single-molecule localisation microscopy (SMLM) is an approach that achieves the detection and localisation of single molecules in intact cells using fluorescence and is achieved by fitting the PSF of fluorophores. Variants of SMLM include STORM, dSTORM (direct stochastic optical reconstruction microscopy), and PALM that make use of reversible saturable optical fluorescence transitions (RESOLFT)378 between two comparably stable states of fluorescent molecules. Here the properties of photo-activatable fluorescent labels are exploited by switching on only sparse subsets of fluorescent molecules whereas the majority of fluorescent labels remain in a dark state. Imaging of a fluorescent subset of activated molecules means that each fluorophore can be localised with high precision.

In STORM a pair of cyanine dyes is used acting as reporter and activator to induce photochemical reactions and generate cycles between on and off states of fluorophores373. The transitions between off and on states are limited to just a subset of available fluorophores that are spatially separated by the distance of the PSF of the microscope, so that the molecules can be located with a precision of 5-50 nm. The process of switching on only a subset of fluorescent molecules is repeated and recorded over many cycles, capturing new molecules turning on and others turning off in a stochastic manner. Super-resolution images can be generated by assembling all localised points of sequentially activated fluorophores that have been collected over thousands of frames. In each frame the centre of each PSF is determined by statistical fitting to a Gaussian function and the centre of the fitted curve reveals the localisation of the molecule in a range of tens of nanometres. The reconstructed super-resolution image then provides structural detail at sub-diffraction resolution.

Two years after the invention of STORM using dye pairs, the Sauer lab improved the technique by eliminating the need to rely on the proximity of two fluorophores in a specific ratio and distance372,374 by using only a single colour of fluorophores and named the technique ‘direct’ STORM (dSTORM)379. Here a single synthetic cyanine dye is combined with an appropriate buffer generating a blinking mode to obtain super- resolution images, localising the molecules of interest to a position of about 20 nm. In dSTORM, different synthetic organic fluorophores379,380 can be used and reversible 41

photoswitching of these fluorophores can be induced. Most Alexa Fluor dyes (available through Invitrogen) and Atto fluorophores (available through ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties implying that the triplet state of these fluorophores is reduced by thiols such as glutathione380. Thus these fluorophores can be switched reversibly between an on and off state if a ‘photoswitching buffer’ with thiol-containing reducing agents such as β- mercaptoethylamine (MEA), dithiothreitol (DTT), or glutathione (GSH)380 is used, greatly facilitating the application of fluorophores in this technique.

PALM makes use of genetically-encoded fluorescent proteins and different photo-physical properties of proteins are available: reversible photo-switchable fluorophores can cycle multiple times from dark to fluorescent states, such as Dronpa; photo-convertible proteins, such as mEOS2 and psCFP2, can be stimulated to convert between two spectrally separated colours. Photo-switching and photo-conversion are achieved by illumination with (near-)ultraviolet light.

Combining TIRF microscopy with the strategy of sequential activation of fluorophores as used in dSTORM and PALM allows the detection of plasma membrane molecules with very high precision. In fact, this strategy is at the forefront of innovation regarding fluorescence microscopy, and facilitates a better understanding of the spatial distribution of proteins, the partitioning of cellular membranes and the architecture of cellular structures.

1.7 Aim

While the respective contribution of lipids and proteins to the compartmentalisation of the plasma membrane into subdomains is still debated, it is now widely accepted that membrane domains do exist and play an essential role in organising cell surface proteins, endocytosis or signalling. Lectins in general and galectin-3 in particular have the potential to delineate membrane domains due to both their ability to bind to glycosylated cell surface proteins and lipids, and to form oligomers. Galectin-3 binds specifically to carbohydrates and in particular

42 polylactosamine structures, which are abundant on the cell surface. Hence, the binding of galectin-3 to cell surface proteins and its subsequent endocytosis can affect the distribution and the turnover rate of these proteins by determining where and when membrane components will be internalised. In this study I focused on galectin-3 and demonstrate its capacity to organise and shape the plasma membrane in HeLa, MEF and T cells, and mediate the internalisation of proteins in T cells. The endocytosis of galectin-3 in T lymphocytes and its localisation and involvement in T cell signalling are currently poorly understood. To illustrate the contribution of galectin-3 to the organisation of plasma membranes and endocytosis, this study intends to address the following three scientific issues:

(1) Galectin-3 is known to bind to glycosylated cell surface molecules and has been implicated in the formation of molecular lattices. The impact and mechanism of galectin-3 in plasma membrane domain formation is not yet fully understood. In this thesis, direct stochastic optical reconstruction microscopy (dSTORM) in combination with total internal reflection (TIRF) was used to investigate the molecular distribution of galectin-3 in the plasma membrane of HeLa, MEF and T cells. The clustering properties of galectin-3 and two of its ligands, CD44 and β1-integrin, were determined and their sensitivity to glycosphingolipids and N-glycosylation was probed. This data provides insight into the ability of galectin-3 to act in membrane partitioning and demonstrates the influence that N-glycans exert on galectin-3 clustering.

(2) Further, this study aims to further understand the internalisation of membrane-bound galectin-3 into intracellular compartments. To explore the endocytic pathway of galectin-3 in T lymphocytes, flow cytometry was applied to test the effect of various pharmacologic inhibitors. Thus the susceptibility of galectin-3 internalisation to inhibitors of known endocytic mechanisms as well as lipids rafts and glycosphingolipids in resting T cells was probed. Additionally, in colocalisation studies known endocytic markers were exploited to determine to which intracellular compartments galectin-3 is transferred after endocytosis.

(3) Lastly, the potential role for galectin-3 at the immunological synapse in activated T lymphocytes was addressed. T cells with internalised galectin-3 were

activated and analysed by confocal microscopy. The position of galectin-3 vesicles in relation to the immunological synapse was determined. Furthermore the localisation of T cell signalling proteins within galectin-3 positive vesicles was assessed.

Following this introductory chapter, material and methods used in this study are presented in chapter 2. Chapters 3, 4, and 5 enclose all experimental data obtained during the investigation of above-mentioned aims; a discussion of the data concludes each of the three results chapters. To conclude, chapter 6 provides a summary of the results obtained during this study, discusses the conclusion and gives an outlook regarding future research directions based on the findings presented in this thesis.

Chapter 2

Materials and Methods

2.1 Tissue culture

Cell lines

All mammalian cell lines were maintained at 37˚C in a humidified incubator under 5% CO2 atmosphere. Foetal bovine serum (FBS) obtained from Sigma-Aldrich was heat-inactivated (HI) at 56˚C for 30 minutes in order to inactivate complement protein.

Hematopoietic cell lines

Jurkat E6.1

Jurkat cells, clone E6.1 (ATCC, TIB-152), are human peripheral blood T lymphocytes established from a male acute T cell leukaemia patient. Cultures were maintained in a growth medium comprising RPMI-1640 medium (Invitrogen, 21870- 076) supplemented with 4 mM L-glutamine and 10% (v/v) HI-FBS. Maintenance density for the culture of Jurkat E6.1 cells was between 0.2 x 106 – 1.5 x 106 cells/ml. For experiments and sample preparation cells were pre-cultured to a density of 0.5 – 1.0 x 106 cells/ml in order to use cells at their log-phase.

Raji

Raji cells (ATCC, CCL-86), are human lymphoblast B lymphocytes established from a male patient with Burkitt’s lymphoma. Cultures were maintained in a growth medium comprising RPMI-1640 medium (Invitrogen, 21870-076) supplemented with 4 mM L-glutamine and 10% (v/v) HI-FBS. Maintenance density for the culture of Raji cells

was between 0.2 x 106 – 2.5 x 106 cells/ml. For experiments and sample preparation cells were pre-cultured to a density of 0.5 – 1.0 x 106 cells/ml in order to use cells at their log-phase.

Epithelial cells and fibroblasts

Epithelial cells line all the major cavities of the body, they line most organs, are found in ducts and glands, and they can specialize into sensory cells. As they form a barrier between the surrounding environment and the interior of the body, they provide protection and they can replicate often in order to replace damaged and dead cells. In certain situations epithelial cells can give rise to fibroblasts, a process termed epithelial-mesenchymal transition. Like other cells of connective tissue, fibroblasts originate from the primitive mesenchyme, and conversely, there is also a process called mesenchymal-epithelial transition, where fibroblasts give rise to epithelial cells, as seen during developmental events, and also in wound healing and tumourigenesis. Unlike epithelial cells, fibroblasts are not restricted by attachment to the basal lamina. Fibroblasts are also able to migrate over substrates, a function that epithelial cells lack.

HeLa

HeLa cells (ATCC, CCL-2) are an adherent continuous cell line. HeLa cells are human epithelial cells derived from a cervical adenocarcinoma. HeLa cultures were maintained in growth medium comprising DMEM high-glucose medium (Invitrogen, 31053-036) supplemented with 4 mM L-glutamine and 10% (v/v) HI-FBS. Maintenance density for the culture of HeLa cells was 80-90% confluence. Cells were then rinsed 3x briefly with phosphate buffered saline (PBS) and detached from the surface with a solution made of 0.05% trypsin and 0.5 mM EDTA in PBS (Invitrogen, 25300-054); then cells were resuspended in fresh DMEM growth medium and re-seeded in a new culture container with fresh DMEM growth medium or retained for experimental preparation.

MEF

Mouse embryonic fibroblasts (MEFs) are isolated from 14 day mouse gestation embryos (mixed male and female), subsequently expanded and inactivated by γ irradiation. MEF cultures were maintained in growth medium comprising DMEM high- 46 glucose medium (Invitrogen, 31053-036) supplemented with 4 mM L-glutamine and 10% (v/v) HI-FBS. Maintenance density for the culture of MEF cells was 80-90% confluence. Cells were then rinsed 3x briefly with PBS and detached from the surface with a solution made of 0.05% trypsin and 0.5 mM EDTA in PBS (Invitrogen, 25300- 054); then cells were resuspended in fresh DMEM growth medium and re-seeded in a new culture container with fresh DMEM growth medium or retained for experimental preparation.

Techniques

Thawing cells from cryopreservation

Cryovials stored in liquid nitrogen vapour-phase storage containers were retrieved and thawed in a waterbath pre-warmed to 37˚C, the vial was rinsed with 80% (w/v) ethanol, and its content immediately transferred to pre-warmed growth media, then centrifuged at 300 x g to remove the cryo-preservative dimethyl sulfoxide (DMSO), then resuspended in pre-warmed growth media and placed in an incubator. Generally cell cultures were passaged at least 3 times weekly.

Determination of cell numbers in culture cells

For cell counting, a CASY counter (Roche, Model TT) determining the cell number by pulse area analysis and the viability of cells by electric current exclusion was used. Culture cells, if adherent, were placed in suspension and disturbed with a pipette to separate cell clumps into single cells. A 50 µl aliquot was diluted in 5 ml of CASYton buffer (Roche, 05651808001) and 400 µl of cell suspension was withdrawn three times by the CASY counting device. Population statistics were determined by the incorporated software using particle gates.

Alternatively, a Neubauer glass chamber was used for cell counting. Here, cell counts from four grids of 1 mm2 size using a simple hand hold register were averaged to determine the concentration of cells, given that the volume contained within each grid is 100 nl.

2.2 Sample preparation using T cells

Cleaning coverslips

Coverslips were submerged alternatingly in beakers containing 10 M NaOH and EtOH for 20 min each in a sonicator bath, and rinsed in between in distilled H2O. Coverslips were dried under N2 stream.

Activation of T cells

Clean glass coverslips were incubated with activating antibodies: mouse anti-human CD3 antibody, clone OKT3 (eBioscience, 16-0037-85) and mouse anti-human CD28 antibody, clone CD28.2 (eBioscience, 16-0289-85) were diluted in PBS to 20 μg/ml, each. Diluted antibody was pipetted into the centre of clean glass coverslips and incubated for 1 hour in a humidified atmosphere at 37℃. Coverslips were then washed three times with PBS.

6 Jurkat E6.1 cells were counted and their concentration adjusted to 1 × 10 cells/ml in growth medium. 0.4 x 106 cells of the cell suspension were applied to fresh antibody- coated glass coverslips and incubated at 37℃ for indicated time points (2-20 minutes). Activated cells were then fixed by adding 4% (w/v) paraformaldehyde (PFA) (freshly prepared in PBS). Cells were returned to the incubator and fixed for 10 minutes at 37˚C. Fixed cells were washed three times in PBS.

Fixation

Fixation of cells occurred in 4% (w/v) paraformaldehyde (PFA), prepared fresh from a 16% (w/v) PFA stock solution (ProSciTech, C004) in PBS. Fixation was carried out for 10 minutes at 37˚C or 20 minutes at room temperature. After this time the PFA solution was removed and the sample was washed three times with PBS.

Coating of coverslips

Poly-L-lysine (PLL)-coated glass slides were prepared by incubation of cleaned glass coverslips at RT for 30 min in a 0.01% (w/v) poly-L-lysine acquired from Sigma.

Resting cells

Jurkat E6.1 cells were fixed in 4% (w/v) PFA for 10 minutes at 37℃. The fixed cells were then deposited onto bare glass coverslips coated with PLL by gentle centrifugation at 145 × g for 2 minutes in a CytoSpin centrifuge (Shandon). The coverslips were then rinsed gently in PBS, twice.

Transfection by Neon Electroporation

Jurkat E6.1 cells were transfected using the Neon electroporation device by Invitrogen (MPK5000) according to the manufacturer’s instructions. Plasmid DNA of high purity prepared using ‘Invitrogen Purelink Hipure Plasmid Filter Maxi-prep’ kits (K2100-17). Cells were counted and centrifuged at 300 x g for 5 minutes at room temperature. Washed twice in PBS, then resuspended in 50 µl Neon Buffer R and mixed with 5 µg plasmid DNA. For loading of cells into the electroporation pipette tip, 10 µl were withdrawn from the cell-buffer mixture. The electroporation tip was then inserted into the Neon pipette station containing Neon Buffer E and an electric potential was applied (1150V, 2 pulses for 30 ms each). Transfected cells were suspended in pre-warmed growth media and returned to the humidified incubator.

2.3 Galectin-3: Expression, Purification & Labelling

A plasmid for the purification of a 6xHis-tagged galectin-3 (pHis2Parallel) was kindly provided by Dr. Christian Wunder from the Johannes lab in Paris, France.

Transformation & Expression of galectin-3

The plasmid for 6xHis-tagged galectin-3 was transformed into Rosetta (DE3) pLysS competent cells as follows. Competent cells (50-100 µl) were thawed on ice. SOC medium was warmed to 37˚C. 5µL of plasmid DNA were added to each vial of competent cells directly and gently mixed by tapping the vial (instead of pipetting up and down). The vial containing the competent bacteria together with the plasmid DNA was incubated on ice for 30 mins. A heat-shock lasting exactly 30 seconds at 42˚C in a water bath was applied (do not shake or mix) and the vial was subsequently placed on 49

ice 10 min. 250 µL of pre-warmed SOC medium was added to the vial, followed by an incubation at 37˚C for 1 hour at 225 rpm in a shaking incubator. 20-200 µl of each transformation reaction of competent bacteria were plated onto pre-warmed LB Agar plates containing the appropriate antibiotic(s), and incubated inverted at 37˚C overnight (O/N). For Rosetta (DE3) pLysS cells, 50-100 µg/ml Ampicillin + 34 µg/ml Chloramphenicol were used.

For the inoculation of the starter culture O/N, conical flasks of 250 ml volume were prepared, 10 ml of 2xYT medium containing antibiotics were added, a single colony was picked from the agar plate with a clean pipette tip to inoculate the media, and the media was vortexed briefly to dislodge the bacterial pellet. Starter culture was incubated O/N at 37˚C, 200 rpm for max. 16 hours. Setting up the main culture: The complete starter culture (10 ml) was added to 500 ml 2xYT medium plus antibiotics in a 2L flask and shaken at 37˚C, 200 rpm for 4-6 hours or until an OD595 of 0.5 was reached. Induction of protein expression was achieved by addition of IPTG to a final concentration of 60 µM followed by a continuous incubation for 16 hours at 21˚C. For the harvest (from now on always working on ice) bacteria were centrifuged at 6000 rpm for 10 mins at 4˚C (centrifuge tubes were weighed to determine the weight of the pellet). At this stage the bacterial pellet were either frozen for later, or bacterial cells were lysed directly.

Column purification of galectin-3

For cell lysis, bacteria were resuspended in 1x Lysis Buffer supplemented with protease inhibitor (for 1 g of bacterial pellet, 10 ml of cold lysis buffer were used). Bacteria were sonicated on ice-water for 2 mins netto time (10 sec pulse 50 sec off, amplitude 65%, i.e. 12 mins for one sonication round, two sonication rounds were preformed) in 50 ml centrifuge tubes. The sample was centrifuged at 70000g for 30 mins at 4˚C and the supernatant was transferred to a new tube. Purification by gravity flow column: An appropriate volume of cobalt resin/beads (0.5ml beads/10ml lysate), was transferred to the column, and column and allowed to drain. Beads were washed with 5ml column volumes of sterile distilled water, and allowed to drain. 5ml column volumes of binding buffer (20 mM Tris HCl, 5 mM Imidazol) were added and allowed to 50 drain, this step was repeated a second time. Binding: Bacterial lysate was added to equilibrated column and incubated for 30-60 minutes at 4˚C with gentle agitation. After incubation lysate was allowed to flow through and 0.5ml-1ml fractions were collected. Column was washed twice with 10 ml of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 10-20 mM imidazol), flow through was collected. Elution: Bound protein was eluted using 10ml native elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250-300 mM imidazol) and 1 ml fractions were collected. Beads were washed twice with 3 bed volumes of elution buffer, flow through was collected. Protein fractions were dialysed O/N at 4˚C in PBS + EDTA, followed by a dialysation step in pure PBS. Protein concentration was measured, protein solution was aliquoted, snap frozen in LN, and stored at -80˚C. Reconstitution of cobalt resin/beads: Resin was washed with distilled water, followed by an incubation with binding buffer for 30 min, and a wash using distilled water. Beads were stored at 4˚C in 20% EtOH.

