(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins II: Intercellular Transfer of Matter (Inheritance?) That Matters.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of the plasma membrane (PM) bilayer by covalent linkage to a typical glycolipid and expressed in all eukaryotic organisms so far studied. Lipolytic release from PMs into extracellular compartments and intercellular transfer are regarded as the main (patho)physiological roles exerted by GPI-APs. The intercellular transfer of GPI-APs relies on the complete GPI anchor and is mediated by extracellular vesicles such as microvesicles and exosomes and lipid-free homo- or heteromeric aggregates, and lipoprotein-like particles such as prostasomes and surfactant-like particles, or lipid-containing micelle-like complexes. In mammalian organisms, non-vesicular transfer is controlled by the distance between donor and acceptor cells/tissues; intrinsic conditions such as age, metabolic state, and stress; extrinsic factors such as GPI-binding proteins; hormones such as insulin; and drugs such as anti-diabetic sulfonylureas. It proceeds either "directly" upon close neighborhood or contact of donor and acceptor cells or "indirectly" as a consequence of the induced lipolytic release of GPI-APs from PMs. Those displace from the serum GPI-binding proteins GPI-APs, which have retained the complete anchor, and become assembled in aggregates or micelle-like complexes. Importantly, intercellular transfer of GPI-APs has been shown to induce specific phenotypes such as stimulation of lipid and glycogen synthesis, in cultured human adipocytes, blood cells, and induced pluripotent stem cells. As a consequence, intercellular transfer of GPI-APs should be regarded as non-genetic inheritance of (acquired) features between somatic cells which is based on the biogenesis and transmission of matter such as GPI-APs and "membrane landscapes", rather than the replication and transmission of information such as DNA. Its operation in mammalian organisms remains to be clarified.

(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins I: Localization at Plasma Membranes and Extracellular Compartments.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript).

Transfer of Proteins from Cultured Human Adipose to Blood Cells and Induction of Anabolic Phenotype Are Controlled by Serum, Insulin and Sulfonylurea Drugs.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are anchored at the outer leaflet of eukaryotic plasma membranes (PMs) only by carboxy-terminal covalently coupled GPI. GPI-APs are known to be released from the surface of donor cells in response to insulin and antidiabetic sulfonylureas (SUs) by lipolytic cleavage of the GPI or upon metabolic derangement as full-length GPI-APs with the complete GPI attached. Full-length GPI-APs become removed from extracellular compartments by binding to serum proteins, such as GPI-specific phospholipase D (GPLD1), or insertion into the PMs of acceptor cells. Here, the interplay between the lipolytic release and intercellular transfer of GPI-APs and its potential functional impact was studied using transwell co-culture with human adipocytes as insulin-/SU-responsive donor cells and GPI-deficient erythroleukemia as acceptor cells (ELCs). Measurement of the transfer as the expression of full-length GPI-APs at the ELC PMs by their microfluidic chip-based sensing with GPI-binding α-toxin and GPI-APs antibodies and of the ELC anabolic state as glycogen synthesis upon incubation with insulin, SUs and serum yielded the following results: (i) Loss of GPI-APs from the PM upon termination of their transfer and decline of glycogen synthesis in ELCs, as well as prolongation of the PM expression of transferred GPI-APs upon inhibition of their endocytosis and upregulated glycogen synthesis follow similar time courses. (ii) Insulin and SUs inhibit both GPI-AP transfer and glycogen synthesis upregulation in a concentration-dependent fashion, with the efficacies of the SUs increasing with their blood glucose-lowering activity. (iii) Serum from rats eliminates insulin- and SU-inhibition of both GPI-APs' transfer and glycogen synthesis in a volume-dependent fashion, with the potency increasing with their metabolic derangement. (iv) In rat serum, full-length GPI-APs bind to proteins, among them (inhibited) GPLD1, with the efficacy increasing with the metabolic derangement. (v) GPI-APs are displaced from serum proteins by synthetic phosphoinositolglycans and then transferred to ELCs with accompanying stimulation of glycogen synthesis, each with efficacies increasing with their structural similarity to the GPI glycan core. Thus, both insulin and SUs either block or foster transfer when serum proteins are depleted of or loaded with full-length GPI-APs, respectively, i.e., in the normal or metabolically deranged state. The transfer of the anabolic state from somatic to blood cells over long distance and its "indirect" complex control by insulin, SUs and serum proteins support the (patho)physiological relevance of the intercellular transfer of GPI-APs.

