Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor

Abstract

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

Single Point Mutation in Bin/Amphiphysin/Rvs (BAR) Sequence of Endophilin Impairs Dimerization, Membrane Shaping, and Src Homology 3 Domain-mediated Partnership

“…Immunoblotting-Immunoblotting was performed exactly as described previously (41). Peroxidase activity using either conjugated secondary antibodies or conjugated StrepTactin was detected by enhanced chemiluminescence using the ECL Plus detection kit (GE Healthcare).…”

Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

“…Studies of membrane recycling in hippocampal synapses using the fluorescent dye FM1-43 also suggest that synaptic vesicles formed by endocytosis can fuse with the plasma membrane directly, without passing through an endosomal compartment (4). In the PC12 neuroendocrine cell line, synaptic vesicles are also derived from two different pathways as follows: directly from the plasma membrane (5,6) as well as from an endosomal intermediate (7)(8)(9)(10). Together these data form an emerging model of multiple pathways to recycle both synaptic vesicle proteins and synaptic vesicle membrane.…”

“…This homogenate was then spun as before (P1) and the supernatants combined (S1). The S1 was spun at 40,000 rpm (66,0000 ϫ g av ) for 15 min in a Beckman (Fullerton, CA) TLA 100.4 rotor (P2) (5). The supernatant (S2) was then used for further analysis.…”

The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation

“…Vesicles were identified by the migration of the synaptic vesicle markers ZnT3, VAMP II, and synaptophysin. All of these markers comigrated in the center of glycerol gradients ( Figure 4A), indicating that synaptic vesicle antigens are targeted to homogeneously sized vesicles whose physical properties were similar to brain SV ( Figure 4A) (Clift-O'Grady et al, 1990;Schmidt et al, 1997). We characterized the SLMV isolated from glycerol gradients by electron microscopy ( Figure 4C).…”