Protein concentration in collected fractions was measured using a Nanodrop spectrophotometer (Thermo Scientific). Fractions were loaded onto a SDS-PAGE gel to evaluate purity. According to concentration and purity certain fractions were pooled. Pooled fractions were dialyzed extensively O/N at 4˚C against PBS-EDTA followed by PBS only.

Fluorescent labelling of galectin-3

Fluorophore-labelling of galectin-3 was performed using Alexa-647 fluorophores (Sigma A20006, 1 mg dry powder). 1 mg powder was dissolved in 20 µl DMSO; the molecular weight of Alexa-648 being 1300 g/mol, this will produce a fluorophore solution with a concentration of 38.46 nmol/µl. To label 1.5 mg of purified galectin-3 protein 10 µl of dye-solution or 384.6 nmol of dye were used. For an optimal labelling reaction the pH of the protein solution was adjusted to pH 8.5-8.7 using 1 M sodium bicarbonate; as a rule of thumb: the necessary amount of 1 M sodium bicarbonate solution should be about 1/10 of the total volume of the protein solution, but this volume should be approached in increments with continuous confirmation of the current pH. The protein solution was mixed with dye solution and incubated for 2-4

hours at 4˚C (shaking). The stop reagent was added to interrupt the labelling reaction to remove loosely bound dyes by incubating for 3-6 hours at 4˚C.

The stop reagent (1.5 M hydroxylamine at pH 8.5) terminates the labelling reaction and removes weakly bound dyes molecules. By adding 0.1 mL of freshly prepared 1.5 M hydroxylamine, pH 8.5, and incubating the hydroxylamine-containing reaction for one hour at room temperature will stop the labelling reaction. For Galectin-3, the stop reagent was added and the reaction was incubated at 4°C for 3-4 hours or O/N. For the preparation of the reagent (MW: 69.49 mol/l or g/l) hydroxylamine hydrochloride was dissolved at 210 mg/mL in distilled water and the pH was adjusted to 8.5 with 5 M NaOH. The resulting 3 M solution was diluted with an equal volume of distilled water to get 1.5 M hydroxylamine, pH 8.5. This reagent was always prepared freshly before use.

The reaction mixture was cleaned via size exclusion columns and aliquoted. The protein aliquots were snap frozen in liquid nitrogen and stored at -80˚C.

2.4 Galectin-3 Assays

Galectin-3 binding and lattice formation

Jurkat cells were incubated in ATP-depletion media for 30-60 minutes. 6 ug/ml of recombinant human galectin-3-Alexa647 were added to Jurkat cells suspended in ATP- depletion media and incubated for 30 mins at 37˚C. Cells were washed to remove unbound Galectin-3, then fixed in 4% paraformaldehyde and spun onto PLL-coated coverglasses.

ATP depletion

ATP depletion media was composed of PBS supplemented with 0.5 mM CaCl2, 0.5 mM MgCl2, 10 mM 2-deoxy-D-glucose, and 10 mM NaN3. Energy depletion of cells was launched 30 min before galectin-3 incubation.

Sucrose and Lactose treatment

Cells were incubated for 20 min at 37˚C with 3 mM β-D-lactose (w/v) or 3 mM D(+)-sucrose (w/v) in PBS.

Galectin-3 internalisation

Jurkat cells were incubated in serum-free RPMI for 30 minutes. 6 ug/ml of recombinant human galectin-3-Alexa647 were added to Jurkat cells suspended in serum-free media. Cells were either incubated at 4˚C for 30 mins prior to endocytosis at 37˚C or immediately incubated at 37 ˚C for respective time points. At each time point cells were removed from 37˚C and incubated in cold PBS in order to stop the uptake reaction. Cells were washed with cold buffer to remove unbound Galectin-3, subsequently cells were fixed in 2% PFA and analysed by flow cytometry (FACS Verse).

Surface staining assay

Cells were washed twice in ice-cold PBS, 1 x 106 cells were resuspended in PBS or flow buffer (HBSS containing 1% BSA and 0.1% sodium azide). Cells were incubated with the respective mAb/pAb for 30 min at 4˚C, then washed twice with PBS or flow buffer, and incubated with the secondary Ab for 30 mins at 4˚C and then washed twice. Subsequently cells were fixed in 2% PFA and analysed by flow cytometry (FACS Verse).

2.5 Pharmacological Inhibitors

Different pharmacological inhibitors were used that interfere with the glycosylation of cellular molecules, disrupt plasma membrane organization, or inhibit endocytosis.

PPMP381 (DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol, Sigma P4194) is a UDP-glucose:ceramide-glucosyltransferase inhibitor. A stock solution of 25 mM of PPMP in pure ethanol was prepared. Before use, stock was diluted to 2.5 mM in PBS and then added at 1/500 dilution to cells to yield a final concentration of 5 µM. Cells were incubated in PPMP for 6 days prior to analysis.

1-deoxymannojirimycin hydrochloride117,382,383 inhibits Golgi α-mannosidase I but not ER α-mannosidase. A stock solution of 400 mM was prepared in DMSO, then diluted in fresh cell culture media supplemented with a reduced concentration of FBS to 5% to avoid interference with the inhibitor, yielding a final concentration of 0.5 mM. Cells were incubated for 48 hours.

Latrunculin A384–386 disrupts microfilament organization and inhibits actin polymerization. A stock solution of 10 mM in DMSO was prepared and used at a working concentration of 10 uM for 30 min in culture media, subsequently cells were either fixed and analysed or kept on ice for flow cytometric analysis.

Cytochalasin D384,386,387 inhibits actin polymerisation. A stock solution of 10 mM in DMSO was prepared and used at 4 uM for 30 min, added to the culture media. Subsequently cells were either fixed and analysed or kept on ice for flow cytometric analysis.

Chlorpromazine388,389 is an inhibitor of clathrin-dependent endocytosis. A stock solution of 10 mM in DMSO was prepared. A working concentration of 20 uM diluted in cell culture media was added to cells for 2 hours.

Dynasore390–392 is an inhibitor of dynamin-dependent endocytosis. A stock solution of 200 mM in DMSO was further diluted to a working concentration of 80 uM and added to cells for 30 min.

Methyl-β-cyclodextrin393 leads to cholesterol depletion. A stock of 500 mM in DMSO was used at 2.5 mM for 20 minutes.

7-ketocholesterol192,193,394 is used to decrease membrane order by disrupting the tight packing of saturated acyl chains of membrane lipids. A stock solution was prepared according to the following protocol, subsequently complexes were quantified using HPLC. A working concentration of 56 uM was used and was added to cells for 1 hour.

2.6 Preparation of sterol-complexed cyclodextrin

Preparation

Aqueous stock solution of 50 mg/ml methyl-β-cyclodextrin (mβCD) complexed to 1.5 mg/ml 7-ketocholesterol were prepared as described previously395. 7-KC (Steraloids #C6970-000) in ethanol was prepared in a volume of 1 ml at a concentration of 15 mg/ml. To prepare 5% mβCD stock, 500 mg MβCD (Sigma #C4555) were dissolved in o 10ml milliQ-H20 and heated to 80 C while stirring. Gradually cholesterol or 7-KC was added to the beaker in small aliquots. No more than 2.5% of 15 mg/ml 7-KC of total MβCD-volume should be added to the beaker at a time, then mixed thoroughly for 5- 10 minutes. The mixture was then left on the hotplate at 80oC for 1 hour or until clear (do not cover). The prepared 7-KC/ MβCD complexes were transferred into a 15 ml falcon tube each and 7KC/ MβCD complexes were frozen at -20oC until HPLC analysis.

Quantification

MβCD-7KC complexes were thawed at room temperature. 15 µl of mβCD-7KC complex were mixed with 1 ml of PBS (in triplicates). 10 µl BHT (200 µM) and 100 µl EDTA (200 Mm) were added to Kimax tubes. Butylated hydroxytoluene (BHT) (2,6-Di- tert-butyl-4-methylphenol) in ethanol (Sigma Aldrich #B1378) and ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) in milliQ water (Sigma Aldrich #ED2SS) were prepared. 500 µl of diluted complex and 390 µl PBS were added to the Kimax tube to obtain a total volume of 1 ml. Then 2.5ml methanol and 5ml Hexane were added to the Kimax tube, and tube was vortexed for 15 seconds, followed by a centrifugation at 2500 rpm for 15 min at 10oC. 4 ml of hexane (top layer) were taken off and added to a Kimble tube, then dried down in the speedivac for 45-60 minutes, followed by a reconstitution in 200 µl of mobile phase (MP210) and transferred to an HPLC vial for analysis. To determine peak retention time and to check the system a standard was used. Standard preparation: 10 µl of 15 mg/ml 7KC were added to Kimble tube, evaporated in the speedivac and reconstituted in 180 µl of mobile phase MP210. Transfer to an HPLC vial for analysis. The reconstituted samples and standard can be frozen at -20oC for later HPLC analysis.

Treatment

Cells were incubated with 56 µM mβCD-7KC complexes in RPMI for 10 - 20 min at 37˚C.

2.7 Immunofluorescence

Using a CytoSpin centrifuge T cells were spun onto PLL-coated glass slides, fixed with 4% PFA, washed with PBS, and permeabilized with 100 µg/ml lysolecithin in PBS. When external membrane-bound proteins were examined, cells were not permeabilized. Cells were blocked with 5% BSA, and immunostaining was performed using primary antibodies according to the manufacturere’s instuctions (EEA1 #3288, Rab11 #5589, , CD82 NBP2-21792, CD63 NBP100-77913, LAMP2 NBP-1-71692 were from Novus Biologicals, golgin-97 A-21270 from Invitrogen, β1-integrin CP26 from Calbiochem, CD44 ab119863 and pCD3ζ ab68235 were from Abcam) followed by incubation with the respective secondary antibody (Dylight 488, 550; Alexa Fluor 647). The pCD3ζ antibody from Abcam detects the phosphorylated amino acid tyrosine (Tyr,Y) located at position 142.

2.8 Quantification of galectin-3 uptake by Flow cytometry

Internalization of galectin-3 labelled with Alexa Fluor 647 by Jurkat E6.1 cells after treatment with inhibitors and control cells was measured by flow cytometry. Cells pre-treated with inhibitors were incubated at 37˚C for 60 minutes in uptake medium (serum-free RPMI). After incubation, cells were washed with chilled PBS and either kept at 4˚C or cells were fixed in 2% PFA, washed and analysed using a Becton Dickinson (BD) FACSVerse flow cytometer. In each sample 20,000 cells were analysed. The mean fluorescence intensity of internalized galectin-3 in control and inhibitor- treated cells was calculated and plotted.

2.9 Confocal Fluorescence Microscopy

Images were acquired on a TCS SP5 confocal laser scanning (LS) microscope (Leica Microsystems) with a 100x oil-immersion objective with a numerical aperture of 1.4. An argon laser was used to excite Alexa Fluor 488, a DPSS laser of 561 nm was used to excite Alexa Fluor 555, and a helium-neon laser of 633 nm was used to excite Alexa Fluor 647. For fixed or live samples imaging was performed at RT or 37˚C, respectively.

2.10 Pearson’s Coefficient

The Pearson’s correlation coefficient (PCC) was used to quantify the degree of colocalisation between fluorophores. The PCC has a range of +1 (indicating perfect positive correlation) and -1 (indicating perfect negative correlation), with 0 denoting an absence of relationship. Colocalisation of confocal images was quantified using the ImageJ Colocalisation JACoP plugin2 in order to obtain the Pearson’s coefficient.

2.11 dSTORM Single Molecule Microscopy

Sample preparation

Cells pre-treated with above described conditions and incubated or not with Alexa Fluor 647 or 555-conjugated galectin-3 were fixed in 4% PFA for 10 min at 37˚C. If required, an immunostaining was performed as described above. Subsequently cells were placed in PBS and kept at 4˚C until imaging.

Imaging conditions and acquisition

Reversible photoswitching of Alexa Fluor 647 fluorophores in dSTORM experiments was performed in a redox imaging buffer composed of 25 mM Hepes and 5% glycerol in PBS, pH 8, supplemented with 75 mM cysteamine and an oxygen scavenger system made of 0.05 mg/ml glucose oxidase, 0.025 mg/ml horseradish

peroxidase, , and 25 mM glucose396. For imaging, 4 mW of a633 nm laser was used for excitation and 30 to 300 μW of a 488 nm laser for conversion of molecules from the dark state.

dSTORM images were acquired on a total internal reflection fluorescence (TIRF) microscope (ELYRA; Zeiss) with a 100x oil-immersion objective with a numerical aperture of 1.46. During image acquisition, 15000-20000 frames were acquired per cell using a cooled, electron-multiplying charge-coupled device (EMCCD) camera (iXon DU- 897D, Andor) with an exposure time of 40 ms.

Data processing

For data processing, raw fluorescence intensity images were analysed using the software Zen 2010D by Zeiss MicroImaging. Correction for thermal drift occurring during image acquisition was done using the model-based drift correction implemented into the ZEN software. Multiple localisations from the same molecule can occur and dSTORM probes can blink on multiple timescales397. To exclude re- blinking from the same molecule grouping (e.g. summing and counting as one) was applied. If consecutive blinking events were detected in the same 100 nm diameter area, these events were grouped and counted as one molecule. Further, an off-gap was defined as the time period in which recurring non-consecutive fluorescent events in the same 100 nm area were counted as one. Correction parameters were kept constant throughout experiments. For x-y coordinates of molecules recorded in dSTORM images, an algorithm identifying the centre of the point spread function (PSF) of each molecule was used398. To calculate the centre of each PSF a Gaussian mask was used. Localisation precision was determined using parameters such as the number of collected photons (N), the width of the PSF (s), and the background variance (b).

Point spread function (PSF) fitting and localization were performed in Zen 2010D. Single molecules were identified as areas with an intensity-to-noise ratio greater than 6.. From each dSTORM image, 4 µm x 4 µm or 3 µm x 3 µm non-overlapping regions were selected for analysis. Events with localization precision worse than 50 nm were

58 discarded. For each analysed region, the molecules distribution was analyzed using the Ripley’s K-function was calculated as:

Kr A ∑ ∑ where δ Equation 1 1 if δ < r, else 0 Where n is the number of points contained in an analysed region of area A (e.g. 4

µm x 4 µm). dij is the distance between two points i and j and r is the analysed spatial scale. Localizations at the edge of the analysed region were weighted to negate edge effects. The K-function is a measure of the number of points encircled within concentric circles of radius r centred on each point and scales with circle area. It is therefore transformed into the L-function to obtain linear scaling with radius r via Equation 2:

() Equation 2 () = Random distributions have L(r)=r at all r (i.e. L(r)-r = 0). We therefore plot L(r)-r versus r. Positive values of L(r)-r at a given r indicate clustering at that spatial scale. 99% confidence intervals (CI) were generated by simulating 100 spatially random distributions.

To generate cluster heat maps, L(r) values at a spatial scale of 50 nm, L(50), were computed for each point individually (Equation 3).

∑ ( ) where Equation 3 (50) = δ = 1 δ < 50, 0

Values of L(50) were interpolated to form a cluster heat map (MATLAB, The Mathworks). By applying a threshold of L(50)=78 to this map, a binary cluster map was created from which cluster statistics (number, size, shape, molecules per cluster) were extracted. This method has previously been demonstrated to extract clustering parameters from PALM and dSTORM data at the plasma membrane396,399,400. Statistical significance of the means of two data sets was assessed with Student t-tests (Prism, GraphPad Software).

Crucial to PALM and dSTORM data analysis is the correct identification of single molecules and avoidance of over-counting molecules whose fluorescence has ceased and re-appeared. To correct for this effect we used an accepted method of analysis first published by Annibale et al.397, and later modified by Rossy et al.396 to include two different time-scales of blinking. Here Gal3-Alexa647 data were fitted to the following equation (4):

( ) ( ) Equation 4 (t) = N 1 + +

Here N(td) is the number of molecular counts at different off gaps (td) where off- gap is the maximum time period a molecule is dark before being identified as a different molecule when its fluorescence resumes; N is the number of fluorescent molecules; nblink1 and nblink2 are the average number of counts a molecule converts to a dark state; toff1 and toff2 are the average lifetimes of the fluorophore.

Alexa647-Gal3 exhibited 0.25 blinks for 0.26 frames (7.8 ms) and 0.77 blinks for 6.2 frames (186 ms) in HeLa control cells, and 0.16 blinks for 0.25 frames (7.5 ms) and 0.44 blinks for 7.7 frames (231 ms) in PPMP treated cells. Alexa647-Gal3 molecules adhered to coverslips displayed 0.19 blinks per molecule for 0.5 frames (15 ms) and 0.2 blinks for 22 frames (660 ms). Based on this analysis, an off-gap threshold of 40 frames was applied.