Biological Role of the Intercellular Transfer of Glycosylphosphatidylinositol-Anchored Proteins: Stimulation of Lipid and Glycogen Synthesis.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs), which are anchored at the outer leaflet of plasma membranes (PM) only by a carboxy-terminal GPI glycolipid, are known to fulfill multiple enzymic and receptor functions at the cell surface. Previous studies revealed that full-length GPI-APs with the complete GPI anchor attached can be released from and inserted into PMs in vitro. Moreover, full-length GPI-APs were recovered from serum, dependent on the age and metabolic state of rats and humans. Here, the possibility of intercellular control of metabolism by the intercellular transfer of GPI-APs was studied. Mutant K562 erythroleukemia (EL) cells, mannosamine-treated human adipocytes and methyl-ß-cyclodextrin-treated rat adipocytes as acceptor cells for GPI-APs, based on their impaired PM expression of GPI-APs, were incubated with full-length GPI-APs, prepared from rat adipocytes and embedded in micelle-like complexes, or with EL cells and human adipocytes with normal expression of GPI-APs as donor cells in transwell co-cultures. Increases in the amounts of full-length GPI-APs at the PM of acceptor cells as a measure of their transfer was assayed by chip-based sensing. Both experimental setups supported both the transfer and upregulation of glycogen (EL cells) and lipid (adipocytes) synthesis. These were all diminished by serum, serum GPI-specific phospholipase D, albumin, active bacterial PI-specific phospholipase C or depletion of total GPI-APs from the culture medium. Serum inhibition of both transfer and glycogen/lipid synthesis was counteracted by synthetic phosphoinositolglycans (PIGs), which closely resemble the structure of the GPI glycan core and caused dissociation of GPI-APs from serum proteins. Finally, large, heavily lipid-loaded donor and small, slightly lipid-loaded acceptor adipocytes were most effective in stimulating transfer and lipid synthesis. In conclusion, full-length GPI-APs can be transferred between adipocytes or between blood cells as well as between these cell types. Transfer and the resulting stimulation of lipid and glycogen synthesis, respectively, are downregulated by serum proteins and upregulated by PIGs. These findings argue for the (patho)physiological relevance of the intercellular transfer of GPI-APs in general and its role in the paracrine vs. endocrine (dys)regulation of metabolism, in particular. Moreover, they raise the possibility of the use of full-length GPI-APs as therapeutics for metabolic diseases.

A targeted bioinformatics approach identifies highly variable cell surface proteins that are unique to Glomeromycotina.

Diversity in arbuscular mycorrhizal fungi (AMF) contributes to biodiversity and resilience in natural environments and healthy agricultural systems. Functional complementarity exists among species of AMF in symbiosis with their plant hosts, but the molecular basis of this is not known. We hypothesise this is in part due to the difficulties that current sequence assembly methodologies have assembling sequences for intrinsically disordered proteins (IDPs) due to their low sequence complexity. IDPs are potential candidates for functional complementarity because they often exist as extended (non-globular) proteins providing additional amino acids for molecular interactions. Rhizophagus irregularis arabinogalactan-protein-like proteins (AGLs) are small secreted IDPs with no known orthologues in AMF or other fungi. We developed a targeted bioinformatics approach to identify highly variable AGLs/IDPs in RNA-sequence datasets. The approach includes a modified multiple k-mer assembly approach (Oases) to identify candidate sequences, followed by targeted sequence capture and assembly (mirabait-mira). All AMF species analysed, including the ancestral family Paraglomeraceae, have small families of proteins rich in disorder promoting amino acids such as proline and glycine, or glycine and asparagine. Glycine- and asparagine-rich proteins also were found in Geosiphon pyriformis (an obligate symbiont of a cyanobacterium), from the same subphylum (Glomeromycotina) as AMF. The sequence diversity of AGLs likely translates to functional diversity, based on predicted physical properties of tandem repeats (elastic, amyloid, or interchangeable) and their broad pI ranges. We envisage that AGLs/IDPs could contribute to functional complementarity in AMF through processes such as self-recognition, retention of nutrients, soil stability, and water movement.

Chip-Based Sensing of the Intercellular Transfer of Cell Surface Proteins: Regulation by the Metabolic State.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are anchored at the surface of mammalian blood and tissue cells through a carboxy-terminal GPI glycolipid. Eventually, they are released into incubation medium in vitro and blood in vivo and subsequently inserted into neighboring cells, potentially leading to inappropriate surface expression or lysis. To obtain first insight into the potential (patho)physiological relevance of intercellular GPI-AP transfer and its biochemical characterization, a cell-free chip- and microfluidic channel-based sensing system was introduced. For this, rat or human adipocyte or erythrocyte plasma membranes (PM) were covalently captured by the TiO2 chip surface operating as the acceptor PM. To measure transfer between PM, donor erythrocyte or adipocyte PM were injected into the channels of a flow chamber, incubated, and washed out, and the type and amount of proteins which had been transferred to acceptor PM evaluated with specific antibodies. Antibody binding was detected as phase shift of horizontal surface acoustic waves propagating over the chip surface. Time- and temperature-dependent transfer, which did not rely on fusion of donor and acceptor PM, was detected for GPI-APs, but not typical transmembrane proteins. Transfer of GPI-APs was found to be prevented by α-toxin, which binds to the glycan core of GPI anchors, and serum proteins in concentration-dependent fashion. Blockade of transfer, which was restored by synthetic phosphoinositolglycans mimicking the glycan core of GPI anchors, led to accumulation in the chip channels of full-length GPI-APs in association with phospholipids and cholesterol in non-membrane structures. Strikingly, efficacy of transfer between adipocytes and erythrocytes was determined by the metabolic state (genotype and feeding state) of the rats, which were used as source for the PM and sera, with upregulation in obese and diabetic rats and counterbalance by serum proteins. The novel chip-based sensing system for GPI-AP transfer may be useful for the prediction and stratification of metabolic diseases as well as elucidation of the putative role of intercellular transfer of cell surface proteins, such as GPI-APs, in (patho)physiological mechanisms.

