Identification of the phospholipid lysobisphosphatidic acid in the protozoan Entamoeba histolytica: An active molecule in endocytosis

Phospholipids are essential for vesicle fusion and fission and both are fundamental events for Entamoeba histolytica phagocytosis. Our aim was to identify the lysobisphosphatidic acid (LBPA) in trophozoites and investigate its cellular fate during endocytosis. LBPA was detected by TLC in a 0.5 R_f spot of total lipids, which co-migrated with the LBPA standard. The 6C4 antibody, against LBPA recognized phospholipids extracted from this spot. Reverse phase LC-ESI-MS and MS/MS mass spectrometry revealed six LBPA species of m/z 772.58–802.68. LBPA was associated to pinosomes and phagosomes. Intriguingly, during pinocytosis, whole cell fluorescence quantification showed that LBPA dropped 84% after 15 min incubation with FITC-Dextran, and after 60 min, it increased at levels close to steady state conditions. Similarly, during erythrophagocytosis, after 15 min, LBPA also dropped in 36% and increased after 60 and 90 min. EhRab7A protein appeared in some vesicles with LBPA in steady state conditions, but after phagocytosis co-localization of both molecules increased and in late phases of erythrophagocytosis they were found in huge phagosomes or multivesicular bodies with many intraluminal vacuoles, and surrounding ingested erythrocytes and phagosomes. The 6C4 and anti-EhADH (EhADH is an ALIX family protein) antibodies and Lysotracker merged in about 50% of the vesicles in steady state conditions and throughout phagocytosis. LBPA and EhADH were also inside huge phagosomes. These results demonstrated that E. histolytica LBPA is associated to pinosomes and phagosomes during endocytosis and suggested differences of LBPA requirements during pinocytosis and phagocytosis.     

Lysosomal degradation of membrane lipids

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes.     

Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells

Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors.     

Unconventional Trafficking of Mammalian Phospholipase D3 to Lysosomes

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Sorting of MHC Class II Molecules into Exosomes through a Ubiquitin-Independent Pathway

Major histocompatibility complex class II (MHC-II) molecules accumulate in exocytic vesicles, called exosomes, which are secreted by antigen presenting cells. These vesicles are released following the fusion of multivesicular bodies (MVBs) with the plasma membrane. The molecular mechanisms regulating cargo selection remain to be fully characterized. As ubiquitination of the MHC-II β-chain cytoplasmic tail has recently been demonstrated in various cell types, we sought to determine if this post-translational modification is required for the incorporation of MHC-II molecules into exosomes. First, we stably transfected HeLa cells with a chimeric HLA-DR molecule in which the β-chain cytoplasmic tail is replaced by ubiquitin. Western blot analysis did not indicate preferential shedding of these chimeric molecules into exosomes. Next, we forced the ubiquitination of MHC-II in class II transactivator (CIITA)-expressing HeLa and HEK293 cells by transfecting the MARCH8 E3 ubiquitin ligase. Despite the almost complete downregulation of MHC-II from the plasma membrane, these molecules were not enriched in exosomes. Finally, site-directed mutagenesis of all cytoplasmic lysine residues on HLA-DR did not prevent inclusion into these vesicles. Taken together, these results demonstrate that ubiquitination of MHC-II is not a prerequisite for incorporation into exosomes.     

Exosomes: Extracellular organelles important in intercellular communication

In addition to intracellular organelles, eukaryotic cells also contain extracellular organelles that are released, or shed, into the microenvironment. These membranous extracellular organelles include exosomes, shedding microvesicles (SMVs) and apoptotic blebs (ABs), many of which exhibit pleiotropic biological functions. Because extracellular organelle terminology is often confounding, with many preparations reported in the literature being mixtures of extracellular vesicles, there is a growing need to clarify nomenclature and to improve purification strategies in order to discriminate the biochemical and functional activities of these moieties. Exosomes are formed by the inward budding of multivesicular bodies (MVBs) and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane (PM). In this review we focus on various strategies for purifying exosomes and discuss their biophysical and biochemical properties. An update on proteomic analysis of exosomes from various cell types and body fluids is provided and host-cell specific proteomic signatures are also discussed. Because the ectodomain of ~ 42% of exosomal integral membrane proteins are also found in the secretome, these vesicles provide a potential source of serum-based membrane protein biomarkers that are reflective of the host cell. ExoCarta, an exosomal protein and RNA database (http://exocarta.ludwig.edu.au), is described.     

Sorting out signals in fly endosomes

Ligands and receptors that mediate cell–cell interactions during development are removed from the cell surface by endocytosis. Subsequently, many of these internalized proteins are detected in multivesicular bodies (MVBs). Recent work in different organisms has elucidated some aspects of MVB biogenesis and trafficking. This review discusses some intriguing links between these findings, the sorting of proteins in endocytic trafficking, and the regulation of signaling pathways in Drosophila .