2.12 Statistical Analyses

Unless stated otherwise, data are presented as mean ± standard error of the mean (SEM) from at least three replicates of a single experiment. Statistical comparisons between two populations were made using Student’s t-test, and multiple comparisons were made by one-way analysis of variance (ANOVA) and Bonferroni post-testing. Statistical analyses were performed using Prism (Graphpad Software).

Chapter 3

Galectin-3 at the cell surface

3.1 Introduction

Interactions of galectins with their glycan ligands are rather complex. Most members of the galectin family bind simple β-galactosides, however, their affinity for simple disaccharides is weak and affinity increases with the complexity of glycans. Factors such as the oligomeric state of the galectins and mode of presentation of N- glycans also contribute to high affinity binding. The preferred ligand for galectins is N- actyllactosamine13,14 and although plenty ligands bearing complex N-glycan structures are available at the cell surface, galectins differ in their specificities for modified oligosaccharides, and structural differences in their CRDs lead to differential ligand binding among galectins8,70,401. For galectin-3 a multitude of cell surface ligands has been identified so far, including common receptors found on many cell types as well as more specific receptors only encountered on T cells33–37. Among the diverse galectin-3 ligands, different integrins have been identified and the binding of galectin-3 to members of the integrin family has been reported and investigated in endothelial and carcinoma cells. In those studies, galectin-3 has been found to regulate integrin- dependent cell adhesion, spreading and migration as well as the clustering of integrin402,403. Galectin-3 binding has been shown to regulate the diffusion rates of integrins and other cell surface receptors75,251, however, the precise functions of the interactions of most of these glycoproteins with galectin-3 are yet to be elucidated.

The association of galectins with specific carbohydrates via their CRDs and their ability to from dimers and pentamers, enabling cross-linking of glycoproteins and - lipids at the plasma membrane, promotes the formation of molecular lattices403. Addition of fluorescently labelled galectin-3 to neutrophils and endothelial cells was 61

shown to form stable oligomers at the cell surface74. Lattice formation has been implicated in the lateral compartmentalization of the plasma membrane and observations in T cells implicated galectin-1 in the induction of CD45 clustering404. The molecular lattice is not only expected to impact on the spatial allocation of plasma membrane molecules but also on turnover times of cell surface receptors as well as on signalling in general and signalling thresholds more specifically. A study using diffuse large B-cell lymphoma (DLBCL) cells demonstrated that cell surface galectin-3 interacted with CD45 and regulated its phosphatase activity, leading to cell survival in the presence of galectin-3 but apoptosis in the absence of galectin-3405. Demetriou et al showed in T cells that the galectin-glycoprotein lattice negatively regulated T cell activation by restricting TCR recruitment to the site of antigen presentation and that the lattice was fortified by N-glycosylation71. This study further indicates that the generation of these N-glycan structures that are necessary for galectin binding is mediated via β1,6-N-acetylglucosaminyl transferase V (Mgat 5). The absence of Mgat 5 facilitated the clustering of TCRs at activation sites and lowered the T cell activation threshold leading to hypersensitive T cells71. Similarly, Chen et al demonstrated that the binding of galectin to N-glycans at the plasma membrane influenced the lateral compartmentalisation of T cell signalling proteins and negatively regulated T cell signalling131. Furthermore, the authors suggest an antagonistic control exerted by the galectin lattice opposing the actin cytoskeleton in T cell regulation131.

GSLs belong to the most diverse and abundant cell surface glycolipids in the outer leaflet of plasma membranes of animals. Through interactions with lectins, GSLs are involved in cellular recognition processes and modulate signal transduction by influencing the distribution of cell surface receptors181,182. The importance of GSLs in plasma membrane compartmentalisation, domain formation and immune cell regulation has been demonstrated in several studies. For instance, galectins-4 as well as -9 have been shown to bind GSLs185,186. Galectin-9 is interacting with Forssman GSLs that are important for apical trafficking in MDCK cells186 and the absence of galectin-9 leads to striking differences in the polarity of these cells. The interaction of galectin-4 with sulfoglycosphingolipids plays a functional role in the clustering of lipid rafts and is also crucial for apical delivery185. Galectins interact with gangliosides, which have

62 shown to be engaged in galectin-dependent cell-cell adhesion, to control cell growth and be involved in signalling processes174,183,184. In neuroblastoma cells, endogenous galectin-1 was demonstrated to be a major binding partner of the lipid raft marker ganglioside GM1. This interaction was carbohydrate-dependent and was relayed by sialidase-dependent alterations172 present on N-glycans of the complex and hybrid types406. In these cells galectin-1 negatively regulates cell growth183. In mammary carcinoma cells galectin-3 interacts with N-cadherin at cell junctions, colocalises with GM1 and forms a molecular lattice that affects lateral diffusion174.

Crystallographic studies provided insights into the interactions that occur between galectins and GSLs. Galectin-3 bound to oligosaccharides of GSLs revealed that the open nature of the galectin-3 CRD binding site can accommodate extensions in GSLs of the lacto- and neolacto series171. Furthermore, colocalisation of galectin-4 with the GSLs SB1a and GM1 was demonstrated at the cell surface of human colon adenocarcinoma cells176. In Chinese hamster ovary (CHO) cells, galectin-8 associated with GSLs175. Using super-resolution microscopy and reconstitution studies, we recently showed a requirement for GSLs for galectin-3-induced clustering and membrane bending for cellular uptake of CLIC cargo187. In summary, these observations indicate that glycosylated lipids are important ligands for carbohydrate binding proteins such as galectins at the surface of mammalian cells. Hence, in this study, the role of glycosylated lipids in the distribution of galectin-3 was investigated. Posttranslational modifications mediate the partitioning of plasma membrane molecules, and this study examined the impact of glycosylated sphingolipids on the clustering of galectin-3.

This chapter is focused on the involvement of N-glycan attachments on glycoproteins in the galectin-3-induced formation of plasma membrane. High mannose-type oligosaccharides comprise a class of N-linked oligosaccharides and are also known intermediates in the biosynthesis of hybrid- and complex-type oligosaccharides. 1-deoxymannojirimycin (dMNJ) and kifunensine are two distinct inhibitors of the Golgi α-mannosidase I preventing the generation of hybrid- and complex-type oligosaccharides114,115. The effect of dMNJ on the galectin-3 dependent

distribution of the glycoprotein CD44 on the plasma membrane in mouse embryonic fibroblasts (MEF) was probed in this study. To further investigate the expression of specific N-glycan structures necessary for galectin binding, the Golgi resident enzyme Mgat 5 was examined. MEF cells from a mouse knock-out model lacking the Mgat 5 enzyme were used to probe for galectin-3 binding. These cells are (1) unable to generate tetra-antennary N-glycans and (2) existing N-glycan branches are not elongated with poly N-acetyllactosamine – two important structures in the creation of a preferred ligand of galectin-3.

Here TIRF microscopy in conjunction with dSTORM imaging was applied to study the distribution of galectin-3 and galectin-3 binding proteins at the plasma membrane of T cells, HeLa, and MEF cells. The single molecule data was analysed with a quantitative cluster analysis. The impact on clustering of certain oligosaccharide chains, i.e. intermediates or specific branches that are generated during intracellular N-glycosylation of proteins and lipids, was also assessed.

3.2 Glycosphingolipid-dependent galectin-3 clustering

GSLs can be found in membranes of all mammalian cell types, and throughout nature in animals, bacteria, plants and fungi. They have been implied to function in many biological processes181,182,407–410 and binding to GSLs has been shown for galectins-1, -3, -4, -8 and -911,171,172,174–176,183–187. In order to address the contribution of GSLs in the cell surface organization of the carbohydrate-binding protein galectin-3 at the plasma membrane, I determined its distribution in mammalian cells depleted of GSLs. Depletion of GSLs was achieved in a 6 day treatment of cells using RPMI cell culture media supplemented with 5 µM of the glyosylceramide synthase inhibitor D,L- threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). Subsequently control and GSL-depleted HeLa cells were placed in ATP depletion buffer

64 supplemented with PPMP and incubated with Alexa Fluor-647 labelled galectin-3 to evaluate its surface distribution.

For data acquisition at the single molecule level dSTORM (direct stochastic optical reconstruction microscopy) was used. Reversible photoswitching of Alexa Fluor 647 fluorophores in fixed cells was induced using a specific redox imaging buffer380,411– 413. For excitation of fluorophores a 633 nm laser was used and for conversion of fluorophores from the dark state a 488 nm laser light was applied. Cells were imaged in TIRF (total internal reflection fluorescence) mode providing a high signal-to-noise ratio and limiting the detection of molecules to a depth of 100 – 150 nm of the glass surface. TIRF is the best suited technique for the detection of molecules located in the plasma membrane. For dSTORM image acquisition 15,000 – 20,000 frames per cell were recorded in order to capture blinking emitted by single molecules.

For x-y coordinates of molecules recorded in dSTORM images, an algorithm identifying the centre of the point spread function (PSF) of each molecule was used398. Localisation precision was calculated using parameters extracted from image frames, such as the number of collected photons, the width of the PSF, and the background variance. As multiple localisations from the same molecule can occur, stringent single molecule detection parameters, such as grouping, were applied (see following paragraph and section in Chapter 2).

In dSTORM multiple localisations from the same molecule can occur, e.g. occasionally dSTORM probes can be switched on again and emit single molecule blinking over a second cycle397. To avoid double-counting of the same molecule, data correction parameters were determined and applied during analysis by grouping of molecules (e.g. summing and counting as one) (Figure 3.2). If blinking events in the same area within 40 frames (off gap) were detected, these events were grouped and counted as one molecule. The off gap was defined as the time period in which recurring fluorescent events in the same area were counted as one. Correction parameters were kept constant throughout experiments.

To quantify the distribution, quantitative cluster analysis based on Ripley K- function was applied and maps of molecular clustering calculated (Figure 3.1). The Ripley K-function is a measure of particle distribution relative to a spatially random distribution. In brief, the Ripley K-function draws a circle around every molecule, counts the number of molecules as a function of the radius, subtracts the number of molecules corresponding to a random distribution at the same density and then assigns a value, L(r), to each point. These values were interpolated into a map and pseudo-colouring was applied as shown in Figure 3.1.B. To plot Ripley K-function curve against the radius, r, the function is linearized to L(r)-r. Experimental data was compared to a simulation of spatially random distributions that provided 99% confidence intervals. To compare two different experimental conditions, a threshold was applied to the cluster maps of L(r) > x. This generated black and white binary cluster maps in which white areas are membrane regions with clustered molecules (Figure 3.1 C). Because the binary cluster map is derived from single molecule images, cluster statistics such as cluster size, percentage of molecules in clusters and clusters per area can be derived399,411,414 (Figure 3.D-I). (For further information on data analysis please refer to Chapter 2.)

A differential organization of galectin-3 in control and GSL-depleted cells was detected and revealed that galectin-3 clustering at the plasma membrane was dependent on GSLs. The radius of galectin-3 clusters became smaller (66.42 ± 1.3 nm in PPMP treated cells versus 74.9 ± 1.99 nm in control cells) and clusters as well as molecules per area were more abundant in cells depleted of GSLs (7.8 ± 0.18 in PPMP treated cells versus 6.7 ± 0.24 clusters per µm2 in control cells and 264.4 ± 15.3 in PPMP treated cells versus 191.2 ± 10.72 molecules per µm2 in control cells). Thus in control cells the presence of GSLs leads to larger clusters but clusters and molecules are less abundant compared to PPMP-treated cells. These findings suggest that less binding of galectin-3 occurs in control cells. Furthermore, in the absence of GSLs galectin-3 was more randomly distributed than in to control cells. In summary these data show that GSLs contribute to galectin-3 cluster size as well as the level of clustering indicating that the cell surface distribution of galectin-3 is dependent on the presence of GSLs.

Figure 3.1. dSTORM imaging and cluster analysis of Gal3-Alexa647 in control and PPMP- treated HeLa cells. (A) dSTORM images of Gal3-Alexa647 in HeLa cells. Cells were pre-treated or not with PPMP, then energy-depleted to prevent endocytosis and incubated with 6 ug/ml of Alexa Fluor-647 labelled galectin-3 for 30 minutes, washed, fixed and imaged using dSTORM. (B) Color-coded cluster maps retrieved from local point pattern analysis of regions (red squares in A). Colour indicates level of clustering, L(r), with low to high clustering coloured blue to red. (C) Maps of Alexa Fluor 647 labelled galectin-3 clusters and molecules inside clusters after applying a threshold to colour-coded maps (as shown in B). (D-I) Quantitative statistical analysis of galectin-3 clustering in MEF: (D) total number of molecules per µm2, (E) number of clusters per µm2, (F) radii of clusters in nm, (G) size of clusters in µm2, (H) L(r)-r indicates the level of clustering, (I) maxima of L(r)-r. Means ±S.E.M. from 10-20 cells, n=3 experiments. Statistical analysis of data: Student’s t test, **** p<0.0001, *** p<0.001, ** p<0.01. Scale bar: 10 µm

Cell Clusters Molecule Cluster Condition Max L(r)-r type per area density radius

HeLa galectin-3 clustering , ± PPMP ↓↓↓↓ ↑↑↑ ↑↑↑ ↓↓ GSL-dependent Table 3.1. Summary of galectin-3 in GSL-depleted HeLa cells. Absence of GSLS modified reduced clustering of galectin-3 and led to smaller clusters. However, the molecule density increased as well as clusters per area.

Figure 3.2. Analysis for the effects of photoblinking. To correct for repeated excitation of the same fluorophore the number of localized Galectin3-Alexa647 molecules in Hela control (A) and PPMP treated (B) cells as well as Alexa647-Gal3 molecules adhered to coverslips (C) was plotted versus the off gap. Experimental counts are shown as dots and data fit is indicated by a solid line. Alexa647-Gal3 exhibited 0.25 blinks for 0.26 frames (7.8 ms) and 0.77 blinks for 6.2 frames (186 ms) in HeLa control cells, and 0.16 blinks for 0.25 frames (7.5 ms) and 0.44 blinks for 7.7 frames (231 ms) in PPMP treated cells. Alexa647 fluorophores in samples of Gal3 adhered to coverslips displayed 0.19 blinks per molecule for 0.5 frames (15 ms) and 0.2 blinks for 22 frames (660 ms). Based on this analysis the off gap of 40 frames conservatively excluded over-counting of molecules due to possible photoblinking at time points beyond 1200 ms; thus observed clustering has not been caused by repeated excitation of the same molecule but by excitation of new molecules. To avoid data bias and prove a random distribution of Gal3, the clustering behaviour of Alexa647-Gal3 was analysed on its own when adhered to a clean coverslip and the level of clustering was analysed and indicated by (D) Ripley’s K-function curve (E) and the Max of L(r)-r showing a very low L(r)-r peak indicative that observed clustering in data sets is indeed significant, and (F) comparison of Ripley’s K-function curves of HeLa control and PPMP treated cells, and Alexa Fluor-647-labelled galectin-3 adhered to a coverslip.

3.3 N-glycan-dependent clustering of galectin-3

To evaluate the organization of galectin-3 at the plasma membrane in the presence or absence of highly branched N-glycans, MEF Mgat5-/- knockout cells were used. These cells lack expression of the enzyme Mgat5, which is responsible for the generation of highly branched N-glycans, particularly tetra-antennary N-glycans that are elongated with poly-N-acetyllactosamine, creating glycan structures with high- affinities for galectin-311.

The contribution of highly-branched N-glycans to plasma membrane compartmentalization was assessed in energy-depleted Mgat5-positive and Mgat5- negative MEF cells incubated with Alexa Fluor-647 conjugated galectin-3 for 30 min at 37˚C. dSTORM experiments and cluster analysis was performed as described above.

Quantification of the molecular distribution of galectin-3 at the plasma membrane revealed a differential organization of galectin-3 in comparison to control cells (Figure 3.3, Table 3.2). The radius of galectin-3 clusters was smaller (from 84.56 ± 2.69 nm to 72.84 ± 1.62 nm) and also the level of clustering decreased (from 125.1 ± 12.43 to 78.16 ± 3.86) in Mgat5 knockout compared to Mgat5-expressing cells. On the other hand clusters and molecules per area were more abundant in cells unable to create tetra-antennary N-glycans (6.83 ± 0.17 in MEF Mgat5-/- cells versus 5.68 ± 0.27 clusters per µm2 in control cells and 200.3 ± 10.4 in Mgat5-/- cells versus 144.9 ± 8.59 molecules per µm2 in Mgat5+/+ cells). To summarize, the distribution of molecules and clusters in Mgat5-/- cells was more abundant per um2 indicating that the organization of cell surface-bound galectin-3 was less clustered and more random due the lack of these N-glycan structures. At the same time the absence of these N-glycans leads to a lower level of clustering and smaller clusters in Mgat5-deficient cells indicating that less high-affinity binding sites impact on the cell surface clustering of galectin-3.

