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BMS 109 > MT2- SNares > Flashcards

MT2- SNares Flashcards

1
Q

Which scientist made big progress researching SNAREs?

A

Rothman 1993- using NSF and SNAP (known SNAREs) he pulled out a 20s complex of other SNAREs.

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2
Q

Rothman 1993 experiment first steps?

A

Using known fusion proteins NSF and SNAP, a 20s complex was pulled out by immunopull down. How?
NSF is epitope tagged to Myc and an anti-myc antibody (IgG) to a bead. Add alpha and Y SNAP and then bovine brain tissue.
When ATP gamma S was added this complex formed (NSF in ATP state stabilised).
Then eluted after addition of MgATP (NSF in ADP form)

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3
Q

Rothman 1993 experiment after complex eluted?

A

Specific eluate after MgATP addition pulled out lots of bands and ran on SDS.
blots cut out and digested with trypsin. High performance liquid chromatography used- separate and sequence.
- Already known peptides identified.

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4
Q

Rothman experiment to prove necessity of NSF?

A

Made an AB raised to the C terminal peptide of SNAP25 and incubate with excess recombinant alpha and gamma SNAP © or without excess recombinant NSF (B)
And separated by centrifugation. Without a 5s complex was pulled out as it was not fully assembled, and with the 20s complex.

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5
Q

As well as the model of the proteins in the complex what also did the Rothman 1993 experiment find?

A

More than one isoform of the proteins were found e.g. Syntaxin A and B. There is no evidence to suggest that these bind and dimerise, so the Rothman lab speculated that these could perform the same job but add specificity to certain vesicles or targets.

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6
Q

What was theorized in the Rothman 1993 paper but not evidenced?

A

How the fusion particle comes together.
It was unknown whether these exist together in a 20s particle or whether separate particles come together to make an attachment site and the 20s is only part.

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7
Q

What did Rothman pull out of the 20S complex?

A

Syntaxin A, B, SNAP25 nd Synaptobrevin.

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8
Q

6 steps in fusion (phrase answer only)

A
  1. Tethering
  2. Docking/Priming
  3. Fusion initiation
  4. Hemifusion
  5. Fusion pore opening
  6. Full collapse
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9
Q

What equipment advancement has helped to understand the fusion process?

A

CryoEM- Freezes sample in liquid nitrogen. Before fixation altered distance between vesicle and Pm enabled differentiation between tethering and docking. Enabled uncovering of the steps.
found that vesicle docking requires Munc13 priming proteins, all SNARES but not synaptotagmin or complexins. (Imig 2014)

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10
Q

Regulation of fusion by what?

A

Calcium- synaptotagmin

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11
Q

Q vs R Snare?

A

R snares- have an arginine in centre which associates with vesicle e.g. synaptobrevin.
Q Snares- Gluatmine in centre e.g. syntaxin and SNAP25.

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12
Q

Structure of Synaptobrevin?

A

(VSNARE) TMD, short carboxyl terminus inside vesicle, alpha helices in a coiled coil, arginine residue in centre which associates with vesicle (R snare)

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13
Q

Syntaxin structure?

A

(T SNARE) TMD through target with carboxyl termini sticking out of cells, Glutamine in centre (Q) where coils of SNARES wrap around (E.g. SNAP25).
Habc domain can fold back and hide SNARE domain.
Open or closed configuation for regulation.

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14
Q

SNAP 25 structure?

A

achored to the PM by 4 palmitolylated cysteine residues in the middle. 2 SNARE motifs, gluatmine containing.

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15
Q

Syntaxin role?

A

On the target membrane. Regulates the fusion of Vesicle with membrane by having a Habc domain- can fold back to obscure the SNARE Motif and go into a closed formation

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16
Q

Munc18 role?

A

/Sec1- binds to Syntaxin in closed configuration (negative regulator) preventing dead ended SNARES ( 2 syntaxins bind to one SNAP25 so no Synaptobrevin binding place. if MUNC18 bound one end, can be 1:1). Positively control fusion also.

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17
Q

What is NSF?

A

Fusion protein, cytosolic ATPase, required for transport vesicle fusion. binds to SNAP which binds to SNARE (requires ATP to dissociate)

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18
Q

What does SNARE stand for?

A

SNAP receptors. Have SNAP binding sites.

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19
Q

SNAP stand for?

A

Soluble NSF attatchment protein.

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20
Q

SNAP forms?

A

Alpha, beta, gamma. Alpha used for ER to Golgi trafficking

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21
Q

What makes up the 20s complex?

