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Flashcards in krueger 3 Deck (53)
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1

transmitter exocytosis needs? (2)

priming

calcium dependent fusion of SV

2

what happens during priming (exocytosis)

- requires?

- main purpose

- SV membrane and plasma membrane are brought close in proximity through interactions of SV membrane proteins and plasma membrane proteins

- required 3-4 SNARE proteins
- NSF and SNAP involved (regulate SNARE)

- main purpose: organize SNARE proteins into correct conformation for membrane fusion

3

Ca dependent fusion of SV

- cooperative Ca binding to the Ca sensory leads to the fusion of closely apposed membranes

4

what is involved to bring SV and plasma membrane close in proximity

- membrane fusion involves SNARE proteins
- 3.4 SNARE, on 2 fusing membranes form SNARE complex

5

what is a SNARE protein

SNAP receptors

- do not bind Ca

6

what is a SNARE complex, how is it assembled?

assembly of SNARE motifs into parallel 4-helical bundle

- assembled by formation of helical bundle of
one SNARE motif of each synaptobrevin and syntaxin
and 2 SNARE motifs of SNAP-25

7

SV exocytosis is mediated by... (3 proteins)

1) SNAREs synataxin (plasma membrane)
2) SNAP-25 (plasma membrane)
3) Synaptobrevin (SV membrane)

8

what inhibits neurotransmitter release by cleaving SNARE proteins?

Clostridial neurotoxins
- responsible for tetanus and botulism (proteases), block neurotransmitter release by cleaving SNAREs

- this inhibition of neurotransmitter release shows the SNARE complex formation is required for synaptic vesicles

9

potential role of the SNARE complex in membrane fusion

- free energy gained during formation of SNARE complex may be utilized for energetically unfavourable membrane fusion

- SNARE complex may be "minimal machinery" sufficient for membrane fusion

- SNARE complex, is not sufficient for kinetically efficient membrane fusion

10

roles for accessory proteins in aiding SNARE complex in fusion (2)

1) keep in position favourable for membrane fusion

2) structurally organize several SNARE complexes that have to act cooperatively

11

Munc18

- 2 functions

- essential for neurotransmitter release
- genetic ablation causes inhibition of both Ca-dependent and spontaneous neurotransmitter release

1) bind to syntaxin in "closed" conformation (inhibiting SNARE complex formation)
- may regulate syntaxin recruitment into SNARE complexes

2) binds tightly to syntaxin complexes
- enhances fusion of synaptobrevin, syntaxin and SNAP-25
- essential part in fusion of machinery
- facilitating SNARE complex mediated fusion

12

disassembly of SNARE complex

- need to be dissasembled after fusion to allow reuse of individual SNAREs
- SNARE complexes are very stable-- a chaparone is needed to dissociate it

13

what is a chaparone?

protein folding enzyme

14

what is the chaparone that dissociates SNARE complexes?

- how does it dissociate?

NSF

- binds to complex via an adaptor protein (a-SNAP)
- unwinds the a-helical bundle of SNARE comples, using ATP as energy source

15

2 proteins that unwind SNARE comples

NSF and a-SNAP

16

Synaptotagmin is made up of

- Ca and phospholipid binding protein

composed of
- short N-terminus intravesicular sequence
- single transmembrane region
2 cytoplasma C2 domains

17

role of synaptotagmin (3)

- both C2 domains bind Ca (Ca binding is cooperative)

- C2 domains also bind to phospholipid membranes in Ca-dependent and Ca in a phospholipid-dependent manner

- also binds to SNARE complexes (in partly Ca-dependent manner)

18

evidence that synaptotagmin is the Ca sensory responsible for Ca-dependent fast transmitter release

- destroy gene, no synaptotagmin left, eliminated fast release, no fusion of SV to plasma membrane

- in synaptotagmin knockout mice, fast transmitter release is absent

- spontaneous AP and Ca-independent release is unchanged--- vesicle fusion not affected

- asynchronous transmitter release unaffected by synaptotagmin, it is caused by SV fusions that are triggered in Ca-dependent manner, with some delay, has to rely on other Ca sensory (not synaptotagmin)

19

how synaptotagmin allows for SV fusion (3)

1) synaptotagmin binds to SNARE complex

2) Ca influx/binding leads to additional charges of C2A and C2B domaines (highly positive charge), attract negative charge of membranes, pull the membranes close together
-

3) energetically unfavourable, promoting membrane fusion (lipid layers fuse, and have fusion pore)

20

endocytosis at presynaptic cell is...

clathrin mediated

21

clathrin mediated endocytosis steps (4)

1) nucleation- assembly of clathrin lattice on patches of plasma membrane to be endocytosed

2) Membrane invagination- generation of clathrin-coated pits through changes in membrane curvature

3) fission- of clathrin coated membrane invagination to create clathrin-coated vesicles

4) uncoating- disassembly of clathrin coat

22

what is nucleation (endocytosis)

- begins with recruitment of adaptor protein AP-2 to plasma membrane region to be endocytosed
- synaptic vesicle proteins containing tyrosine-based AP-2 binding motif recruiting them

AP180 (neuron specific protein), binds AP-2 and clathrin
- required for assembly of clathrin coats

- patches of AP-2 and AP180 recruit clathrin (triskelion structure), oligomerize into lattice with characteristic ultrastructure

23

lattice of clathrin made up of.. (2)


- structure name

heavy chain

light chain

- triskelion structure (3-legged appearance)

24

2 things involved in fission of clathrin-coated vesicles from plasma membrane

Dynamin and Actin

25

role of dynamin in membrane fission

dynamin= GTPase
- absence causes arrest of SV endocytosis at stage of deeply invaginated pits
- with endophilin and amphiphysin, dynamin oligomerizes into stacks of rings around the membrane stalk of coated pits

- pinches off synaptic vesicle

26

role of actin in membrane fission

- perturbation of actin function inhibits endocytosis, leading to accumulation of coated puts with a wide neck

- dynamin interacts with proteins that participate in regulation of actin cytoskeleton= syndapin

- actin is localized to coated pits immediately following the recruitment of dynamin

- helps dynamin pinch off vesicle

- exist in 2 forms
1) g-actin
2)f-actin - generated to force to pinch off SV

27

process of uncoating of clathrin

- 2 things required

- uncoating follows fission of vesicles from plasma membrane

Required: chaparone (unfolding protein)= Hsc70 and cofactor auxilin

- auxilin binds clathrin and AP-2 and targets Hsc70 to clathrin coat

- subsequent removal of the adaptor proteins is Hsc70-independent

28

regulation of endocytosis at presynaptic terminals

- endocytosis in neurons is highly regulated and coupled to exocytoss

- phosphatidylinositides (photpholipids containing inositol head groups), may represent the regulative link between endocytosis and exocytosis

29

PI(4,5)P2

phosphatidylinositol-4,5-bisphosphate

- plasma membrane is enriched with it
- generated by PIPK1y
- PIPK1y activated by neuronal stimulation

- PIP(4,5)P2 dephosphorylated by synaptojanin
- synaptojanin= phosphatase localized to clathrin- coated membranes

30

why generation and degradation of PIP(4,5) P2 is important for endocytosis (2 reasons)

1) absence of PIPK1y leads to delayed endocytosis

2) presynaptic terminals deficient in synaptojanin show large increase in number of coated vesicles and decrease in SV available for exocytosis