Figure 3.3. Clustering of galectin-3 in the presence or absence of Mgat5 (A) dSTORM images of energy-depleted MEF Magt5+/+ and MEF Mgat5-/- cells incubated with Alexa Fluor-647 labelled galectin-3 (B) cluster map of the red square outlined in A. The degree of clustering is colour-coded and indicates areas with low (blue) to high (red) clustering. (C) Thresholded cluster-maps in black and white showing the outline of clusters. (D-I) Quantitative statistical analysis of galectin-3 clustering in MEF. (D) total number of molecules per µm2, (E) number of clusters per µm2, (F) radii of clusters in nm, (G) size of clusters in µm2, (H) L(r)-r indicates the level of clustering, (I) maxima of L(r)-r. Means ±S.E.M. from 10-20 cells, n=3. Statistical analysis of data: Student’s t test, **** p<0.0001, *** p<0.001. Scale bar: 10 µm

Cell Clusters Molecule Cluster Condition Max L(r)-r type per area density radius

MEF galectin-3 clustering, ± Mgat5 ↓↓↓↓ ↑↑↑ ↑↑↑ ↓↓↓ Mgat 5-dependent Table 3.2. Summary of Mgat5-dependent clustering of galectin-3 at the plasma membrane of MEF. Absence of Mgat5 modified N-glycans induced less clustering of galectin-3 as well as smaller clusters. However, molecule density increased as well as clusters per area.

3.4 Galectin-3 nanodomains in T cells

To obtain a more detailed picture of the clustering behaviour of galectin-3 at the surface of mammalian cells, the distribution of galectin-3 molecules on the plasma membrane of resting T cells was examined.

Although expression of galectin-3 in T lymphocytes has been shown65, Jurkat E6.1 cells - a cell line originally established from a T cell leukaemia patient by Schneider et al.415 - proved to be galectin-3 deficient416. Hence Jurkat cells provided a suitable cell line to study the impact of externally added galectin-3 as no endogenous galectin-3 lattice exists.

To prevent endocytotic processes and maintain a molecular lattice constituted of galectin-3 molecules, Jurkat E6.1 cells were energy-depleted using an adenosine triphosphate (ATP)-depletion buffer that was supplemented with nutrients necessary for the maintenance of cell viability. Subsequently Jurkat cells were incubated in Alexa Fluor 647-conjugated galectin-3 at a concentration of 6 µg/ml in the presence of ATP- depletion buffer for 30 min at 37˚C. The galectin-3 concentration used here did not exceed physiological concentrations417–419 and was in agreement with previous studies403,420–423. Subsequently cells were washed and fixed in 4% PFA.

In Jurkat E6.1 cells, free of ATP, galectin-3 localised to and remained at the plasma membrane (Figure 3.4). The accumulation of galectin-3 at the plasma membrane of energy-depleted cells indicates that binding of galectin-3 to the plasma membrane is an energy-independent process. Despite the plethora of available ligands for galectin-3 at the surface of T cells34–37,41,42 galectin-3 was not evenly distributed at 71

the plasma membrane but accumulated in distinct domains forming patchy islands of 52.28 ± 2.41 nm in radius, which we termed nanodomains.

Figure 3.4. Cluster analysis of Gal3-Alexa647 nanodomains in Jurkat E6.1 T cells. dSTORM images of Gal3-Alexa647 in Jurkat cells. Cells were ATP-depleted, and incubated with 6 ug/ml of Gal3-Alexa647 for 30 min, then fixed and imaged under TIRF illumination. Single molecule dSTORM images were generated with the software Zen (Zeiss) as described in Chapter 2. (A) Reconstructed dSTORM image of Gal3-Alexa647. (B) Color-coded cluster maps retrieved from local point pattern analysis of regions (red squares in A). Colour indicates level of clustering, L(r), with low to high clustering coloured blue to red. (C) Thresholded cluster-map in black and white showing the outline of clusters. (D) Radii of Gal3-Alexa647 clusters in nm. Means ±S.E.M. from 10-20 cells, n=3 independent experiments. Scale bar: 10 µm

3.5 Galectin-3 dependent surface expression of β1- integrin

To evaluate the distribution of β1-integrin cell surface receptors in the presence or absence of galectin-3, resting T cells were incubated or not with Alexa Fluor-555 conjugated galectin-3 for 30 min at 37˚C. Here labelled galectin-3 was used as a control to analyse only galectin-3 positive cells. Subsequent to washing and fixation in 4% PFA, immunocytochemistry was performed by staining against a primary β1-integrin

72 antibody followed by incubation using an Alexa Fluor-647 labelled secondary antibody. Cells were imaged as described above.

β1-integrin molecules at the plasma membrane of control cells (Figure 3.5, Table 3.3) displayed an increased molecular density compared to galectin-3 treated cells (39.26 ± 4.4 versus 20.52 ± 2.4 molecules per µm2 in galectin-3 treated cells). Cluster analysis showed that more clusters per area were formed in control cells than in galectin-3-treated cells (6.5 ± 0.64 to 4.23 ± 0.42 clusters per µm2). Cluster size, however, was not influenced by galectin-3 treatment. Plots of Ripley’s Κ-functions showed that β1-integrin in non-treated cells were clustered to a similar level than in galectin-3 treated cells. In summary the data suggest a galectin-3 mediated downregulation of β1-integrin at the cell surface of T lymphocytes.

Figure 3.5. Integrin-β1 clusters on the plasma membrane of T cells. (A) dSTORM images of resting Jurkat E6.1 cells incubated with or without galectin-3 and immunostained with anti- integrin-β1 antibodies. (B) Cluster map of the red square outlined in A. The degree of clustering is colour-coded and indicates areas with low (blue) to high (red) clustering. (C) Thresholded cluster-map in black white white area outlining clusters. (D-I) Quantitative statistical analysis of integrin-β1 clustering in resting T cells: (D) total number of molecules per µm2, (E) number of clusters per µm2, (F) radii of clusters in nm, (G) size of clusters in µm2, (H) L(r)-r indicates the level of clustering, (I) maxima of L(r)-r. In D-I, each symbol represents one image regions from 10-20 cells from 3 independent experiments. Horizontal and vertical bars represent mean and standard deviation from 10-20 cells, n=3 experiments. Statistical analysis of data: Student’s t test, **** p<0.0001, *** p<0.001, ** p<0.01, ns = not significant. Scale bar: 10 µm

Cell Clusters Molecule Cluster Condition Max L(r)-r type per area density radius

T β1-integrin clustering, ± gal-3 ≈ ↓↓ ↓↓ ≈ cells galectin-3 dependent Table 3.3. Summary of galectin-3-dependent β1-integrin clusters at the plasma membrane of T cells. The maxima of the level of clustering (Max L(r)-r) or the cluster size was not changed but the molecular density of β1-integrin and clusters per area decreased in the presence of galectin-3.

β1-integrin is one of the most predominant galectin-3 interaction partner as shown by several studies33–37 and other galectins have been reported to bind integrins424–426. Interactions of galectin-3 with N-glycans on β1-integrin at the cell surface of T cells lead to differential clustering, thereby regulating domains at the plasma membrane. Galectin-3 may counteract the dimerization of integrins and the aggregation with other surface molecules. Also, galectin-3 may cause a downregulation of β1-integrin. This is in accordance with previous reports of galectin-3 being involved in the formation of molecular lattices at the cell surface leading to the compartmentalization of the plasma membrane, and indicates that galectin-3 influences the distribution of integrin-β1 and residency time at the surface of T cells.

3.6 Galectin-3 dependent clustering of CD44

CD44 is a glycoprotein containing 5 N- and at least 7 O-glycosylation sites at its extracellular domain427,428 and has been shown to be a ligand for galectin-3 at the cell surface187. To examine the effects of N-linked glycosylation regarding galectin-3 induced clustering of CD44, the inhibitor 1-deoxymannojirimycin (dMNJ) which interferes with the processing of N-linked glycans was applied to MEF cells. Application of dMNJ inhibits the synthesis of hybrid- and complex-type oligosaccharides114,115 and leads to an accumulation of high-mannose-type oligosaccharides. Hence, the use of this inhibitor may reveal whether the availability of certain N-glycans impacts on the allocation of cell surface receptors in the presence of galectin-3.

MEF were incubated in RPMI cell culture media containing a reduced concentration of FBS of 5% supplemented with 0.5 mM dMNJ for 48 hours at 37˚C. 75

Then cells were incubated with galectin-3 for 30 min at 37˚C in the presence of the inhibitor. Subsequently cells were washed, fixed in 4% PFA, stained with Alexa-647- labelled anti-CD44 antibodies and dSTORM images acquired (Fig 3.6).

Figure 3.6. Effects of altered N-glycosylation on the clustering of CD44 in the presence of galectin-3. (A) dSTORM images Alexa-647-labelled anti-CD44 antibodies in control and dMNJ- treated MEF cells. (B) Cluster map of the red squares outlined in A. The degree of clustering is colour-coded and indicates areas with low (blue) to high (red) clustering. (C) Thresholded cluster-map in black and white showing the outline of clusters. (D-I) Quantitative statistical analysis of galectin-3 dependent CD44 clustering in MEF treated or not with dMNJ: (D) total number of molecules per µm2, (E) number of clusters per µm2, (F) radii of clusters in nm, (G) size of clusters in µm2, (H) L(r)-r indicates the level of clustering, (I) maxima of L(r)-r. Means ±S.E.M. from 10-20 cells, n=3 experiments. Statistical analysis of data: Student’s t test, **** p<0.0001, ** p<0.01. Scale bar: 10 µm 76

Cell Clusters Molecule Cluster Condition Max L(r)-r type per area density radius

MEF CD44 clustering in the presence of galectin-3, ± dMNJ ↓↓↓↓ ↓↓↓↓ ↓↓↓↓ ↓↓ dMNJ dependent Table 3.4. Summary of dMNJ-dependent galectin-3-induced CD44 clusters at the plasma membrane of MEF. DMNJ treatment abrogated the generation of hybrid- and complex-type oligosaccharides and caused a reduction of the maximum degree of CD44 clustering and CD44 cluster size, as well as the molecular density and clusters per area.

Modification of N-glycosylation using dMNJ reduced the average density of CD44 molecules in clusters to 209.9 ± 42.99 molecules per um2 (from 501.8 ± 43.22 molecules per um2 in untreated cells), the number of clusters per area was also reduced (to 3.4 ± 0.15 from 4.7 ± 0.23 clusters per µm2) (Figure 3.6 D,E). Analysis of cluster radii indicated a reduction of CD44 cluster size to 35.34 ± 1.83 nm (from 40.72 ± 1.6 nm in untreated cells) (Figure 3.6 F,G). Collectively, in the absence of specific N- glycans the density of molecules and clusters, the sizes of clusters, as well as the level of clustering were reduced. These results indicate that galectin-3 impacts generally on the surface distribution of CD44 and furthermore that hybrid- and complex-type oligosaccharides are necessary for galectin-3 induced clustering in a direct or indirect fashion.

3.7 Discussion

The protein distribution of galectin-3 and galectin-3 ligands at the cell surface of mammalian cells was investigated in this chapter. In this context the contribution of complex N-glycans and GSLs to the clustering at the cell surface was examined. The quantification of the galectin-3 distribution itself and its influence on the distribution of CD44 and on the surface expression of β1-integrin was achieved using dSTORM. Spatial point patterns were analysed and cluster maps from dSTORM images were generated in order to obtain various statistics regarding the spatial distribution of molecules and features of clusters. This analysis revealed that galectin-3 and other examined proteins in this study are not evenly distributed, but instead form clusters at 77

the cell surface of HeLa, MEF and T cells. Additionally, various properties of these clusters were affected by different conditions (Table 3.5).

Cell Clusters Molecule Cluster Condition Max L(r)-r type per area density radius

HeLa galectin-3 clustering , ± PPMP ↓↓↓↓ ↑↑↑ ↑↑↑ ↓↓ GSL-dependent MEF galectin-3 clustering, ± Mgat5 ↓↓↓↓ ↑↑↑ ↑↑↑ ↓↓↓ Mgat 5-dependent T galectin-3, domain 52.28 cells formation ± 2.41 T β1-integrin clustering, ± gal-3 ≈ ↓↓ ↓↓ ≈ cells galectin-3 dependent MEF CD44 clustering in the presence of galectin-3, ± dMNJ ↓↓↓↓ ↓↓↓↓ ↓↓↓↓ ↓↓ dMNJ dependent Table 3.5. Summary of galectin-3 and galectin-3-induced clusters at cellular membranes of different mammalian cells (MEF, HeLa, T cells). Complex N-glycans and glycosphingolipids impacted on galectin-3 as well as galectin-3-dependent clustering at the plasma membrane of MEF, HeLa, and T cells.

GSLs have a crucial impact on galectin-3 clustering

It has been shown previously that several different galectins, including galectin-3, bind to GSLs11,171,172,175,176,185–187. While protein mediated clustering of GSLs at the plasma membrane results in the formation of microdomains429, GSLs, in turn, organise protein function by forming raft-like glyco-signalling domains where specific proteins cluster and interact430,431. In the present study GSL-dependent galectin-3 clustering properties were examined and showed that GSLs contribute to galectin-3 clustering. Indeed, in GSL deficient cells galectin-3 molecules were more randomly distributed across the plasma membrane compared to control cells. Additionally, galectin-3 clusters were smaller but clusters were more abundant and more galectin-3 binding was detected compared to control cells (Figure 3.1, Table 3.5). Increased binding of galectin-3 in the absence of GSLs was an unexpected finding and suggests that the absence of the glycans on these lipids could allow access to previously masked galectin-3 binding sites.

In conclusion, glycolipids such as GSLs impact on the cell surface distribution of galectin-3 and change its clustering properties. These findings are part of a recently published study, which demonstrated that GSLs were required for the clustering of membrane bound galectin-3 and the formation of galectin-3 induced clathrin- independent carriers (CLICs) mediating cell surface receptor uptake187.

N-glycan-dependent clustering of galectin-3

N-glycans at the cell surface have a crucial impact on the membrane architecture and on the distribution of membrane associated proteins, and consequently on many cellular processes. A recent study demonstrated that glycan-mediated interactions restrict the mobility of cell surface receptors to distinct membrane regions432. Galectins have a higher affinity for N-glycans generated by Mgat5 than for structures that are less-branched and not elongated11. In previous studies using Mgat5-/- cells, the absence of tetra-antennary and elongated N-glycans reduced the binding of galectin-3 to several cell surface receptors, such as the T cell receptor in T lymphocytes71 as well as the EGF receptor and the TβRII in epithelial cells19. The present study investigated galectin-3 clustering in cells lacking these highly branched and elongated N-glycan structures. Mgat5-/- MEF cells displayed a more random distribution of galectin-3 molecules, smaller but more clusters and more galectin-3 binding compared to control cells (Figure 3.3, Table 3.5). The differential distribution of galectin-3 illustrates how important these highly branched structures are for the formation of membrane domains by galectin-3. The increased binding of galectin-3 in the absence of preferred ligands is an interesting finding. It suggests that numerous alternative galectin-3 binding sites are available at the cell surface but are inaccessible to galectin-3 in the presence of highly branched N-glycan structures.

In summary, highly branched structures for which galectin-3 has a high affinity are required for the assembly of large galectin-3 clusters and thus represents an essential contribution in the organisation of the plasma membrane by the galectin lattice.

Galectin-3 nanodomains in T cells

In Jurkat T cells that were depleted of ATP, galectin-3 bound to and remained at the plasma membrane without internalisation, forming a molecular lattice. However, despite the plethora of available ligands for galectin-333–37, the distribution of galectin- 3 molecules at the plasma membrane was not even or random. In contrast, dSTORM imaging revealed the formation of distinct nanodomains at the plasma membrane. While the formation of a molecular lattice composed by oligomerised galectin-3 molecules bound to subsets of glycosylated cell surface molecules has been suggested by others433, this study provides an unprecedented insight into the structure of this lattice through super-resolution microscopy. In a conventional, diffraction limited image, many nanodomains may appear as a continuous lattice. Here, dSTORM imaging allowed the detection of sub-resolution galectin-3 nanodomains of 52.28 ± 2.41 nm radius at the cell surface of T cells. The data support the notion that galectin-3 compartmentalizes the cell surface by cross-linking specific cell surface molecules (Figure 3.4).

Galectin-3 regulates β1-integrin surface expression

Integrins are N-glycosylated molecules and different oligosaccharide structures have been identified on integrins; β1-integrin, for instance, contains twelve potential asparagine-linked (N-linked) glycosylation sites434,435. It has been previously shown that galectin-3 associates with various glycosylated cell surface proteins and receptors including integrins among many others33–37. Galectins interact with integrins, and it was demonstrated that galectins in general and galectin-3 in particular bind to N- acetyllactosamine sequences located on N-glycans on different integrins, including integrin-β1434,436. The experiments presented here aimed at establishing the role of galectin-3 in the distribution of β1-integrin at the surface of in T cells. Unexpectedly, exposure of Jurkat cells to exogenous galectin-3 had no direct effect on the clustering of β1-integrin, suggesting that other influences, such as lipid domains or the actin cytoskeleton drive this clustering (Figure 3.5, Table 3.5). However, β1-integrin surface

80 expression was clearly down-regulated by exogenous galectin-3, as demonstrated by the decreased molecular density and clusters per area in galectin-3 treated cells. Combined with the previous observation demonstrating that highly branched N-glycan structures actually drive galectin-3 clustering187, these data suggest a model in which pre-clustered glycosylated surface proteins may recruit galectin-3 to facilitate their endocytosis through its membrane bending abilities.

