Jan-Willem Slot

Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation.

Exosomes are 60–100-nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane. Here we determined the protein and lipid compositions of highly purified human B cell-derived exosomes. Mass spectrometric analysis indicated the abundant presence of major histocompatibility complex (MHC) class I and class II, heat shock cognate 70, heat shock protein 90, integrin α4, CD45, moesin, tubulin (α and β), actin, Giα2, and a multitude of other proteins. An α4-integrin may direct B cell-derived exosomes to follicular dendritic cells, which were described previously as potential target cells. Clathrin, heat shock cognate 70, and heat shock protein 90 may be involved in protein sorting at multivesicular bodies. Exosomes were also enriched in cholesterol, sphingomyelin, and ganglioside GM3, lipids that are typically enriched in detergent-resistant membranes. Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-containing buffers. Multivesicular body-linked MHC class II was also resistant to CHAPS whereas plasma membrane-associated MHC class II was solubilized readily. Together, these data suggest that recruitment of membrane proteins from the limiting membranes into the internal vesicles of multivesicular bodies may involve their incorporation into tetraspanin-containing detergent-resistant membrane domains.

Recycling Compartments and the Internal Vesicles of Multivesicular Bodies Harbor Most of the Cholesterol Found in the Endocytic Pathway

We employed our recently developed immuno‐electron microscopic method (W. Möbius, Y. Ohno‐Iwashita, E. G. van Donselaar, V. M. Oorschot, Y. Shimada, T. Fujimoto, H. F. Heijnen, H. J. Geuze and J. W. Slot, J Histochem Cytochem 2002; 50:u200343–55) to analyze the distribution of cholesterol in the endocytic pathway of human B lymphocytes. We could distinguish 6 categories of endocytic compartments on the basis of morphology, BSA gold uptake kinetics and organelle marker analysis. Of all cholesterol detected in the endocytic pathway, we found 20% in the recycling tubulo‐vesicles and 63% present in two types of multivesicular bodies. In the multivesicular bodies, most of the cholesterol was contained in the internal membrane vesicles, the precursors of exosomes secreted by B cells. Cholesterol was almost absent from lysosomes, that contained the bulk of the lipid bis(monoacylglycero)phosphate, also termed lysobisphosphatidic acid. Thus, cholesterol displays a highly differential distribution in the various membrane domains of the endocytic pathway.

Concentration of rafts in platelet filopodia correlates with recruitment of c‐Src and CD63 to these domains

Summary.u2002 The molecular mechanism that causes non‐adhesive, discoid platelets to transform into sticky dendritic bodies that form blood clumps is a complex series of events. Recently it has become clear that lipid microdomains—also known as rafts—play a crucial role in this process. We have used a non‐cytolytic derivative of perfringolysin‐O, a cholesterol binding cytolysin, that binds selectively to cholesterol‐rich membrane domains, combined with confocal‐ and immunoelectron microscopy to visualize cholesterol‐raft dynamics during platelet adhesion. In resting platelets cholesterol was uniformly distributed on the cell surface and confined to distinct intracellular compartments (i.e. multivesicular bodies, dense granules, and the internal membranes of α‐granules). Upon interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells. Stimulation with thrombin receptor activating peptide (TRAP) resulted in a similar redistribution of cholesterol towards filopodia. The adhesion‐dependent raft aggregation was accompanied by concentration of the tyrosine kinase c‐Src and the tetraspanin CD63 in these domains, whereas glycoprotein Ib (GPIb) was not selectively targeted to the raft clusters. c‐Src, the tetraspanin CD63, and GPIb were recovered in biochemically isolated low‐density membrane fractions. Disruption of rafts by depleting membrane cholesterol had no effect on platelet shape change but inhibited platelet spreading on fibrinogen and TRAP‐induced aggregation. Our results demonstrate that cholesterol rafts in platelets are dynamic entities in the membrane that co‐cluster with the tyrosine kinase c‐Src and the costimulatory molecule CD63 in specialized domains at the cell surface, thereby providing a possible mechanism in functioning as signaling centres.

[26] Immunocytochemical localization of platelet granule proteins

Publisher Summary This chapter emphasizes the potential of post-embedding techniques for the immunocytochemistry of platelet-granule proteins and discusses the problems encountered with their uses. Immunocytochemical techniques can be classified according to the accessibility of an antigen. Pre-embedding techniques are most suitable for the antigens that are directly accessible to antibodies. These techniques can also be used for the localization of intracellular antigens after the cellular membranes have been made permeable for antibodies by chemical or mechanical treatment. In post-embedding techniques, the immunoreaction is performed on the surface of ultrathin sections of an embedded tissue. An important advance in recent years has been the introduction of ultracryomicrotomy in which ultrathin cryosections are immunolabeled, which can be embedded after the immunoreaction to protect the structures against shrinking while drying. Most of the data on the immunocytochemical localization of platelet-granule proteins have been obtained with ultrathin cryosections.

Kiyoteru Tokuyasu: a pioneer of cryo‐ultramicrotomy

In July 2015 Professor K.T. Tokuyasu passed away in San Diego giving us the opportunity to reflect on the contribution this electron microscopist made to the field of immunocytochemistry. His work provided a sensitive, minimally invasive approach to producing thin sections of biological material for labeling with antibodies. His approach has been applied to a wide range of biological applications and provided important information on cellular processes.