Sept 24 - Exocytosis and Secretion Flashcards
Constitutive secretion
Secretory pathway is required in all cells (for instance, albumin, ECM proteins, collagen)
Regulated secretion
Found in specialized cell types and used on demand (neurotransmitter)
Regulated secretion is in response to a stimuli
Synaptic vesicles vs Large dense-core vesicles
Large dense core vesicles are bigger (20 nm vs 50 nm), secrete slower (milliseconds), secrete peptides, affect cells in surrounding area, duration of effect is longer lived, high frequency stimulation, require more Ca to secrete, and lack local vesicle recycling.
George Palade + Claude + de Duve
Combined electron microscopy and differential centrifugation to make fundamental discoveries about structural and functional organization of the cell
Axioms of the regulated secretory pathway
- Regulated secretory material must be concentrated in the correct compartment.
- Processing enzymes must be included in ER, golgi, AND mature secretory vesicle (why the last one?)
- Cell must recognize shared targeting signals. ??
How is insulin processed?
Pro-insulin in the ER membrane.
Once packaged it is cleaved and generates secretory insulin.
Proteolytic processing usually occurs within the secretory vesicle.
Where does proteolytic processing usually occur?
within the secretory vesicle
What are 2 models for how things get sorted for exocytosis?
- Sorting at entry
- Sorting by retention
In entry-based models, sorting occurs in the TGN either by the intrinsic ability of regulated pathway proteins to aggregate or by the presence of a sorting signal on the prohormones themselves or by one or more TGN sorting receptors. By contrast, in the retention model, sorting is largely a post-TGN event, occurring in the clathrin- and AP-1-coated ISGs that support the proteolytic maturation of prohormone molecules. This model relies on the ability of granule proteins to condense into a core, leaving nongranule proteins in the lumenal periphery where they are removed from the maturing granule apparently by clathrin-based coats.
In reality, both mechanisms probably contribute.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1424219/
Saponin, digitonin
Detergent use to permeabilize cells
Staph alpha toxin
Streptolysin O
Toxins used to permeabilize cells
Effect of priming in chromaffin cells?
If you have magnesium, calcium does not make any difference between primed and unprimed vesicles.
If you take away ATP and Mg, then primed vesicles release much faster.
2 sequential steps to secretion?
- Priming
2. Triggering release of content
What’s one way to measure secretion?
Measure by capacitance using caged Ca2+. (NP-EGTA complexed with Ca2+) Basically you illuminate it, dissociate the complex, and release calcium inside the cell. You have it voltage clamped. Capacitance increases when calcium is released. (Maybe bc membrane is getting bigger?)
Using this we see that secretion occurs in 2 phases - a first fast phase and a slower phase.
How can you study fusion using TIRFM / Evanescent wave microscopy ?
Excite molecules close to the plasma membrne; when the fluorophore labeled molecule gets close, observe an increase in the fluorescence intensity as the vesicle approaches the glass. Then if the vesicle fuses there is diffusion along the plasma membrane and intensity gets diffused along the membrane.
2 phases of secretion?
Population 1 fuses - rapid release
Population 2 fuses (new arrivals) - slow component - older, more distant from cell surface, replenishes depleted secretory vesicles
Which granules fuse first?
The population close to the plasma membrane - the readily releasable pool
Kiss and run fusion?
The vesicles fuse, release a little, fuse, release a little
What is pHluorin?
Mutagenized GFP that is sensitive to different pHs. When phluorin is inside granules, you see no fluorescence. When you fuse with the membrane you see a signal. So you can use it to measure kiss and run dynamics.
Which vesicles are secreted first?
Newly synthesized vesicles are secreted first. They used a time sensor GFP to test this - over time as the protein matures, it emits yellow, then red.
Botulinum toxin
Cleaves SNAP-25
What prevents spontaneous membrane exocytosis?
A long list of proteins, SNARE regulators
Munc-13-1
Soluble protein that binds snares
Priming
SNARE is zippered almost all the way. But calcium hasn’t been added.
Docking
Vesicle is nearby
Synaptotagmin
Has calcium binding/sensing domain. In absence of calcium, it binds SNARE proteins. Binds both syntaxin and SNAP-25 in a calcium dependent manner (presumably causing fusion)
Syntaxin
In the membrane
VAMP
On the vesicle
Complexin I and II
Highly enriched in neurons, interact selectively with ternary SNARE complex but not with individual SNAREs.
Complexin and synaptotagmin act as a switch to control fast synaptic vesicle exocytosis
FM1-43
Fluorescent dye that only stains the lipids of the plasma membrane. When vesicle fuses, all the lipids get exposed.
When endosome forms in the presence of this dye, its internal leaflet contains dye molecules, trapped inside. If extracellular dye is washed away, labeled endosomes remain as the fluroescence signal. If the endosome undergoes exocytosis, dye is spilled and fluorescence disappears.
Unconventional regulated protein secretion
This is another way protein secretion occurs - only a few specialized cells and a few proteins do this. IL-1-beta does it this way. Multivesicular bodies.