In summary, galectin-3 assembles in nanodomains on the plasma membrane of T cells. These domains appear to have no impact on the distribution of cell surface molecules, such as integrin-β1, but contribute to facilitate their internalization.

Galectin-3-dependent clustering of CD44

CD44 is expressed in numerous mammalian cell types and is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion and cell migration. While it has a predicted molecular weight of 37kDa, CD44 extensive glycosylation (5 potential sites for N-linked glycosylation and 10 sites for O-linked glycosylation) brings its molecular mass to 90kDa428,437. The impact of CD44 glycosylation and its uptake in MEF cells in the presence of galectin-3 has been investigated in a recent study I contributed to, which shows that CD44 endocytosis relies on galectin-3 and N-glycosylation187. In the study at hand, I asked whether galectin-3 influences the distribution of CD44 at the plasma membrane via specific oligosaccharide residues located at the extracellular domain of CD44. Comparison of dMNJ treated cells to control cells revealed that the absence of hybrid- and complex-type oligosaccharides clearly impaired the ability of galectin-3 to induce CD44 clustering (Figure 3.6). However, CD44 molecule density and clusters per area were also decreased, suggesting a higher internalization of CD44 when its binding to galectin-3 is impaired. This is in contrast to the results obtained with β1-integrin and opens the possibility of a differential control of cell surface protein internalization by galectin-3, based on the glycosylation pattern of these proteins. Because the function of the extraordinarily complex glycosylation of cell surface proteins remains rather poorly understood to date, this hypothesis would have to be the scope of a whole different study. 81

In summary, CD44 hybrid- and complex-type oligosaccharides serve as ligands for galectin-3. Upon binding to these structures galectin-3 increases CD44 clustering and regulates its surface expression.

Conclusion

The aim of this chapter was to assess the distribution of galectin-3 and galectin-3 receptors in the presence or absence of certain glycan structures located on proteins and lipids at the plasma membrane and the influence on plasma domain formation. The data revealed that galactin-3 forms nanoclusters at the surface of various cell types. More importantly, the experiments performed in this chapter demonstrate that Galectin-3 clustering strongly relies on N-glycans attached to cell surface proteins. In HeLa and MEF cells, highly branched and elongated N-glycans contributed to the overall level of galectin-3 clustering and specifically the formation of large galectin-3 clusters. The data further suggest that the interactions with these N-glycans lead to a common architecture of galectin-3 nanoclusters, which are rather consistently 50 – 80 nm in size in all three cell types that were examined in this study. As a matter of fact, large galectin-3 clusters were fragmented into smaller units in cells lacking glycosylated lipids. Hence, interaction of galectin-3 with N-glycans not only regulates galectin-3 clustering, but also determines the very size of these clusters. Additionally, the changes in CD44 clustering resulting from the ablation of the specific oligosaccharide mediating its interaction with galectin-3 illustrates that galectin-3 is susceptible to control the distribution of some glycosylated surface proteins. Thus, the relationship of galectin with the cell surface is bidirectional: the availability of N- glycans controls galectin-3 clustering, which in turn determines the distribution of glycosylated proteins. Such a two-way interdependence is strongly reminiscent of lipid- protein interplay ruling the organisation of the plasma membrane, typically observed during endocytic processes438. Finally, the decrease in the surface expression of β1- integrin in Jurkat cells treated with galectin-3 strongly suggests that galectin-3 mediates internalisation processes in these cells. This hypothesis is the topic of the next chapter.

Chapter 4

Galectin-3 internalisation

4.1 Introduction

The previous chapter evaluated the distribution of galectin-3 and galectin-3 dependent surface molecules at the plasma membrane of HeLa, MEF and T cells. Functions for extracellular galectin-3 other than lattice formation have been demonstrated before, and these involve the activation of various types of inflammatory cells35, mediation of cell-cell interactions402,436, as well as endocytic events187. In this chapter the binding, endocytosis and intracellular localization of exogenous galectin-3 in the human T lymphocytic cell line Jurkat E6.1 were examined.

Galectin-3 is a small (30 kDa) soluble lectin that is widely distributed in different tissues and is expressed by many cells of the immune system. It is expressed by activated but not resting T lymphocytes65 and exerts unusual dual properties exhibited by its presence both in the cytoplasm and the extracellular space. Studies measuring the levels of circulating galectin-3 in cases of cancer compared to healthy people have shown that its concentration can be drastically increased in sera of cancer patients417,418. Secretion of galectin-3 via non-classical pathways leads to its release into the extracellular space41,55 where it can bind to the plasma membrane of cells in an autocrine or paracrine fashion. Many effects induced by extracellular galectin-3 have been described so far, such as cytokine release439, chemotaxis440, signal induction441, as well as its participation in cancer, such as immune evasion in cancer cells442, tumour angiogenesis and metastasis36,38,71,76,443–445. The carbohydrate- dependent interaction of galectin-3 at the cell surface leads to cross-linking of membrane glycoproteins and thus lattice formation15,37,74,77–79,132; similarly cell surface- bound galectin-3 can enter cells via endocytosis thereby influencing endocytic 83

events187, residency time of cell surface receptors187, and trafficking of cell surface proteins187,446.

Internalisation of molecular cargo occurs by a variety of different mechanisms and sorting of internalised material is an essential process necessary for cellular functions. Incoming molecules need to be processed and directed to a variety of different destinations. The early endosome (EE) is not only the first but also the key sorting station and is in charge of routing incoming molecules to different destinations. Receptors and proteins can be recycled back to the plasma membrane either directly from the EE or indirectly via recycling endosomes; other molecules are transported to the late endosome/lysosome, or travel the retrograde route to the trans-Golgi network (TGN). Small GTPases of the Rab family are associated with different endosomal compartments ensuring their function and identity, such as Rab4 for endocytic recycling, Rab5 for early endosomes, and Rab11 for recycling endosomes447,448. The toxic plant lectin ricin binds to a variety of lipids and proteins at the plasma membrane, and becomes internalised by different endocytic mechanisms259,320,449. Ricin is known to enter the Golgi apparatus on its way to the ER and cytosol, and has previously been used to investigate the pathway from the early endosome to the Golgi apparatus320,450– 452.

Similarly to ricin, galectin-3 can bind to a variety of cell surface proteins33–36,39,42 and lipids171,174,183, and the binding of exogenous galectin-3 to the cell surface has been demonstrated131,295–300,433,453,454. Previous studies have established that the extracellular binding of galectin-3 requires the activity of the carbohydrate recognition domain (CRD) whereas the ability to oligomerise resides in the N-terminal domain of galectin-36,32. Endocytosis of galectin-3 has been observed in several cell types295,297– 300,454, although its entry mechanisms and endocytic pathways are not fully understood and particularly in hematopoietic cells not well characterised. Binding and endocytosis of galectin-3 does not appear to follow the same pathways in all cell types. In previous studies evaluating the internalisation of galectin-3 in various cell lines, uptake of galectin-3 was found to be carbohydrate-dependent in the majority of investigated cell lines298, but in certain macrophage-like cell types internalisation of galectin-3 was demonstrated to occur independently of carbohydrates via its N-terminal domain298. 84

Other galectins also depend on interactions with cell surface glycans: binding and intracellular sorting of galectin-8 relied on the interaction of the N-terminal CRD with sialylated saccharides455, and binding and uptake of galectin-1 occurred in a carbohydrate-dependent manner303. In all previously investigated cell types internalisation of galectin-3 occurred rapidly295–297, but mechanisms of endocytosis differed297,298,300. Endocytic routes of galectin-3 have so far not been investigated in detail but revealed a presence of galectin-3 in early endosomes and in recycling endosomes299,301, also more than one uptake route has been discovered in endothelial cells299. Similarly, internalisation of galectin-1 followed dual pathways and occurred by clathrin- and raft-dependent endocytosis303. The present study investigated the binding, endocytosis and intracellular localisation of galectin-3 in resting T lymphocytes.

4.2 Binding and uptake of galectin-3

Plasma membrane binding of galectin-3

Confocal microscopy and flow cytometry were used to examine whether the endocytosis of galectin-3 depends on the binding to carbohydrates at the plasma membrane of T cells. Galactose is an essential carbohydrate moiety for ligand binding of all galectins and binding affinity increases if galactose is attached to other saccharides, e.g. N-acetylglucosamine forming N-acetyllactosamine11. Studies investigating carbohydrate-binding activity and specificity of galectin-3 described N- acetyllactosamine (LacNAc) as its preferred ligand13,55 and there are numerous structurally and functionally diverse ligands for galectin-3 at the plasma membrane. Galectin-3 has a single CRD coupled to an N-terminal domain that mediates multimerisation in the presence of N-glycans70. Interaction of galectin-3 with cell surface ligands can be inhibited by lactose, a competitive inhibitor interfering with the association of the galectin-3 CRD with its ligands.

The following section determined whether the association of galectin-3 with the plasma membrane of T cells is dependent on the binding of its CRD to N-glycans of

glycosylated cell surface molecules. To investigate this, Jurkat E6.1 T lymphocytes were pre-treated with 3 mM of the disaccharides β-D-lactose or D(+)-sucrose followed by an incubation with 6 µg/mL Alexa Fluor 647-conjugated galectin-3 for 60 min at 37˚C. After fixation in paraformaldehyde (PFA), the distribution of galectin-3 was analysed by confocal microscopy and the fluorescence intensity of galectin-3 was quantified by flow cytometry. The data show that galectin-3 binding to Jurkat cells pre-treated with the competing disaccharide lactose was prevented. As a consequence of binding- inhibition, little or no endocytosis of galectin-3 into cellular structures occurred (Figure 4.1). Using flow cytometry the mean fluorescence intensity of galectin-3 binding and uptake was quantified (Figure 4.1, B). Lactose treatment almost completely abolished galectin-3 binding (p<0.0001). In contrast, sucrose as a control treatment did not compete with galectin-3 binding. In untreated control cells as well as sucrose-treated control cells, constant endocytosis of galectin-3 into cytoplasmic structures occurred (Figure 4.1). In conclusion, treatment of cells with lactose prevented binding of galectin-3 to the cell surface indicating that the CRD of galectin-3 mediates the binding to the plasma membrane.

Figure 4.1. Galectin-3 binding and uptake in T cells is carbohydrate-dependent. To examine the N-glycan dependent binding and endocytosis of galectin-3, cells were pre-treated with 3 mM lactose or 3 mM sucrose as control for the lactose treatment. 6 µg/ml Alexa Fluor 647- labelled galectin-3 was and incubated for 60 min at 37˚C. Subsequently cells were washed, fixed and analysed by confocal microscopy as well as flow cytometry. (A) Confocal images of Jurkat cells treated with lactose or sucrose that were incubated with galectin-3 Alexa Fluor-647 for 60 min. (B) For flow cytometry, 20,000 cells were measured in each sample. The fluorescence intensity in control cells was set to 1 and data was normalized. Mean values ± SEM were obtained from 3-6 separate experiments carried out in duplicates or triplicates. Statistical analysis of data: One-way ANOVA, **** p<0.0001, ns = not significant.

Endocytosis of galectin-3 is energy-dependent

It has been described previously that galectin-3 can cross-link cell surface proteins thereby forming a lattice15,36,37,74,77–79,132; coincidentally, galectin-3 can also be rapidly endocytosed upon binding to the plasma membrane. In resting Jurkat cells, galectin-3 labelled with Alexa Fluor 647 bound to the plasma membrane and was readily endocytosed within minutes into cytoplasmic endosomal compartments (Figure 4.2). Here, a high spatial resolution was achieved using SIM imaging, demonstrating a clear separation of the plasma membrane from intracellular membranes.

Figure 4.2. Confocal SIM image of galectin-3 vesicles in Jurkat T lymphocytes. Alexa Fluor 555-labelled galectin-3 was added to cell culture media of Jurkat cells and allowed to internalize for 40 min. Five minutes prior to fixation, the plasma membrane was stained with the lipophilic fluorescent membrane stain DiD. After fixation, cells were imaged on a SIM microscope to achieve a higher spatial resolution (see Materials and Methods section). Galectin-3 vesicles (green) are visible below the plasma membrane (red) in the intracellular space.

The distribution of galectin-3 molecules incubated with Jurkat cells depleted of cellular adenosine triphosphate (ATP) was examined. For depletion of ATP stores, Jurkat cells were incubated with ATP-depletion buffer for 20 min at 37˚C, then cells were incubated with 6 µg/ml galectin-3 conjugated to Alexa Fluor 647 in the presence of ATP-depletion buffer. Galectin-3 was absent from cytoplasmic compartments in energy-depleted Jurkat cells (Figure 4.3). Here galectin-3 localised only to the plasma membrane, accumulating at the outer leaflet of the plasma membrane forming patchy islands or nanodomains (Figure 4.3 A, right panel, and Chapter 3, Figure 3.1). The accumulation of galectin-3 at the plasma membrane of energy-depleted cells indicates that N-glycan based binding of galectin-3 to cell surface proteins is an energy- independent process, whereas endocytosis seems to be dependent on ATP. Indeed, in the presence of ATP, endocytosis of galectin-3 began shortly after binding to cells and galectin-3 was internalised via endocytic vesicles and accumulated over time in

cytoplasmic structures as seen in confocal images as well as mean fluorescence intensity of galectin-3 measured by flow cytometry (Figure 4.3).

Figure 4.3. Confocal images and flow cytometry analysis of galectin-3 binding and endocytosis in energy-depleted Jurkat T lymphocytes. Alexa Fluor 647 labelled galectin-3 was added to Jurkat cells that were pre-treated or not with ATP depletion buffer for 20 min at 37˚C (for buffer composition please refer to Materials and Methods section). In control cells, galectin-3 was internalised into endocytic vesicles, and in ATP-depleted cells galectin-3 remained at the plasma membrane without internalisation. (A) Confocal images showing the distribution of Alexa Fluor 647-labelled galectin-3 in control cells and ATP-depleted Jurkat cells. (B) Mean fluorescence intensity measured by flow cytometry analysis of control and ATP depleted cells. 20,000 cells were measured per sample. The fluorescence intensity was normalised to control cells. Mean values ± SEM were obtained from 3-6 separate experiments carried out in duplicates or triplicates. Statistical analysis of data: Student’s t test, **** p<0.0001.

To evaluate galectin-3 endocytosis over an extended period of time, the cell culture medium of Jurkat cells was supplemented with 6 µg/ml Alexa Fluor 647- labelled galectin-3 and cells were incubated for 6 hours. At several time points a subset of cells was removed, washed, and fixed. Subsequently the fluorescence intensity of galectin-3 was quantified by flow cytometry and plotted against time (Figure 4.4). Long-term incubation of T cells with galectin-3 indicated that galectin-3 was continuously endocytosed by these cells and suggested that galectin-3 accumulated in the cytoplasmic space.

Figure 4.4. Time course of galectin-3 uptake by Jurkat cells. Alexa Fluor 647-labelled galectin- 3 was added to cell culture media of Jurkat cells and allowed to be continuously internalised for 360 min. Mean fluorescence intensity was analysed by flow cytometry. Mean values ± SEM obtained from 3 separate experiments carried out in duplicates.

4.3 Mechanisms of galectin-3 endocytosis

Dynamin and clathrin in galectin-3 endocytosis

Dynamin is a 100 kDa GTPase which plays an essential role in cellular membrane fission during vesicle formation and is thus required for clathrin- and caveolae- mediated endocytosis278,456. To determine whether there is an involvement of dynamin in the endocytosis of galectin-3, the effects of the inhibitor dynasore were explored. Dynasore is a specific inhibitor of the GTPase activity of dynamin and was reported to lead to a reduction of endocytosis391.The mean fluorescence intensity of Jurkat cells treated with 80 µM dynasore for 30 min, and subsequently incubated with Alexa Fluor 674-labelled galectin-3 in the presence of dynasore, was measured by flow cytometry. After 60 min of galectin-3 uptake, flow cytometric measurements showed a decrease in galectin-3 endocytosis to 14.5% compared to untreated control cells (Figure 4.5). The data also confirmed that fluorescent galectin-3 was endocytosed by Jurkat cells.

Since dynamin GTPase activity is a hallmark of clathrin-mediated endocytosis, I asked whether there is a functional role for clathrin in galectin-3 entry into T cells and examined the inhibitory effect of chlorpromazine on galectin-3 endocytosis.

Chlorpromazine (CPZ) is a known inhibitor that prevents assembly of clathrin-coated pits at the plasma membrane457. Treatment of Jurkat cells with 20 µM CPZ for 2 h significantly reduced internalisation of galectin-3 to 17.5% as analysed by flow cytometry (Figure 4.5). Taken together, these results indicate an involvement of both dynamin and clathrin in the endocytosis of galectin-3 by Jurkat T cells.

Figure 4.5. Effects of endocytic inhibitors on galectin-3 uptake. Flow cytometry data of Alexa Fluor 647-labelled galectin-3 internalisation in Jurkat cells. Cells were pre-treated with 80 µM dynasore (DNS), 20 µM chlorpromazine (CPZ), 10 µM latrunculin A (Lat A), or 4 µM cytochalasin D (Cyto D) at 37˚C for times indicated in the main text. Subsequently galectin-3 was added to the media containing the inhibitors and after further 60 min of incubation at 37˚C, cells were analysed by flow cytometry. 20,000 cells were measured in each sample. The fluorescence intensity was normalised to control cells. Mean values ± SEM were obtained from 3-6 separate experiments carried out in duplicates or triplicates. Statistical analysis of data: One-way ANOVA, **** p<0.0001.

