Exocytosis Flashcards
Exocytosis - Introduction
- secretion of soluble content of vesicles or large pleomorphic carriers (LPC)
- delivery of specific proteins or lipids to the plasma membrane
- enlargement of the PM (growth, change of shape e.g. during phagocytosis)
- enlargement and sealing of PM during cytokinesis
- repair of PM
Exocytosis - types of compartments
- vesicles or large pleiomorphic carriers (LPC) derived from TGN
- vesicles derived from recycling Endosomes
- secretory lysosomes / MVB (Multivesicular body)
- conventional lysosomes
Temporal control of exocytosis
Constitutive exocytosis - regulated exocytosis
- constitutive exo. observed only for TGN/endosome derived material - regulated exo. observed for all different membrane sources
- regulated exocytosis
- prominent in several secretory cells (Mast cells, chromaffige cells, neural synapses)
- basic fusion cycle is similar to ordinary constitutive fusion events
- always triggered by local increase of Ca2+
-regulated exocytosis may start already 100 µs after the Ca2+ signal -> too fast to allow a complete docking and fusion reaction
-> between Docking and Fusion a new step, called “priming” must exist, were vesicles rest
-> additional factors are involved, that control passage from priming to fusion
Types of regulated secretion of TGN- and endosome - derived vesicles
- secretion of secretory granules which contain enzymes or hormones in glands
- secretion of neurotransmitters
- delivery of specific membrane proteins
- delivery of membrane patches
Secretory granules (dense core secretory vesicles)
One cell can harbor more than one type of DCSG -> specific sorting into the specific DCSG
Secretory granules - models for biogenesis
sorting during maturation by entry? or by retention?; direct entry regulation (cytoplasmic signals of membrane proteins) or indirect (luminal proteins binding membrane receptors); retention by aggregation of luminal proteins?
Sorting to DCGR -> Variety of signals
- cytoplasmic tail (in some membrane proteins)
- luminal signatures (membrane-associated alpha-helix; acidic cluster; disulfide constrained loop; paired basic AA)
- formation of aggregates with other DCGR proteins
The average synaptic vesicle - content
- about 40 nm and 16 MaD
- vesicle membrane about 70 % phospholipids and 25 % membrane helices
- but vesicle surface to about 60 % covered with protein domains
- about 80 different membrane proteins
- at least 13 different SNAREs (not only those needed for SV-PM fusion!)
- up to 33 different Rabs (= about 50 % of all Rab-species of mammals ?!)
- only v-ATPase is present in 1-2 copies per vesicle; all other proteins essential for fusion with PM or reload with neurotransmitters are present in 10-70 copies per SV
Models for synaptic vesicle recycling
- single fusion events last only several (approx. 6) ms -> too fast for Clathrin-dependent endocytosis of the fused membrane area
Hypothesis:
-> In addition to the classical exocytosis-endocytosis cycle with a complete collapse of the vesicle with its target membrane there is a “kiss and run” pathway without an intense membrane mixing
kiss and run (=CME independent)
CME-free versions “kiss and run” - “kiss and stay”
REUSE (“kiss and run”)
1) NT uptake
2) Docking
3) Priming
4) Fusion pore opening
5) Endocytosis
REUSE (“kiss and stay”)
1) NT uptake
2) Priming
3) Fusion pore opening
Calcium-triggered fusion - full cycle
- specific components are needed for tight temporal and spatial regulation: e.g. Munc13 (CATCHR-family), complexin, synaptotagmin
- synaptotagmin is the minimal component required for Ca2+-dependence of regulated exocytosis in neurons
Munc18a is Blocks unproductive side reactions stabilizing Syntaxin1 in a pre-bundle form. Munc13 addition alters the Munc18a effect allowing the formation of a partially assembled SNAR-bundle stabilized by Munc18a.
Priming: arrest of a partially zippered SNARE complex, (e.g. with bound Munc18, Munc13 and synaptotagmin) and binding of complexin
Calcium influx triggers binding of synaptotagmin to the SNARE complex and to the plasma membrane (involving PI(4,5)P2), associated with displacement of complexin and (possibly) Munc18a.
Triggering pulls the vesicle closer via synaptotagmin-mediated cross-linking, resulting in full SNARE assembly, associated with full opening of syntaxin and re-orientation of Munc13.
Involvement of chaperons in regulated exocytosis
- special HSPs (CSP with DnaJ domain) directly involved in fusion
- regulation of calcium channel (HSC70)
- stabilization of monomeric SNAREs (HSC70)
- recycling of Rab3a and GDI form the PM to new vesicles (HSC70 and HSP90)
Components involved in regulated secretion in different systems - examples
SYNAPTIC VESICLES
- present in neurons
- V-SNARE: VAMP1/Synaptobrevin
- t-SNAREs: Syntaxin 1A or 1B - SNAP25
- SM: Munc18a
- others: Munc13, Synaptotagmin(s)
LARGE DENSE CORE VESICLES
- present in beta-cells
- V-SNARE: VAMP2
- t-SNAREs: Syntaxin 1A, SNAP25
- SM: Munc18a
- others: Munc13, Synaptotagmin
GLUT4 - CONTAINING VESICLES
- present in Adipocyte/muscle cell
- V-SNARE: VAMP2
- t-SNAREs: Syntaxin4, SNAP23
- SM: Munc18c
- Others: Synip; tomosyn, CDP138
ZYMOGEN GRANULA
- present in acinar pancreas cell
- V-SNARE: VAMP8/endobrevin
- t-SNAREs: Syntaxin2, SNAP23
- SM: Munc18b
- Others: Slp4-a/granuphilin
Porosome - a supramolecular semi-stable docking platform at the PM for secretory vesicles?
Proposed structure may be composed of cytoskeletal components and t-SNAREs
Entry of BoNT and TeNT into neurons
1) Membrane binding
2) Internalization
3) L chain translocation
4) Intracellular activity (SNARE cleavage)
