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BIOM3014 > Module 6 - exo and endocytosis > Flashcards

Module 6 - exo and endocytosis Flashcards

1
Q

Type 1 diabetes

A

Autoimmune, destroys beta cells via immune reaction of body

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2
Q

Type II diabetes effects

A

Obesity, genetics&raquo_space; insulin resistance&raquo_space; hyperinsulinemia&raquo_space; composition - normal glucose tolerance&raquo_space; impaired&raquo_space; b-cell failure > type II diabetes
increased HGO
Loses second phase of glucose response

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3
Q

Complications of diabetes

A

macrovascular - brain (cerebrovascular disease), heart (CAD, MI, CS, heart failure), extremities (peripheral VD)

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4
Q

Treatment - type I

A

insulin injections
insulin pumps
not cures

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5
Q

Stimulus-secretion coupling in B-cell

A

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.

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6
Q

Fusion: docking and priming

A

Granule with SNARE proteins approaches celll membrane with SNAP25 and syntaxin-1. Granule approaches cell membrane and SNAREs interact&raquo_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&raquo_space; fusion.

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7
Q

Secretion

A

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.

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8
Q

Db/db model

A

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.

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9
Q

Db/db loss of insulin secretion

A
  1. Loss of GLUT2 transporters, shown via histochemical staining. Loses about 80% capacity to transport glucose.
  2. Reduced ATP production due to reduced glucose uptake.
  3. Decreased insulin content
  4. Changes in calcium signalling - rising phase is lower in db/db compared to control (very rapid)

Major deficits: GLUT2 and exocytosis

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10
Q

Calcium signalling in db/db

A

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

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11
Q

Exocytosis is targeted

A

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)

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12
Q

ELKS

A

scaffolding protein, links calc channels with site selected exocytosis. also assoc w/ vasculature in b-cells.

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13
Q

Model for targeted secretion of insulin

A

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

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14
Q

Exocytic pathway

A
  1. Secretory proteins translated on ER-bound ribosomes
  2. Polypeptide chains inserted into ER lumen
  3. Correctly folded proteins move into cis-golgi, folded within ER
  4. Cisternal progression from cis to trans
  5. mature proteins move from golgi via secretory vesicles to cell surface
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15
Q

Constitutive vs. regulated

A

Constitutive: soluble proteins, growth factors, continuously secreted

Regulated: hormones, neurotransmitters, stimulated release/secretion

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16
Q

SNARE hypothesis

A

vesicular SNAREs guide vesicles to the right target membrane by docking with an appropriate target SNARE - forms SNARE complex. Success = vesicle fusion

17
Q

SNAREs

A

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)

18
Q

How were SNAREs discovered?

A

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

19
Q

Endocytotic pathway of BoNTs

A

Step 1. Binding to neurons

  1. Internalisation - endocytosed to terminal
  2. Translocation. Proton pumped into endosome induces a conformational chain which releases light chain into the cytosol
  3. Blockade - SNARE proteins removed, blocks NT
20
Q

BoNT targets

A

BoNTA - causes most of neurotoxic effects. Mainly targets SNAP25 (so does C and E)
The rest: VAMP/synaptobrevin or syntaxin

21
Q

Synaptotagmin 1

A

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

22
Q

Ca-activation of membrane fusion complex

A
  1. absence of Ca, weak interaction between synaptotagmin and t-SNARES in plasma membrane
  2. Ca2+ = rapid penetration of C2 domains into plasma membrane, increased affinity for binding to t-SNARES
  3. membranes drawn together, 4 helix bundle SNARE complex, fusion of both membrane
  4. fusion pore expansion&raquo_space; complete fusion
23
Q

Synaptotagmin - alternate theory

A

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

24
Q

Types of endocytosis

A 
Clathrin-dependent
Caveolin-dependent
Macropinocytic
GEEC pathway
Flotillin-dependent
25
Q

Endocytosis is for…

A

Nutrient uptake, migration, receptor signalling, receptor downregulation, neurotransmission, pathogen entry

Required in neurons to replenish vesicle pools&raquo_space; neurotransmission

26
Q

Types of endocytosis in neurons

A 
Kiss and run (clathrin-independent)
Full fusion (clathrin dependent)
Bulk retrieval (clathrin-independent)
27
Q

Kiss and run

A

neurotransmitter is released in little puffs, fusion pore-like structures open and release, and then shut

28
Q

Full fusion

A

clathrin mediated. vesicle fuses with membrane, releases, and gets recycled again

29
Q

Bulk endocytosis

A

large membranous structures in nerve terminals. vesicles pinch off and recycled to replenish vesicle pools. Can be measured via membrane capacitance. Only observed in high frequency stimulation

30
Q

Clathrin

A

triskelion, 3 heavy chains, 3 light chains

forms polyhedral lattice, surrounds vesicle, acts as scaffold for vesicle formation

31
Q

AP-2

A

Adaptor protein 2
a, B2, u2, o2 subunits
a targets membrane and target sites for amphipysin and AP180. B2 binds clathrin through clathrin box motif, u2 binds PI’s and tethers to membrane

32
Q

AP180

A

interacts w/ clathrin and AP2. also with PIP2. recruitment of it and clathrin to PIP2 domains on PM promotes clathrin assembly

33
Q

Clathrin-mediated endocytosis

A

AP180 acts on PM, acts as binding site for AP2. Recruits clathrin to form basic 3 protein complex. Endocytosis continues with invagination of membrane. Complex occurs over and over until clathrin cage is formed and covers vesicle fully. Ready to be pinched off. Dynammin is recruited to neck of membrane and performs final pinching step. After full internalisation, Hsc70 recruited to clathrin cage and disassembles it, releasing vesicle

34
Q

Dynamin

A

large GTPase
Three isoforms
hydrolysis (N terminal) causes coil to squeeze and membrane pinches off “pinchase”
Middle domain - dynamin assembly
PH domain - binding to phospholipids of PM
C terminal effecter domain - dynamin assembly
PRD - interacts with SH3 domains of amphiphysin, endophillin and actin-binding proteins

35
Q

Dynamin, flies and temperature

A

Drosophila shibire mutants
temp sensitive mutation of dynamin
normal at lower tem ~ 19 C
paralysed at higher temp ~ > 29
block in endocytosis-mediated vesicle recycling&raquo_space; vesicle depletion
Recruitment of dynamin occurs but mutation in GTPase domain prevents final clipping step. Vesicles are not released, therefore no replenishment and further rounds of NT = paralysis

36
Q

Dyngo4a - dynamin inhibitor

A

produces same results as mutation, blocks GTPase domain therefore pinching off
Does not affect rate of high frequency induced synaptic depletion
Inhibits recovery of NT release after depletion. Bulk endocytosis occurs but no pinching = stretched out membrane tubule

BIOM3014 (9 decks)

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