Effect of actin-depolymerizing agents on galectin-3 endocytosis

The plasma membrane is linked to the cell cortex forming a functional integration with the underlying actin cytoskeleton. Vesicular traffic derived from the plasma membrane requires active actin rearrangements, thus cytoskeletal structures are dynamically organised to allow the formation and inward movement of endocytic vesicles. Evidence obtained through studies in budding yeast has demonstrated the involvement of actin in endocytic events458. Furthermore, experiments in mammalian

90 cells revealed the presence of actin as well as actin-regulatory proteins at endocytic sites293,459–461.

To investigate the involvement of actin in the formation and endocytosis of galectin-3 positive vesicles in T cells, two inhibitors whose modes of action are complementary were used. Latrunculin A and cytochalasin D are both known to disrupt the organisation of microfilaments by depolymerisation of actin385,387. Cytoachalasin D binds to plus ends of F-actin filaments preventing further addition of G-actin, latrunculin A on the other hand binds G-actin which as a consequence cannot be added to actin filaments462. Exposure of cells to either 10 µM latrunculin A or 4 µM cytochalasin D for 30 min prior to incubation with labelled galectin-3 exerted inhibiting effects on galectin-3 uptake (Figure 4.5). Internalisation was reduced to 27.3% for latrunculin A and 26.1% for cytochalasin D compared to control cells. This demonstrates that actin, most likely cortical actin, is involved in the endocytosis of galectin-3, as well as the formation and movement of galectin-3-positive vesicles.

Lipids and microdomains in galectin-3 endocytosis

Lipid rafts represent heterogeneous and functionally distinct membrane domains463. Raft-dependent endocytosis is commonly defined by clathrin- independence, dynamin-dependence, and sensitivity to cholesterol depletion393. Raft- associated proteins may not only serve in the lateral segregation of the plasma membrane but may also provide domains with differential endocytic capacities. It has been shown that raft-like domains mediate the internalisation of a variety of endocytic cargo212,464–467. Interactions between lipid microdomains and endocytotic complexes have been demonstrated by others468,469. Therefore I examined the role of raft-like microdomains as well as GSLs in the endocytosis of galectin-3 by T cells.

Oxysterols (oxidized derivatives of cholesterol) are structurally and biologically important lipid components in cellular membranes. They regulate the biophysical properties of lipid rafts192,394,470 and alter the lipid and protein composition in plasma

membrane domains192 influencing the endocytic capacity of the plasma membrane. The oxysterol 7-ketocholesterol (7-KC) prevents tight lipid packing due to a protruding ketone group. Thus incorporation of the oxysterol 7-KC reduces membrane order471 and prevents the formation of raft-like domains193. To determine whether lipid raft disruption has an impact on the endocytosis of galectin-3, T lymphocytes were enriched with 7-KC. According to previous reports 7-KC was prepared as a water- soluble sterol472 and Jurkat cells were treated with 56 µM 7-KC for 10 min at 37˚C. Subsequently cells were incubated with Alexa Fluor 647-conjugated galectin-3 in the presence of 7-KC. After 60 minutes of galectin-3 uptake, the mean fluorescence intensity of cells was analysed by flow cytometry. The measurement revealed that endocytosis of galectin-3 was reduced by 73.8% (Figure 4.6). This suggested a dependence of galectin-3 uptake on lipid rafts or membrane order, and that highly condensed microdomains play a role in the endocytosis of galectin-3.

In another approach for lipid raft disruption, methyl-β-cyclodextrin (mβCD) was applied, leading to cholesterol depletion of the plasma membrane. Jurkat cells were treated with 2.5 mM mβCD for 20 minutes at 37˚C, incubated with fluorescently labelled galectin-3 for 60 minutes, and analysed by flow cytometry. Similarly to 7-KC, treatment with mβCD significantly reduced the endocytosis of galectin-3 (Figure 4.6). Sensitivity of galectin-3 to different lipid raft-disrupting agents such as 7-KC and mβCD suggests a function for plasma membrane domains in the endocytosis of galectin-3.

SLs play important roles in membrane structure and cell function, and studies have focused on examining the requirements of SLs in various endocytic mechanisms473–476. As described in Chapter 3, depletion of glycosphingolipids (GSL) has an impact on the formation of galectin-3 domains at the plasma membrane. Furthermore, GSLs are required for galectin-3-induced uptake as recently shown187. Synthesis of most GSLs begins with glycosylation of ceramides to form glycosylceramide (GlcCer) which is the precursor-molecule for a multitude of different GSLs477. This cerebroside is synthesised by the glucosyltransferase GlcCer synthase from UDP-glucose and ceramide. In 1999, Abe et al. published a range of specific inhibitors of GlcCer synthase478. Here I have used the inhibitor 1-phenyl-2-

92 hexadecanoylamino-3-morpholino-1-propanol (PPMP) to selectively inhibit cellular GlcCer formation.

To examine the role of GSLs in galectin-3 endocytosis, Jurkat cells were treated for 6 days with PPMP that was added to the culture media. Subsequently cells were incubated with Alexa Fluor 647-labelled galectin-3. Galectin-3 uptake was measured by flow cytometry and the mean fluorescence intensity was plotted (Figure 4.6). After 60 min of incubation at 37˚C, uptake in PPMP-treated cells was reduced by 82.6% in comparison to untreated cells indicating a dependence on GSLs in galectin-3 endocytosis by Jurkat cells.

In conclusion, the uptake of galectin-3 is affected by cholesterol depletion, disruption of lipid rafts and the absence of GSLs suggesting that certain lipid environments are crucial for the endocytosis of galectin-3.

Figure 4.6. Effects of lipids on the endocytosis of galectin-3. Flow cytometry data of galectin-3 internalisation in Jurkat cells. Cells were pre-treated with 56 µM 7-KC for 30 min, 5 µM PPMP for 6 days or 2.5 mM mβCD for 20 min, all at 37˚C. Subsequently Alexa Fluor 647-conjugated galectin-3 was added at a concentration of 6 µg/ml and after 60 min cells were washed, fixed and analysed by flow cytometry. 20,000 cells were measured in each sample. Fluorescence intensity in control cells was set to 1 and data was normalized. Mean values ± SEM were obtained from 3-6 separate experiments carried out in duplicates or triplicates. Statistical analysis of data: One-way ANOVA, **** p<0.0001.

4.4 Subcellular distribution of galectin-3

The experiments presented thus far in this chapter were performed in fixed cells to quantify total levels of galectin-3 in Jurkat cells after internalisation. For real time observation of extracellular galectin-3 binding and endocytic vesicle formation, live cell confocal imaging was performed (Figure 4.7). Jurkat cells were allowed to settle onto poly-L-lysine coated coverslips and were subsequently incubated with Alexa Fluor 647- labelled galectin-3 at 37˚C in phenol red-free cell culture media. Images were taken every 15 s over a time period of 40 min. Live cell experiments in Jurkat cells revealed that within the first minutes of incubation galectin-3 gradually accumulated at the plasma membrane (Figure 4.7, first row); subsequently galectin-3 molecules were endocytosed into vesicles. Within 5 minutes of incubation, galectin-3 was located in vesicular structures just below the plasma membrane (Figure 4.7, second row). Over time, galectin-3 positive vesicles accumulated in endosomal structures in the peri- nuclear region (Figure 4.7, third and fourth row).

Figure 4.7. Live cell imaging of galectin-3 binding and endocytosis in resting T cells. Fluorescently conjugated galectin-3 was added to live T cells and was observed to accumulate at the plasma membrane. Fluorescence intensity increased as more galectin-3 molecules bound to the plasma membrane (top row). After 5 minutes of galectin-3 binding to the cell surface, the formation of vesicles that were pinching off the intracellular leaflet was observed (second row, white arrowheads). After removal of galectin-3 from the culture media (third row, second image), fluorescence intensity at the plasma membrane gradually decreased as galectin-3 internalisation continued. This decrease in fluorescence was partially due to bleaching and partially due to the removal of surface bound galectin-3 by internalisation (third and fourth row). Galectin-3-positive vesicles travelled toward a central region in the cytoplasm where galectin-3 accumulated (fourth row, empty arrowheads). Observations were obtained from 2 independent experiments. Scale bar: 10 µm.

To examine the spatial distribution of internalised galectin-3 molecules, Alexa Fluor 647-conjugated galectin-3 was incubated with T cells at 37˚C for 10 min, 20 min, and 40 min. Confocal imaging of fixed cells that were stained for various endocytic 95

markers was performed (Figure 4.8 A). Colocalisation of galectin-3 with markers of endosomal compartments in confocal images was determined with Pearson’s coefficient (Figure 4.8 B). EEA1 is a Rab5-effector containing two Rab5-binding domains, mediating the docking of Rab5-positive endocytic vesicles and is an established marker for early endosomal compartments479,480. At early time points, galectin-3 colocalised with EEA1 and was located below the plasma membrane in early endosomal vesicles (Figure 4.8 A, top row). This indicated a recruitment of recently internalised galectin-3 into early organelles of the endosomal pathway. Rab11 is a member of the Rab-family of docking proteins that regulates traffic through the recycling endosome481 and is a marker for this compartment. Vesicular galectin-3 colocalised with Rab11 in T cells (Figure 4.8 A, second row). The lysosomal glycoproteins LAMP-1 and LAMP-2 have previously been identified and characterised482,483. Their primary residency in the lysosomal membrane484 makes them suitable markers for this compartment485. At late time points, galectin-3 colocalised with LAMP-1 indicating an association of galectin-3 with lysosomes (Figure 4.8 A, third row). The small G-protein Arl1 interacts with golgin-97 and is responsible for its recruitment to the TGN486,487. Due to this localisation, golgin-97 is frequently used as an indicator for the TGN488. In order to examine whether galectin-3 positive vesicles overlap with the Golgi apparatus, antibodies against golgin-97 were applied (Figure 4.8 A, last row) and colocalisation of galectin-3 with the TGN was confirmed. Taken together these results indicate that galectin-3 is endocytosed through a common endocytic pathway passing through different endosomal compartments including the Golgi. On the other hand, colocalisation of galectin-3 with LAMP-1 indicates that a fraction of galectin-3 molecules seems to be targeted for degradation and is directed to late endosomes/lysosomes.

B Figure 4.8. Analysis of the subcellular distribution of galectin-3. (A) Jurkat cells were incubated with Alexa Fluor 555- or 647- conjugated galectin-3, fixed at different time points (10 min for early endosomes, 20 min for recycling endosomes, 40 min for lysosomes and Golgi) and subjected to immunofluorescence analysis with antibodies against EEA-1, Rab11, LAMP-1 or Golgin-97. Yellow in merged images indicates colocalisation. Scale bar: 10 µm. (B) Bar graph shows the Pearson’s coefficient for colocalisation of galectin-3 and endosomal markers. Data was obtained from images acquired by confocal fluorescence microscopy. Quantification shows mean ± SEM of three independent experiments with 10 cells in each experiment.

4.5 Discussion

Confocal microscopy and flow cytometry were used to address the binding, endocytosis and intracellular localisation of exogenous galectin-3 in the human T cell line Jurkat E6.1. The results described above show that binding of galectin-3 to the outer leaflet of the plasma membrane was carbohydrate-dependent, and that this carbohydrate-dependent binding was also a prerequisite for the endocytosis of galectin-3 (Figure 4.1 and 4.7). I further show that galectin-3 internalisation was mediated by classical endocytic pathways involving common and known proteins and lipids of the endocytic machinery such as dynamin, clathrin, and actin, as well as plasma membrane domains (Figures 4.5 and 4.6). The data suggest a synergy of these endocytic mechanisms in galectin-3 uptake, as interference with individual components of these endocytic pathways only partially suppressed uptake of galectin- 3. The subcellular distribution of galectin-3 within cytoplasmic organelles was examined and further confirmed that galectin-3 followed different endocytic routes – it was either transported to the Golgi or targeted to late endosomes and lysosomes (Figure 4.8). These results support a previous report in vascular endothelial cells299 and thus demonstrate that uptake and transport of galectin-3 by different endocytic routes may be a common feature in many cell types.

Binding of Galectin-3 to the Plasma Membrane

Treating Jurkat cells with physiological levels403,417,420–423 of galectin-3, I found that the association of exogenous galectin-3 with the plasma membrane occurred rapidly upon addition to the culture media. After binding to the plasma membrane galectin-3 was internalised via endocytic vesicles (Figures 4.2, 4.7, and 4.8). Endocytosis of galectin-3 was an ongoing process that continued for hours without reaching a plateau (Figure 4.4). Inhibition of galectin-3 binding after treatment with the competing disaccharide lactose revealed that binding to the plasma membrane was dependent on glycosylated molecules at the plasma membrane. Treatment with

98 the non-competing disaccharide sucrose had no effect on either binding or endocytosis (Figure 4.1). The carbohydrate-dependent binding of galectin-3 to Jurkat cells is in agreement with previous studies in other cell types such as breast carcinoma cells297, MDCK cells301, macrophage-like cell types298, and vascular endothelial cells112. Carbohydrate-independent binding of galectin-3 to the plasma membrane of T cells was not observed (Figure 4.1). Taken together, these results suggest that carbohydrate-dependent binding of exogenous galectin-3 to T cells is required for its attachment to the plasma membrane, and that this binding is a prerequisite for its endocytic uptake.

In cells that were energy depleted, galectin-3 bound to the plasma membrane but could not be internalised (Figure 4.3). The association of galectin-3 with various glycosylated cell surface proteins and receptors, including CD98, CD47, TGF-β, VEGF and EGF, CD45 and integrins, among others, has already been shown33–37. Despite the plethora of available galectin-3 ligands at the cell surface, galectin-3 did not bind uniformly to the plasma membrane. Instead galectin-3 formed distinct patchy islands resembling nanodomains that were unevenly distributed across the cell surface (Figure 4.3 A, and Chapter 3, Figure 3.1). Several studies report the existence of a molecular lattice formed by oligomerised galectin-3 bound to specific subsets of glycosylated cell surface molecules and regulating their spatial distribution15,37,71,74,79,132. The formation of galectin-3 domains on the cell surface of MEF, HeLa and T cells, shown in this Chapter and the previous Chapter, supports the notion that galectin-3 contributes to the compartmentalisation of the plasma membrane37,74,77–80,132. In summary, binding of galectin-3 to the plasma membrane of T cells is an energy-independent process and resulted in the formation of galectin-3 nanodomains. Endocytic events cannot proceed without the availability of ATP; accordingly galectin-3 remained bound to the cell surface in energy-depleted cells.

Mechanisms of endocytic galectin-3 uptake

To profile the mechanism of galectin-3 uptake in T lymphocytes, I applied pharmacologic inhibitors of known endocytic pathways. Dynasore and chlorpromazine, which inhibit dynamin- and clathrin-dependent pathways, respectively391,457. Both led to a partial reduction of galectin-3 endocytosis (Figure 4.5). In order to further explore the role of actin in the mechanisms of galectin-3 endocytosis, I tested the impact of the depolymerizing agents latrunculin A and cytochalasin D on galectin-3 endocytosis (Figure 4.5). Both substances impaired the endocytosis of galectin-3 in T cells indicating that actin dynamics, most likely cortical actin, are required for the formation and transport of galectin-3-positive endocytic vesicles away from the plasma membrane.

Lipid raft pathways mediate the internalisation of a multitude of different proteins212,464–467. Lipid-mediated endocytosis of galectin-3 has been investigated in the present study. MβCD depletes cellular cholesterol and hence disrupts lipid rafts393, 7-KC interferes with the lipid packing and thus interferes with the formation of highly condensed membrane domains193,472, and PPMP acts as an inhibiting agent of GSL synthesis478 (Figure 4.6). All three compounds lead to a partial inhibition of galectin-3 endocytosis, demonstrating that lipid organisation of the plasma membrane plays a central role in galectin-3 internalisation. It should be noted that flow cytometry records overall galectin-3 levels per cell and does not reveal whether galectin-3 binding and/or uptake is affected. Hence for this particular experiment confocal images would show whether obtained results are due to a reduction of galectin-3 uptake, or due to a lack of galectin-3 binding to the plasma membrane of treated cells. Although Figure 3.1 in Chapter 3 shows that galectin-3 binding to PPMP treated cells still occurs, a more complete data set would include confocal images to discriminate between impaired binding and impaired internalization of galectin-3.

Collectively, these results show that galectin-3 is susceptible to enter the cells via multiple pathways. These mechanisms include clathrin-dependent and -independent pathways, dynamin-dependence as well as a requirement for raft-like plasma membrane domains. Cargoes for clathrin-independent endocytosis (CIE) are diverse and different studies have shown that the same endocytic cargo may be internalised by different mechanisms in different cell types, or may switch endocytic pathways in a single cell type depending on the conditions256,314,490–492. Hence, redundant clathrin- 100 independent endocytic mechanisms can be used by some cargo molecules256. Although the observation that inhibition of one pathway results in the upregulation of alternative endocytic pathways has been described previously493–496, in the current study no upregulation has been observed. However, the question arises if the remaining endocytic activity is caused by the disrupted pathway itself or caused by another endocytic pathway. Further experiments, to discern the uptake mechanisms of galectin-3 in T lymphocytes could reveal more details and a better understanding of the machinery by which this protein is internalized.

