Secretion machinery at the cell plasma membrane

Abstract

Secretion is a fundamental cellular process involving the regulated release of intracellular products from cells. Physiological functions such as neurotransmission, or the release of hormones and digestive enzymes, are all governed by cell secretion. Three critical activities occur at the cell plasma membrane to ensure secretion. Membrane-bound secretory vesicles dock, fuse, and expel their contents to the outside via specialized and permanent plasma membrane structures, called porosomes or fusion pores. In recent years, significant progress has been made in our understanding of these three key cellular activities required for cell secretion. The molecular machinery and mechanism involving them is summarized in this article.

Figure 1. Porosomes at the plasma membrane in pancreatic acinar cells and the neuron

(A) AFM micrograph depicting ‘pits’ (yellow arrow) and ‘porosomes’ within (blue arrow), at the apical plasma membrane in a live pancreatic acinar cell. (B) To the right is a schematic drawing depicting porosomes at the cell plasma membrane (PM), where membrane-bound secretory vesicles called zymogen granules (ZG), dock and fuse to release intravesicular contents. (C) A high resolution AFM micrograph shows a single pit with four 100–180 nm porosomes within. (D) An electron micrograph depicting a porosome (red arrowhead) close to a microvilli (MV) at the apical plasma membrane (PM) of a pancreatic acinar cell. Note association of the porosome membrane (yellow arrowhead), and the zymogen granule membrane (ZGM) (red arrow head) of a docked ZG (inset). Cross section of a circular complex at the mouth of the porosome is seen (blue arrow head). (E) The bottom left panel shows an electron micrograph of a porosome (red arrowhead) at the nerve terminal, in association with a synaptic vesicle (SV) at the presynaptic membrane (Pre-SM). Notice a central plug at the neuronal porosome opening. (F) The bottom right panel is an AFM micrograph of a neuronal porosome in physiological buffer, also showing the central plug (red arrowhead) at its opening. It is believed that the central plug in neuronal porosomes may regulate its rapid close-open conformation during neurotransmitter release. The neuronal porosome is an order of magnitude smaller (10–15 nm) in comparison to porosome in the exocrine pancreas.

Figure 2. t-SNAREs and v-SNARE in opposing bilayers interact in a circular array resulting in the formation of conducting channels in presence of calcium

(A) Docked v-SNARE vesicles at t-SNARE-reconstituted lipid membrane. (B) Dislodgement of v-SNARE-reconstituted vesicles from the t-SNARE-reconstituted lipid membrane, exposes the pore-like (C,D) t-/v-SNARE ring complexes. (C,D) Three dimension AFM micrographs of SNARE-ring complexes, at low (C) and high resolution (D). A channel at the center of the t-/v-SNARE ring complex is clearly visible (Scale bar=100nm). In presence of calcium, recombinant t-SNAREs and v-SNARE in opposing bilayers drive membrane fusion. (E) When t-SNARE vesicles are exposed to v-SNARE reconstituted bilayers in the presence of calcium, vesicles fused. Similarly, vesicles containing nystatin/ergosterol and t-SNAREs when added to the cis compartment of a v-SNARE reconstituted bilayer, fusion of the t-SNARE containing vesicles with the v-SNARE membrane is demonstrated as current spikes due to conduction via the nystatin channels. To confirm membrane stability, the transmembrane gradient of KCl is increased to 3M, allowing gradient driven fusion of the nystatin-associated vesicles. (F–H) The size of the t-/v-SNARE complex is directly proportional to the size of the SNARE-reconstituted vesicles. (F) Schematic diagramdepicting the interaction of t-SNARE- and v-SNARE-reconstituted liposomes. (G) AFM images of vesicle before and after their removal using the AFM cantilever tip, exposing the t-/v-SNARE ring complex at the center. (H) Note the high correlation coefficient between vesicle diameter and size of the SNARE complex.