Interaction of Full-Length Glycosylphosphatidylinositol-Anchored Proteins with Serum Proteins and Their Translocation to Cells In Vitro Depend on the (Pre-)Diabetic State in Rats and Humans.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), which are anchored at the surface of mammalian cultured and tissue cells through a carboxy-terminal GPI glycolipid, are susceptible to release into incubation medium and (rat and human) blood, respectively, in response to metabolic stress and ageing. Those GPI-APs with the complete GPI still attached form micelle-like complexes together with (lyso)phospholipids and cholesterol and are prone to degradation by serum GPI-specific phospholipase D (GPLD1), as well as translocation to the surface of acceptor cells in vitro. In this study, the interaction of GPI-APs with GPLD1 or other serum proteins derived from metabolically deranged rat and humans and their translocation were measured by microfluidic chip- and surface acoustic wave-based sensing of micelle-like complexes reconstituted with model GPI-APs. The effect of GPI-AP translocation on the integrity of the acceptor cell surface was studied as lactate dehydrogenase release. For both rats and humans, the dependence of serum GPLD1 activity on the hyperglycemic/hyperinsulinemic state was found to be primarily based on upregulation of the interaction of GPLD1 with micelle-like GPI-AP complexes, rather than on its amount. In addition to GPLD1, other serum proteins were found to interact with the GPI phosphoinositolglycan of full-length GPI-APs. Upon incubation of rat adipocytes with full-length GPI-APs, their translocation from the micelle-like complexes (and also with lower efficacy from reconstituted high-density lipoproteins and liposomes) to acceptor cells was observed, accompanied by upregulation of their lysis. Both GPI-AP translocation and adipocyte lysis became reduced in the presence of serum proteins, including (inhibited) GPLD1. The reduction was higher with serum from hyperglycemic/hyperinsulinemic rats and diabetic humans compared to healthy ones. These findings suggest that the deleterious effects of full-length GPI-APs following spontaneous release into the circulation of metabolically deranged rats and humans are counterbalanced by upregulated interaction of their GPI anchor with GPLD1 and other serum proteins. Thereby, translocation of GPI-APs to blood and tissue cells and their lysis are prevented. The identification of GPI-APs and serum proteins interacting within micelle-like complexes may facilitate the prediction and stratification of diseases that are associated with impaired cell-surface anchorage of GPI-APs, such as obesity and diabetes.

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway.

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

Mechanisms of Carrier Formation during Clathrin-Independent Endocytosis.

Clathrin-independent endocytosis (CIE) mediates the cellular uptake of many extracellular ligands, receptors, and pathogens, including several life-threatening bacterial toxins and viruses. So far, our understanding of CIE carrier formation has lagged behind that of clathrin-coated vesicles. Impediments have been the imprecise definition of some CIE pathways, the lack of specific cargoes being transported and of exclusive cytosolic markers and regulators. Notwithstanding these limitations, three distinct molecular mechanisms by which CIE carriers form can be defined. Cargo capture by cytosolic proteins is the main mechanism used by fast endophilin-mediated endocytosis (FEME) and interleukin 2 receptor (IL-2R) endocytosis. Acute signaling-induced membrane remodeling drives macropinocytosis. Finally, extracellular lipid or cargo clustering by the glycolipid-lectin (GL-Lect) hypothesis mediates the uptake of Shiga and cholera toxins and receptors by the CLIC/GEEC pathway. Here, we review these mechanisms and highlight current gaps in knowledge that will need to be addressed to complete our understanding of CIE.

A dual mechanism of cellulose deficiency in shv3svl1.

SHAVEN3 (SHV3) and its homolog SHAVEN3-like 1 (SVL1) encode glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) that are involved in cellulose biosynthesis and hypocotyl elongation in Arabidopsis thaliana. In a recent report, we showed that the cellulose and hypocotyl elongation defects of the shv3svl1 double mutant are greatly enhanced by exogenous sucrose in the growth medium. Further investigation of this phenomenon showed that shv3svl1 exhibits a hyperpolarized plasma membrane (PM) proton gradient that is coupled with enhanced accumulation of sucrose via the PM sucrose/proton symporter SUC1. The resulting high intracellular sucrose concentration appears to favor starch synthesis at the expense of cellulose synthesis. Here, we describe our interpretation of these results in terms of 2 potential regulators of cellulose synthesis: intracellular sucrose concentration and a putative signaling pathway that involves SHV3-like proteins.