Intracellular localisation of galectin-3

Analysis by confocal microscopy of galectin-3 revealed a strong signal in distinct vesicular structures (Figures 4.2, 4.7 and 4.8). Detection of galectin-3 in early and recycling endosomes as well as lysosomes and the Golgi (Figure 4.8) indicated that it was not mainly targeted to degradative compartments and may enter a pool of recyclable endocytic goods which is in agreement with findings by others298,299,454. Sorting of galectin-3 into different endocytic pathways and various subcellular compartments is underscored by previous observations that galectin-3 is bound by different ligands33–37,41,42. The cell surface ligand bound by galectin-3 may be responsible for the choice of a particular endocytic pathway. The trans-golgi-network (TGN) sorts proteins for different destinations and can be considered a traffic hub that is not only limited to forward transport but also receives proteins by retrograde transport via endosomes307. Observation of galectin-3 translocating to the Golgi demonstrates that a fraction of internalised galectin-3 was transported retrogradely to the Golgi apparatus. Galectin-3 thus displayed similarities in trafficking with shiga toxin, cholera toxin B and the lectin ricin, which are endocytosed at least in part through a clathrin-independent mechanism212,314,320,490 and are also transported from the plasma membrane to the Golgi319,452,497,498.

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Conclusion

In conclusion, the data presented in this chapter demonstrate that galectin-3 is endocytosed in a carbohydrate-dependent manner in Jurkat T lymphocytes, and that its endocytosis is mediated by different endocytic mechanisms. The differential endocytosis is also reflected in the localisation of galectin-3 to different endocytic compartments. Plasma membrane domains have been implicated to play a role in endocytosis499–504. Assemblies of clathrin-coated pits form domains at the membrane in cultured cells499–504. Similarly, domain formation in CIE has been demonstrated to facilitate endocytosis505,506 and endocytic domains necessary for endocytosis mediated by pathogens284,507 and lipids have been shown508–510. Hence, galectin-3 may act as a primer for endocytosis of glycoproteins gathering cargo through its ability to oligomerise. Subsequently, selection of the endocytic route is determined by the nature of cargo receptors or by their direct environment within the plasma membrane. Thus galectin-3 may be involved in downregulation and molecular recycling of cell surface ligands.

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Chapter 5

Galectin-3 in T cell activation

5.1 Introduction

Galectin-3 is expressed in various cells and tissues of the immune system including T lymphocytes and regulatory T cells56–64. Galectin-3 was first identified at the extracellular leaflet of the plasma membrane in murine macrophages44. Expression of galectin-3 in CD4+ and CD8+ T cells depends on the activation status of these cells, and certain cytokines can enhance its expression levels351. Constitutive expression of galectin-3 has been confirmed in human regulatory T cells and CD4+ memory T cells416,511,512. Galectin-3 expression can be induced by different stimuli, such as in response to bacterial pathogens513, by the presence of appropriate antigens for tumour infiltrating T cells514, and upon differentiation of naïve T cells512,513,515. Since galectin-3, similarly to all other galectins, does not contain a classical signal sequence, the presence of galectin-3 at the outside of cells is due to its secretion by a non- classical pathway. The ability of galectin-3 expressing cells to secret it differs widely from none to nearly all of newly synthesised galectin-341. Galectin-3 secretion seems to depend on development and differentiation41, and can be stimulated by heat shock or calcium ionophores53,55. Upon secretion, galectin-3 can bind to the cell surface in an autocrine or paracrine fashion. Galectin-3 exerts extracellular functions by binding to glycosylated cell surface molecules due to its lectin activity, as discussed in the previous chapters. Extracellular galectin-3 has not only been shown to bind to the plasma membrane of a variety of cell types but is also internalised by different mechanisms295,297–300,302,303,454. In consistency with its cytoplasmic presence, intracellular functions for galectin-3 have been described329,516–518. Galectin-3 was also found to participate in the sorting of glycoproteins on their way to the plasma

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membrane, and these observations indicate binding of galectin-3 to certain intracellular glycoproteins129. Hence the convergence of glycobiology including cellular glycoproteins and carbohydrate binding proteins, and immunobiology suggests a participation of galectin-3 in T cell regulation at the extracellular and intracellular level71,131,352,433.

It is commonly accepted that T cell signalling relies on the spatiotemporal organisation of signalling proteins in plasma membrane domains519–521. The lateral organization of signalling proteins within the plasma membrane can be achieved by preferential partitioning of proteins into lipid domains, as stated in the lipid raft hypothesis522, or by specific protein-protein interactions, as regulated by the superfamily of tetraspanins523. In order to sustain and regulate TCR signalling at the immunological synapse, active transport of signalling molecules to and within the synapse is crucial524. In this context, the tetraspanin CD82 for example is localised to the plasma membrane in resting T cells but is transported to the periphery of the immunological synapse upon contact of the T cell with the antigen presenting cell (APC)525.

Cellular signalling cascades initiated by cell surface receptors and intracellular trafficking pathways are linked by endosomes to intracellular trafficking pathways, which can then be viewed as intracellular platforms for signal transduction526,527. Evidence implies that endosomal activities contribute to responses generated by the innate and adaptive immune system. It has been shown that the TCR is recycled between the plasma membrane and endosomes528,529 and that this recycling promotes delivery of the TCR to the immunological synapse and maintains signalling530. A number of different components of the T cell signalling machinery can be found in endosomal vesicles that are transported to the site of initial TCR engagement531. The transmembrane component linker for activation of T cells (Lat) is a scaffolding molecule aiding in the assembly of the TCR signalling complex532 and is an essential component of the TCR signalling cascade. Super-resolution microscopy investigating the recruitment of Lat to the immunological synapse, has shown that Lat-positive vesicles near the plasma membrane are delivered to the cell surface upon initiation of early signalling events414,533. In fact, TCR signalling depends on the vesicular trafficking 104 machinery, as the kinase Lck, which initiates T cell signalling by phosphorylating CD3 ITAM domains, is delivered via vesicles to the immunological synapse. Lck vesicles have been shown to accumulate in the peri-centrosomal area534 in recycling endosomes that are translocated to mature synapses indicating that intracellular Lck recycles back to the synapse providing an additional source of phosphorylation535.

It has been previously demonstrated that extracellular galectin-3 can have a negative impact on T cell functions71,352,439, and Mgat 5-deficient mice displayed an increased propensity to TCR activation due to the absence of branched glycans71. Further, endogenously expressed galectin-3 has been shown to be recruited to the immunological synapse where it promotes TCR down-regulation at the pSMAC, contributing to the downregulation of T cell activation352. In Chapter 4 of the present study, I established that galectin-3 is internalized in T cells. This finding led to the investigation of the effects of galectin-3 during T cell activation, the intracellular location of galectin-3 in activated T cells upon internalisation, and whether internalised galectin-3 associates with proteins that are part of the TCR signalling machinery.

5.2 Endogenous galectin-3 in TCR clustering

In order to evaluate the intracellular distribution of galectin-3 expressed in activated T cells, Jurkat cells were transfected with a peCFP-galectin-3 construct. After 24 hours of incubation to allow for gene expression and protein translation, Jurkat cells were activated on antibody-coated coverslips for 20 min at 37˚C. Formation of an immunological synapse at the coverslip is induced by coating glass coverslips with functional grade antibodies that induce T cell activation. In the present study, glass coverslips were coated with functional antibodies against the co-stimulatory receptor CD28 and the subunit CD3ε of the T cell for 1h at 37˚C. Subsequently cells were allowed to settle onto these antibody-coated coverslips to induce activation for 10 minutes at 37˚C. Confocal analysis of galectin-3 distribution revealed the presence of galectin-3 throughout the cell including the nucleus, the cytoplasm and membrane ruffles formed due to the formation of an activation site (Figure 5.1).

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Figure 5.1 Expression of intracellular galectin-3 in activated T cells. Jurkat cells were transfected with peCFP-galectin-3 and cultured for 24 h to allow for galectin-3 biosynthesis. Cells were then activated for 20 min at 37˚C on antibody-coated coverslips. Distribution of galectin-3 in activated T cells was analysed by confocal microscopy.

To evaluate the impact of endogenous galectin-3 in T cell activation and TCR clustering, cells co-transfected with peCFP-galactin-3 as well as CD3ζ-PSCFP2 were activated on coverslips as described above. To determine the distribution of the T cell receptor in the absence or presence of endogenous galectin-3, cells transfected or not with peCFP-galectin-3 were fixed, washed, placed on a TIRF microscope and imaged using PALM for single molecule imaging (Figure 5.2). Subsequent image analysis (as described in Chapter 2 and 3) revealed a similar expression level of CD3ζ-PSCFP2 (as expected), a more random distribution of CD3ζ-PSCFP2 in the presence of galactin-3 (lower maximum level of L(r)-r), and more TCR clusters per area and smaller clusters in the presence of galectin-3 compared to control cells (Figure 5.2). Hence, intracellular expression of galectin-3 affects the distribution of the TCR at the cell surface.

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Figure 5.2. Clustering of CD3ζ-subunit in the presence or absence of intracellular galectin-3. (A) dSTORM images CD3ζ-PSCFP2 in cells expressing or not intracellular galectin-3. (B) Cluster map of the red squares outlined in A. The degree of clustering is colour-coded and indicates areas with low (blue) to high (red) clustering. (C) Thresholded cluster-map in black and white showing the outline of clusters. (D-I) Quantitative statistical analysis of CD3ζ clustering with or without expression of galectin-3 (D) total number of molecules per µm2, (E) number of clusters per µm2, (F) radii of clusters in nm, (G) size of clusters in µm2, (H) L(r)-r indicates the level of clustering, (I) maxima of L(r)-r. Means ±S.E.M. from 10-20 cells, n=3 experiments. Statistical analysis of data: Student’s t test, **** p<0.0001, *** p<0.001, ** p<0.01, ns = not significant. Scale bar: 10 µm

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5.3 Exogenous galectin-3 in TCR clustering

The T cell receptor is a multi-protein complex consisting of the TCRα and β chains as well as several CD3 subunits forming γε, δε and ζζ dimers (for details on the TCR see Chapter 1). Several studies have demonstrated that galectin-3 binds to the TCRβ chain18,35,76,131. In this section it was investigated whether extracellular galectin-3 changes the clustering properties of the T cell receptor in activated T cells. To enable binding of galectin-3 to the cell surface of Jurkat cells but prevent the internalisation of galectin-3, Jurkat cells were placed on ice and incubated, or not, with Alexa Fluor 555- conjugated galectin-3 for 1 h at 4˚C. Here, labelled galectin-3 was used in order to identify cells that were galectin-3 positive. Following this incubation, the temperature was shifted to 37˚C and cells were allowed to settle onto antibody-coated activating coverslips for 10 min at 37˚C in the presence of galectin-3. To detect activated CD3ζ, cells were fixed in 4% PFA, permeabilised and stained with a phospho-specific antibody. The primary antibody was detected using an Alexa Fluor 647-conjugated secondary antibody. dSTORM imaging was performed to analyse the distribution of phospho-CD3ζ molecules on a single molecule level (Figure 5.3).

Incubation of Jurkat cells with exogenous galectin-3 changed the distribution of phosphorylated TCR complexes, leading to a modest albeit significative diminution of its clustering as illustrated by a lower Max of L(r). However, the molecular density and the number of clusters of phosphorylated CD3ζ increased, while their size was not affected. These data suggest that galectin-3 does not directly contribute to CD3ζ clustering but instead promotes its phosphorylation and stabilizes its surface expression. Here it is interesting to note that galectin-3 mediated stabilization of surface proteins expression has already been described for CD44 in the 3rd chapter of this study.

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Figure 5.3. Clustering of phosphorylated CD3ζ in the absence or presence of extracellular galectin-3. (A) dSTORM images anti-phospho-CD3ζ labelled with Alexa Fluor 647 secondary antibody in control and galectin-3-treated T cells. (B) Cluster map of the red squares outlined in A. The degree of clustering is colour-coded and indicates areas with low (blue) to high (red) clustering. (C) Thresholded cluster-map in black and white showing the outline of clusters. (D-I) Quantitative statistical analysis of anti-CD3ζ clustering in T cells treated or not with extracellular galectin-3 (D) total number of molecules per µm2, (E) number of clusters per µm2, (F) radii of clusters in nm, (G) size of clusters in µm2, (H) L(r)-r indicates the level of clustering, (I) maxima of L(r)-r. Means ±S.E.M. from 10-20 cells, n=3 experiments. Statistical analysis of data: Student’s t test, *** p<0.001, ** p<0.01, ns = not significant. Scale bar: 10 µm

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5.4 Galectin-3 at the immunological synapse

A previous study examining the distribution of endogenous galectin-3 expressed in T cells has shown that endogenously expressed galectin-3 accumulates at the immunological synapse upon T cell activation131. In the present study Jurkat cells were exposed to extracellular galectin-3 for 30 min at 37˚C, fixed and analysed by confocal microscopy. In the majority of analysed cells recently endocytosed galectin-3 was present throughout the cytoplasm in vesicular structures. Also, galectin-3 vesicles were detected at the immunological synapse of all activated T cells (Figure 5.4) implying a role for endocytosed galectin-3 in activation events of T cells.

Figure 5.4. Immunological synapse with galectin-3 positive vesicles. Jurkat cells were incubated with 6 µg/ml Alexa Fluor 647-conjugated galectin-3 for 20 min at 37˚C, and then activated on antibody coated coverslips for 10 min at 37˚C. After fixation in 4% PFA, cells were imaged under confocal microscopy and z-stacks generated. Upper panel shows a middle section through the cell with cytosolic galectin-3 vesicles arranged around the nucleus. Lower panel displays a section of the immunological synapse at the glass coverslip with galectin-3 positive vesicles. Image is a representative for 57 cells from 6 different experiments. Scale bar: 10 µm

To determine whether certain galectin-3 binding proteins as well as proteins involved in T cell signalling localise together with galectin-3 to the immunological synapse, their localisation was analysed relative to the position of galectin-3 positive vesicles (Figure 5.5). CD82 and CD63 are both implicated with signalling events at the immunological synapse525,536. Additionally, a proteomic analysis performed by the Johannes lab in Paris in order to identify galectin-3 ligands at the surface of Jurkat E6.1 110 cells pinpointed CD82 and CD63 as high hits in the list of galectin-3 ligands (unpublished data). Thus the localisation of CD82 and CD63 was analysed in galectin-3 positive and activated T cells.

Experimental preparation involved labelling of Jurkat cells with galectin-3 conjugated to Alexa Fluor-555 for 20 min at 4˚C to allow binding but prevent endocytosis. Subsequently cells were placed on antibody-coated coverslips at 37˚C. The shift in temperature allowed for activation of T cells when settling onto activating antibodies and also enabled the endocytosis of galectin-3. After 10 min at 37˚C, cells were fixed in situ, permeabilised and an immuno-labelling performed (Figure 5.5). Antibodies against CD63 and CD82 were used to indicate the intracellular localisation of these proteins in relation to galectin-3. Subsequently the distribution of Alexa Fluor- 555 labelled galectin-3 and antibody-labelled proteins was analysed by confocal microscopy. Co-localisation of proteins is indicated in yellow. Confocal analysis revealed that galectin-3 positive vesicles were present at the immunological synapse (Figure 5.5). In the majority of cells, some of the galectin-3 positive vesicles colocalised with CD63 or with CD82, respectively. This suggests that CD82 and CD63 occupy a subset of galectin-3 positive vesicles at the activation site of T cells.

Figure 5.5. Analysis of galectin-3 positive vesicles at the immunological synapse. Jurkat cells were incubated with Alexa Fluor 555-conjugated galectin-3 for 20 min at 4˚C, subsequently cells were allowed to settle onto activating glass coverslips at 37˚C and were activated for 10 min. Then cells were fixed in 4% PFA, permeablised and subjected to immunofluorescence analysis with antibodies against CD63 (upper panel) and CD82 (lower panel). Yellow in merged images indicates colocalization. Image is a representative for 30 cells from 3 different experiments. Scale bar: 10 µm

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5.5 Discussion

In the present Chapter the impact of endogenous and exogenous galectin on the clustering of the T cell receptor complex was investigated in activated T cells (Table 5.1). For endogenous galectin-3, T cells were transiently transfected with peCFP- galectin-3 enabling intracellular galectin-3 expression and CD3ζ-PSCFP2 in order to investigate the distribution of the T cell receptor complex by single molecule imaging using PALM. Expression of galectin-3 in Jurkat T lymphocytes revealed the presence of galectin-3 in the nuclear compartment as well as the cytoplasmic space including membrane ruffles caused by cell spreading (Figure 5.1). This finding is in agreement with the expression of intracellular galectin-3 in other cell types67,537. Previously galectin-3 was reported to shuttle between the cytoplasm and the nucleus67. For exogenous galectin-3 T cells were incubated with galectin-3 at 4˚C to prevent internalization and to form a galectin-3 lattice at the plasma membrane prior to T cell activation. Prevention of galectin-3 endocytosis at 4˚C has been confirmed by confocal microscopy (data not shown). A temperature shift to 37˚C is necessary for T cell activation and at the same time fosters internalisation of galectin-3. However, the presence of galectin-3 in the surrounding cell culture media should maintain the effects of galectin-3 during T cell activation. Subsequently cells were fixed and imaged using dSTORM. Data revealed an impact of endogenous as well as exogenous galectin- 3 on the distribution of the TCR (Figure 5.2, 5.3). In both conditions the presence of galectin-3 lead to a more random distribution of TCR clusters, while increasing the cluster number per area. Whereas endogenous galectin-3 did not alter the expression level of CD3ζ, exogenously added galectin-3 enhanced the phosphorylation of CD3ζ when T cells were stimulated with adhesive antibodies as described above. This suggests that galectin-3 has an indirect effect on CD3ζ phosphorylation, possibly via the phosphatase CD45 or the kinase Lck bond to the co-receptor CD4, which are both glycosylated and which both control the phosphorylation levels of TCR (see chapter 1). Taken together, galectin-3 influences TCR distribution at the synapse. Further experiments are required to answer the pending questions in order to understand the precise function of galectin-3 at the synapse: what is the actual contribution of

112 galectin-3 to TCR endocytosis and recycling, which proteins are responsible for the increase in CD3ζ phosphorylation triggered by galectin-3?

