Matrix-Dependent Local Retention of Secretory Vesicle Cargo in Cortical Neurons

Abstract

Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent fusion events. Transient events represented full release followed by cargo diffusion or incomplete release followed by vesicle retrieval, as previously observed in neuroendocrine cells. Unexpectedly, we also observed that certain cargo, such as Semaphorin 3A (Sema3A), was delivered at the cell surface as stable deposits. Stable deposits and transient events were observed for single cargo and both were SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and calcium dependent. The ratio between stable and transient events did not depend on cargo size, subcellular localization (synaptic vs extrasynaptic secretion), or the presence of the extracellular matrix. Instead, the ratio is cargo specific and depends on an interaction with the vesicle matrix through a basic domain in the cargo protein. Inhibition of this interaction through deletion of the basic domain in Sema3A abolished stable deposits and rendered all events transient. Strikingly, cargo favoring transient release was stably deposited after corelease with cargo favoring stable deposit. These data argue against cargo diffusion after exocytosis as a general principle. Instead, the vesicle matrix retains secreted signals, probably for focal signaling at the cell surface.

Trafficking and Surface Expression of Hyperpolarization-activated Cyclic Nucleotide-gated Channels in Hippocampal Neurons

“…This might be a result of the more viscous properties of the membrane compared with the cytoplasm, of the action of anchoring complexes, or both. Such an activity-dependent relationship has been established for Semaphorin 3A (42,43) and for the ion channel TRPC5, which was FIGURE 7. Glutamate-dependent surface expression of HCN1 channels.…”

Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

“…Both the fulllength (ϳ95 kDa) and N terminus of processed Sema3A (ϳ65 kDa) could be observed in this extract. However, it is important to note that the Sema3A remains bound at physiological conditions (50 mM TBS) and is retained at the cell surfaces as described in several in vitro studies (25,35).…”

Neuromodulatory function of neuropeptides in the normal CNS

“…Nonetheless, cargo size apparently influences the ratio between complete and incomplete LGV release events (Perrais et al, 2004). For example, compared with the fast kinetics of neuropeptide Y (NPY) release, that of BDNF is slow (de Wit et al, 2009) offering the possibility for an additional way to modulate biological effects in vivo.…”

“…Release of LGVs occurs anywhere at terminal membrane, including (less frequently) the active zones (Buma, 1988;De Camilli and Jahn, 1990;Karhunen et al, 2001;Zhu et al, 1986). It has been recently calculated that a surprisingly large proportion of release events, about 50%, indeed occurs at extra-synaptic locations (de Wit et al, 2009). …”