Module 6 - exo and endocytosis Flashcards
(36 cards)
Type 1 diabetes
Autoimmune, destroys beta cells via immune reaction of body
Type II diabetes effects
Obesity, genetics»_space; insulin resistance»_space; hyperinsulinemia»_space; composition - normal glucose tolerance»_space; impaired»_space; b-cell failure > type II diabetes
increased HGO
Loses second phase of glucose response
Complications of diabetes
macrovascular - brain (cerebrovascular disease), heart (CAD, MI, CS, heart failure), extremities (peripheral VD)
Treatment - type I
insulin injections
insulin pumps
not cures
Stimulus-secretion coupling in B-cell
Responses to glucose via GLUT2 transporter in membrane. Enters down concentration gradient and acts as a fuel inside. ATP production increased, decreased ADP. KAPT (K+ channel) closes. Another channel must bring in Na via Na or Na/K pump and cause depolarisation. Calcium influx results in exocytosis and release of insulin granules.
Fusion: docking and priming
Granule with SNARE proteins approaches celll membrane with SNAP25 and syntaxin-1. Granule approaches cell membrane and SNAREs interact»_space; docking. Priming occurs - calcium dependent. Coils bring two membranes together and waits for Synaptotagmin (calcium sensor) to change its conformation and pull them together»_space; fusion.
Secretion
1st phase - vesicles docked, ready to go any time, not that many
2nd phase - recruit granules from reserve pool and move to cell membrane, undergo docking, priming and fusion. A few hundred. Continuous if glucose present, almost indefinitely.
Db/db model
Rats with leptin receptor impaired/absence. Spontaneous mutation/deletion. Used to induce Type II Diabetes-symptoms.
No satiety - always hungry. Develops insulin resistance, inefficient beta cells. Loses insulin secretion.
Db/db loss of insulin secretion
- Loss of GLUT2 transporters, shown via histochemical staining. Loses about 80% capacity to transport glucose.
- Reduced ATP production due to reduced glucose uptake.
- Decreased insulin content
- Changes in calcium signalling - rising phase is lower in db/db compared to control (very rapid)
Major deficits: GLUT2 and exocytosis
Calcium signalling in db/db
Possibly due to sites of exocytosis and calcium signalling - loses association. Hugh calc signal in control, no differentiation of calcium. Spread out instead therefore not as much focused/concentrated exocytosis. Eventually calcium builds up and second phase recruits granules to cell membrane
Exocytosis is targeted
looking at islet cells - some cells don’t secrete insulin, even with high concs of glucose. Looking at 3D model shows clustering of exocytic events - not evenly distributed across cell. Closely associated with vasculature (blood stream)
ELKS
scaffolding protein, links calc channels with site selected exocytosis. also assoc w/ vasculature in b-cells.
Model for targeted secretion of insulin
Defect in diabetes - exocytosis defect, sites of calcium signalling and exocytosis are separate, decreased events. Perhaps machinery of granule docking and calcium channels is disrupted
Exocytic pathway
- Secretory proteins translated on ER-bound ribosomes
- Polypeptide chains inserted into ER lumen
- Correctly folded proteins move into cis-golgi, folded within ER
- Cisternal progression from cis to trans
- mature proteins move from golgi via secretory vesicles to cell surface
Constitutive vs. regulated
Constitutive: soluble proteins, growth factors, continuously secreted
Regulated: hormones, neurotransmitters, stimulated release/secretion
SNARE hypothesis
vesicular SNAREs guide vesicles to the right target membrane by docking with an appropriate target SNARE - forms SNARE complex. Success = vesicle fusion
SNAREs
syntaxin and SNAP-25 (t-snare, on membrane)
VAMP/synaptobrevin (v-snare, on vesicle)
SNARE core domains on each SNARE interacts to form complex - 4 helices bundle (SNAP has 2x)
How were SNAREs discovered?
Researchers looking at botulinum neurotoxin (extremely toxic, flacid paralysis and death. heavy chain and light chain) targets terminals of motor neurons
removes snare proteins
blocks exocytosis – NT release
Endocytotic pathway of BoNTs
Step 1. Binding to neurons
- Internalisation - endocytosed to terminal
- Translocation. Proton pumped into endosome induces a conformational chain which releases light chain into the cytosol
- Blockade - SNARE proteins removed, blocks NT
BoNT targets
BoNTA - causes most of neurotoxic effects. Mainly targets SNAP25 (so does C and E)
The rest: VAMP/synaptobrevin or syntaxin
Synaptotagmin 1
has N-terminal transmembrane domain that it inserts into vesicle.
2x cytoplasmic C2 domains - mediate Ca-dependent binding to -ve charged membranes.
Trigger for Ca-induced exocytosis
C2 domains insert into lipid bylayer membrane upon Ca binding
Also binds to core of SNARE complex at the same time
membrane interactions pulls both vesicle and plasma membrane together
Ca-activation of membrane fusion complex
- absence of Ca, weak interaction between synaptotagmin and t-SNARES in plasma membrane
- Ca2+ = rapid penetration of C2 domains into plasma membrane, increased affinity for binding to t-SNARES
- membranes drawn together, 4 helix bundle SNARE complex, fusion of both membrane
- fusion pore expansion»_space; complete fusion
Synaptotagmin - alternate theory
Instead of coiling motion
Promotes fusion via buckling of plasma membrane under synaptic vesicle. Brings membranes together and induces further curvature stress, increasing probability of membrane fusion
Types of endocytosis
Clathrin-dependent Caveolin-dependent Macropinocytic GEEC pathway Flotillin-dependent