Cell Cluster Molecule Cluster Condition Max L(r)-r type density density radius

T TCR clustering ± endogenous ↓↓ ↑↑↑ ≈ ↓↓↓↓ cells galectin-3-dep. gal-3

T pTCR clustering ± exogenous ↓↓ ↑↑↑ ↑↑↑ ≈ cells galectin-3-dep. gal-3 Table 5.1. Summary of TCR and pTCR clustering in the absence or presence of galectin-3. Treatment of activated T cells with or without endogenous or exogenous galectin-3 impacted on the clustering TCR and pTCR, respectively.

Galectin-3 positive vesicles locate to the immunological synapse indicating a function for galectin-3 in T cell activation events (Figure 5.4). CD82 and CD63 have previously been shown to locate to the immunological synapse during T cell activation525,536, and the finding that a subset of synaptic galectin-3 vesicles contained CD82 and CD63 molecules suggest a contribution of galectin-3 in the localisation of these proteins to the synapse (Figure 5.5). Galectin-3 may function in the translocation and delivery of these proteins to the immunological synapse, it may be involved in the recycling of these proteins, and galectin-3 positive vesicles may act as carriers of signalling proteins, if not of the TCR, to or from the synapse.

The observation that endocytosed galectin-3 co-localised with certain proteins of the T cell signalling machinery suggests a screening for other T cell signalling proteins that may become associated with galectin-3. If they can be isolated, the analysis of galectin-3 positive vesicles will allow access to their molecular content and reveal which proteins of the TCR signalling pathway they transport. This will also help to determine if galectin-3 vesicles can serve as signalling endosomes, as compartments for the recycling machinery, or if galectin-3 contributes to signal downregulation. Furthermore, a comparative analysis of galectin-3 vesicles between activated and

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resting T cells may reveal differential uptake patterns of galectin-3 depending on the activation state of the cell.

Further future experiments involve the formation of immunological synapses using antigen presenting cells (APCs) such as Raji B cells. Here Raji cells loaded with the superantigen staphylococcal enterotoxin E (SEE) can act as surrogate APCs. Immunological synapses generated ex vivo using cell-cell conjugates will provide more accurate physiological conditions in comparison to other experimental strategies of T cell activation such as antibody-coated coverslips or lipid bilayers. Advantages of activated interfaces created by two cells (compared to a cell on a flat surface) are the geometry and morphology of the cell. Furthermore, activation experiments using cell- cell conjugates will allow determining whether galectin-3 molecules at the immunological synapse are transferred from the T cell to the APC.

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Chapter 6

Conclusion and outlook

6.1 Conclusion

In this thesis I have addressed the fundamental question of how extracellular galectin-3 organises the plasma membrane and delineates membrane domains in mammalian cells. I asked whether and how these domains become internalised and investigated the endocytic properties of galectin-3 in resting and activated T lymphocytes. Additionally, I have established a connection between the membrane- organising ability of galectin-3, its endocytic properties and T cell signalling.

Galectin-3 is implicated in the formation of molecular lattices15,37,74,77–79,538 at the cell surface. Galectin-glycan lattices participate in the organisation of glycoprotein and glycolipid assemblies in the plasma membrane. The C-terminal CRD of galectin-3 is responsible for the binding to glycans298 and a plethora of galectin-3 binding partners was identified so far33–37,41,42. Binding to galectins was suggested to recruit additional galectin molecules and to induce multimerisation of galectins via the N-terminal domain70,78,539–541. Galectin-3 mutants that lack the N-terminal domain fail to multimerise126,540. Previous studies demonstrated that galectin-glycan lattices organise lipid rafts43,230–232,234 and regulate signalling thresholds at the cell surface71,132,133. The role of galectins is not restricted to the cell surface, and galectin-3 also functions in endocytic events295,297–299,303,454 and mediates the endocytosis of cell surface receptors187,297. Endocytosis of galectin-3 occurs via different pathways and several mechanisms of galectin-3 internalisation have been reported; these include cholesterol-enriched rafts297, clathrin-mediated298 and clathrin-independent

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endocytosis300. In some cell types it was shown that several different endocytic mechanisms can be used simultaneously for galectin-3 internalisation298,299 indicating the versatility of the mechanisms mediating its internalisation.

Plasma membrane organisation and endocytosis are essential to immune cell functions339,542. In T cell signalling a critical process is the formation of signalling microclusters in the plasma membrane339,542. Consequently, extracellular galectins have been implicated in signalling events in several immune cells and extracellular galectin-3 is implied in various immunmodulatory activities and inflammatory responses44,53,335,439. Galectin-1 and -3 have been shown to prime neutrophils by inducing an oxidative burst543 and galectin-3 activates naïve and primed neutrophils inducing inflammatory responses544. Regulation of T cell functions and signalling43,71,132,133 by galectin-3 was previously shown and its expression in T cells is induced upon T cell activation65,439. Evidence clearly point towards a role in plasma membrane organisation for galectin 3 in T cells. Lattice formation by galectin-3 was implied in restricting lateral diffusion of surface receptors in T lymphocytes, that impacts on the activation threshold71,131. Galectins also support the formation of clusters at the plasma membrane43,133.

The complex architecture of cellular membranes contains numerous proteins and lipids whose different functions require tight spatiotemporal control and coordination. Lateral segregation and partitioning of cellular components into various subdomains is necessary as a cellular organisational scheme. Such domains have been observed and visualised before, for instance in plasma membrane domains specialised for clathrin-mediated endocytosis504. Diverse groups of membrane proteins assume differential distributions and assemble in distinct domains at the cell surface. In a study examining membrane domains in yeast, the distribution of several membrane proteins has been mapped and reveals a non-homogenous distribution of these proteins displaying different domain formations from patches to networks545. Lipids have also been implicated to act as membrane organisers creating functional raft- domains155,188,198,206,235,546–548. Collectively, various studies investigating domain formation and molecular distributions at the cell surface suggest that the entire plasma membrane is a mosaic of microdomains463,549. Glycan-mediated interactions 116 and formation of molecular lattices at the cell surface represent an additional level of organisation to the plasma membrane. Bulky constituents of the glycocalyx can physically influence receptor organisation and activity at the plasma membrane550, and multivalent glycan-binding proteins of the lectin family are capable of cross-linking glycosylated cell surface molecules15,33,37,79,551,552.

In the present study, the investigation of extracellular galectin-3 at the cell surface of mammalian cells has established that binding of galectin-3 to the plasma membrane does not occur in a random fashion, confirming previous findings that galectin-3 clusters at the cell surface resembling a molecular lattice15,37,71,74,77–79,132. Galectin-3 clustering properties such as cell surface binding, cluster size, randomness of distribution, and cluster formation change when highly-branched elongated N- glycans were depleted or in the absence of GSLs. Similarly the presence of galectin-3 and changes of glycosylation patterns of galectin-3 ligands led to dramatic changes in cell surface distribution of membrane receptors. This observation demonstrates that information stored in carbohydrates of glycoproteins and glycolipids is indeed accessible and exploitable by galectin-3. Modifications in glycosylation patterns impacted on galectin-3 binding and distribution, and may ultimately affect downstream functions. The importance of glycosylation patterns in cellular events is exemplified by a study investigating the function of galectin-1 on T lymphocytes, which showed that galectin-1-induced apoptosis was abrogated in cells whose surface receptors were deficient of O-branched glycan structures that are generated by the core-2-β-1,6-N-acetylglucosaminyltransferase122. Hence, the differential binding of galectins, which is determined by the presence or absence of certain oligosaccharide structures on surface proteins, influences not only the architecture of the plasma membrane and the clustering of cell surface receptors but also leads to drastic alterations of cellular events.

In cells depleted of GSLs via PPMP treatment, uptake of galectin-3 is greatly reduced as shown in Chapter 4, Figure 4.6. Absence of GSLs negatively affects endocytosis, nevertheless, the binding of galectin-3 is increased. Similar results showing increased galectin-3 binding have been observed in Mgat5-depleted cells. These data suggest that binding sites for galectin-3 at the cell surface are plentiful despite the loss of 117

either a major binding partner due to depletion of GSLs, or the loss of Mgat5, an enzyme generating a preferred complex sugar for galectin-3 binding. Lectins are known to have affinities for a plethora of carbohydrates from simple to complex sugars. Although binding affinities of galectins are proportional to the content and branching of N-glycans and affinities increase with the number of N-acetyllactosamine units per N-glycan, galactose is essential for galectin binding and the most basic recognition unit for galectin is lactose12, a simple disaccharide consisting of galactose and glucose (see Chapter1, 1.1 Lectins and Galectins, page 3). Hence in the absence of complex sugars galectin-3 can bind to simple sugars at the cell surface and in the study at hand it was found that binding sites were more abundant. This finding suggests that the absence of bulky complex sugars is exposing more binding sites probably made of simple sugars and that makes binding to simple sugars more favourable when complex carbohydrate ligands are sparse.

An interesting experiment would be the evaluation of galectin-3 uptake in Mgat5 depleted cells, i.e. whether the absence of complex sugars– similar to GSL depleted cells - leads to a decrease in endocytosis. Equally it would be interesting to find out whether galectin-3 monomers actually assemble into oligomers on the surface of GSL and Mgat5-depleted cells and if there is oligomer formation, whether those oligomers consist of pentamers or rather a mixture of monomers as well as some galectin-3 assemblies below the pentameric state. Only from these results could one draw further conclusions regarding details of galectin-3 binding and the effect of binding sites on the functionality of galectin-3. Do complex sugar moieties facilitate properties in galectin-3 that simple sugars cannot? What is the effect of the sugar bearing molecule on galectin-3? Does the property of the sugar unit influence the ability of galectin-3 to oligomerise and to induce endocytosis? These and many other questions still remain to be answered.

Findings on galectin-3-dependent clustering of GSLs and CD44 made in this study have contributed to a recent publication by the Johannes lab in Paris187. In this study, the authors demonstrated that galectin-3, GSLs, and branched N-glycosylation are required for the clustering and uptake of CD44, as well as for the uptake of β1-integrin. Furthermore, CLIC (clathrin-independent carrier) formation and CD44 uptake, initiated 118 by galectin-3-induced membrane bending, was supressed by a un-glycosylated CD44 mutant, demonstrating that the information stored in carbohydrate attachments is crucial for the endocytosis mediated by galectin-3. Thus, galectin-3 oligomerisation acts as a priming factor for domains where cargo receptors and GSLs co-cluster and are subsequently endocytosed. Domain formation by galectin-3 induces mechanical stress on the plasma membrane and creates endocytic invaginations initiating the first step in CLIC endocytosis. This study exemplifies how galectin-3 can operate as a driving force in membrane domain formation and an inducer of endocytosis.

CD44 was previously demonstrated to be a cargo protein of CLIC mediated endocytosis553, however, galectin-3 must not be exclusively viewed as an inducer of CLIC endocytosis. Galectin-3 was shown to be endocytosed by a variety of pathways in T cells in the present study and previously in endothelial cells489. Considering the multitude of different ligands at the plasma membrane to which galectin-3 can bind, diverse endocytic pathways used by galectin-3 gives rise to the following hypothesis (Figure 6.1): galectin-3 binding and clustering of specific cell surface proteins primarily act as the driving force in generating membrane curvature, which will consequently lead to endocytosis. In this model, galectin-3 intervenes first by generating invaginated membrane domains that are ready for endocytosis. The endocytic phase is eventually determined by the clustered cargo receptor itself. The intracellular domain of the receptor recruits the molecular machinery to the site of endocytosis, thus determining the endocytic pathway. Thus, the glycocalyx is a key structural element that holds fundamental information for cellular homeostasis, impacting on cellular events such as signalling, endocytosis, survival and apoptosis.

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Figure 6.1. Model of galectin-3 driven membrane bending and subsequent endocytosis. Galectin-3 monomers are recruited to the plasma membrane and bind to N-glycosylated cell surface receptors and probably GSLs. Membrane bound galectin-3 forms pentamers via its N- terminal domain co-clustering more receptors and GSLs. Galectin-3 oligomerisation and clustering causes mechanical stress on the plasma membrane and induces membrane bending. The clustered cargo receptor determines the endocytic pathway by recruiting the endocytic machinery through its intracellular domain.

Galectin-3 is involved in setting signalling thresholds71,554 at the plasma membrane and is holding other functions important in immune cell regulation331–335. Its influence on TCR clustering during signalling events and its presence in vesicles at the immunological synapse underscore the relevance of galectin-3 in signalling of T cells. Thus it may be involved in the up- or downregulation of proteins that participate in the signalling cascade and may play a role in endosomal signalling. Further analyses are required to elucidate the precise function of galectin-3 vesicles and its contribution to signalling.

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6.2 Future outlook

Based on previous data and the findings described here, further studies addressing the role of complex sugars and glycosylation are necessary to elucidate the function of cabohydrates and carbohydrate-binding proteins. These future findings will contribute to the understanding of plasma membrane architecture and compartmentalization. Glycosylation is one of the most common, most complex and least understood modifications in cellular proteins. Alterations in glycosylation impact greatly on cellular functions and have been shown to be key characteristics in the progression of certain diseases including cancer.

Glycan structures display a great diversity and complexity and their interaction mode with cellular molecules appears to be finely tuned and small changes can have great impact on cellular proceedings. A comprehensive understanding of glycan structures and functions is sketchy and incomplete. Techniques of glycan profiling are still in their infancy and major developments and improvements are necessary. Nevertheless, several tools for glycomics are available and are being further improved. Glycan microarrays such as high-density lectin microarrays can be used to detect specific binding of glycoproteins to certain lectins and to verify protein glycosylation. A more profound knowledge of glycosylation patterns of proteins and lipids is necessary to understand events initiated at plasma membrane and will furthermore help to improve the diagnostics of diseases.

121

Figure 6.2. Different models of endosomal signalling. Binding of a ligand (green) to cell surface receptor (orange) leads to an intracellular signal (red), most commonly a phosphorylation event. (A) A signal generated at the plasma membrane can be either downregulated after being incorporated into an endosome or sustained in the endosome. Hence signal duration is determined by the composition of the endosome, making it a crucial element in the overall regulation of signalling. (B) In this scenario, signal initiation occurs in endosomes and not at the plasma membrane. (C) Changes in endosomal pH process the receptor by introducing conformational changes or cleavage. This results in a signalling competent receptor that is only found in the endosome. (D) Different subsets of signalling proteins are available in different endosomes and thus the signalling outcome (yellow versus blue) is modulated by the endosome.

Increasing amounts of evidence point at the importance of the complex interplay between signalling at the plasma membrane and the endosomal network. Signals propagated by endosomes can originate at different sites. They can be initiated at the plasma membrane and subsequently be internalized in order to be conveyed within the endosome en route to intracellular effector sites. Alternatively, signalling can occur independently of the plasma membrane and originate in the endosome itself (Figure 6.2). There are several unique physical characteristics in an endosomal membrane that are advantageous for signalling and different from the characteristics of the plasma membrane. Endosomal features include: 1) compartmentalisation between the plasma membrane and endosomes is accompanied by a change in lumenal pH that can facilitate recruitment of proteins and regulate enzymatic activity (Figure 6.2C); 2) a confined space within an endosome provides a compartment with clear boundaries and restricted membrane area so that cargo may be protected from cytosolic and plasma membrane-associated phosphatases; and 3) vesicular structures comprise a distinctly different lipid composition compared to other cellular membranes as well as 122 different key signalling components than those residing in the plasma membrane (Figure 6.2D). Endosomes provide temporal control of signalling complexes generated at the plasma membrane as well as the transduction of signals through differential sorting and trafficking of activated receptors. A comprehensive picture of signalling endosomes in general and specifically at the immunological synapse is lacking and many basic questions remain to be answered. What are the scaffolds, adaptors and tethering molecules involved in endosomal sorting and signalling at the immunological synapse? Which endosomal populations are involved in TCR signalling, what are they composed of, and how are they interacting with signalling molecules? In particular, the determination of the subcellular localization of signalling components and their interaction with various endosomal subpopulations would provide valuable insights into signalling networks.

Much of the latest advances in our understanding of signalling at the plasma membrane and the contribution of endosomes in cell signalling are due to high- and super-resolution imaging technologies. Ongoing development of super-resolution microscopy, especially a gain in temporal resolution, will provide crucial information about internal membrane fusions and fissions and hence will reveal details about the complex system of signalling networks regulated by the plasma membrane and endosomal compartments.

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