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Electrophysiological studies in some secretory and non-secretory cells have identified an extensive form of calcium-induced exocytosis that is rapid (hundreds of milliseconds), insensitive to tetanus toxin and distinct from regulated secretion. We have now identified a marker of the process, desmoyokin-AHNAK, in a clonal derivative of the neuronal cell line, PC12. In resting cells, desmoyokin-AHNAK is localized within the lumen of specific vesicles, but appears on the cell surface during stimulation. Desmoyokin-AHNAK-positive vesicles exist in a variety of cells and tissues and are distinct from the endoplasmic reticulum, Golgi, trans-Golgi, endosomes and lysosomes, and from Glut4 and constitutive secretion vesicles. They seem to be involved in two models of plasmalemma enlargement: differentiation and membrane repair. We therefore propose that these vesicles should be called 'enlargosomes'.

The triggering receptor expressed in myeloid (TREM) cells 2, referred to with the acronym TREM2, is a single-spanning membrane receptor of an immunoglobulin/lectin-like family. The members of this family operate through the interaction with a common coupling protein, DNAX-activation protein of 12 kDa (DAP12) which upon phosphorylation induces the activation of various intracellular signalling pathways (tyrosine kinases, phospholipase Cc). Activation of other Received December 20, 2008; revised manuscript received February 11, 2009; accepted April 21, 2009. Abbreviations used: BSA, bovine serum albumin; DAP12, DNAXactivation protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hHsp60, human heat shock protein 60; HRP, horseradish peroxidase; Hsp60, heat shock protein 60; PBS, phosphate-buffered saline; PLOSL, polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy; rHsp60, rat heat shock protein 60; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; siRNA, short interfering RNA; TREM, triggering receptor expressed in myeloid cells. AbstractTriggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu-Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2-binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2-dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down-regulation, the Hsp60-induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding (K d = 3.8 lM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain. Keywords: receptor binding, surface-harboured agonist, western and far-western blotting, phagocytosis, polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy.

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