A

NSF-SNAP-SNARE form a stable 20S complex which
requires ATP hydrolysis to dissociate
Snares= syntaxin and synaptobrevin

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22
Q

SNAREs involved in vesicle fusion?

A

7s complex. (No SNAP or NSF in complex like 20s). (SNAP25 is though, along with syntaxin and synapobrevin)

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23
Q

NSF role?

A

required to dissociate trans and cis SNARE complexes and thereby regenerate free SNAREs for the
next round of vesicle docking and fusion

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24
Q

How do the SNAREs associate with each other?

A

They coil around each other. But Rab proteins stop full zippering(fusion) so they are just docked.

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25
Q

Adapter SNARE complex made by..?

A

SNAP25 and syntaxin, so synaptobrevin can bind.

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26
Q

Which SNARE isnt TM?

A

SNAP25

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27
Q

Why don’t vesicles spontaneously fuse with the membrane?

A

Because the heads of the phospuholipid bilayers of both are negative so the repel each other.

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28
Q

The first step of vesicle fusion is? Details:

A

Tethering- SNAP25 and syntaxin dimer to create an acceptor complex to the V SNARE Synaptobrevin. Also the Rab protein on the vesicle binds to the Rab effector on the target membrane e.g. Rabaptin, bringing the membranes close together. (Rab 5 to APPL1 on endosomes.)

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29
Q

Dead end complexes what? can be broken up by what?

A

2 Syntaxins to one SNAP so Synaptobrevin cant bind, NSF can break these up.

Syntaxin is in a closed formation where the Habc domain hides the SNARE motif, with munc 18 bound.

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30
Q

After tethering next step in vesicle fusion?

A

Docking/ Priming:Pull membranes v close and move other contents out as SNARES wind up from the amino terminus to carboxyl, making a Trans-SNARE complex.

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31
Q

What is superpriming?

A

Complexin binds to increase this priming =fusion clamp. proteins bind to stop full zippering and calcium is needed for fusion in regulated secretion, or this is sufficient for fusion in constitutive secretion.

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32
Q

Function of the Rab proteins? Bind to? example?

A

GEF (guanine exchange factor) recruits Rab proteins to the membrane and alters the conformation of it such that GDP falls off and GTP can bind, activating it. The Rab protein is incorporated into the vesicle. These act as an address signal to the target.
The Rab protein on the vesicle binds to the Rab effector on the target membrane e.g. Rabaptin, bringing the membranes close together. (Rab 5 to APPL1 on endosomes.)

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33
Q

What proteins act as an address signal for vesicles?

A

Rab proteins, Recruited by GEF and activates it into the GTP conformation.

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34
Q

What is fusion initiation?

A

This can be calcium induced (regulatory), Ca binds to synaptotagmin. Synaptotagmin C2a domain pushes complexin arm out of the way to allow fusion? Bend PM up to vesicle, as Ca+ release the clamp to allow full zippering of the SNARES.

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35
Q

What is the role of Munc13?

A

Munc13 dissociates Munc18 from this Syntaxin binding spot so it only interacts with it and opens syntaxin up. (move from Habc domain to N peptide)

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36
Q

What is hemifusion?

A

This is fusion of the outer leaflets of the vesicle and the target membrane. There is no passage through.

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37
Q

What experiment tests for hemifusion?

A

Lipid mixing, if lipids between the vesicle and membrane are exchanged, but content mixing not evidenced hemifusion alone may have happened.

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38
Q

Fusion pore opening is..?

A

Transformation of the Trans-SNARE complex into a Cis (when all snares are found on one membrane-PM) allows the inner leaflets fuse and create a pore between. (test by contents mixing).

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39
Q

Trans-SNARE complex vs Cis?

A

Trans is at the stage of docking beore fusion. Cis is where all SNAREs are found on the same membrane e.g. after fusion when vesicle bilayer fuses to be part of the target membranes. This still has a bulb off so can pinch off again and simply ‘kiss’ the membrane.

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40
Q

Step after fusion pore opening?

A

Full collapse- No vesicle bulb off of the target membrane but now flat and completely fused.
Disassembly is catalysed by NSF ATPase, using ATP and it’s adapters (SNAPS). After vesicle fusion the Rab proteins are hydrolysed into the GDP form.

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41
Q

Why does some cargo need ful collapse and others not?

A

Depends on the size of the cargo, if larger like inuslin may need full collapse.

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42
Q

From docking to full collapse time taken?

A

1ms

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43
Q

Method of quickly recyling SNARE proteins etc?

A

Only using the ‘kiss and run’ technique of vesicle- fusion pore opening. Can pinch off again after transfer cargo.

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44
Q

Rothman (1980) wrongly theorized that…

A

ATP was required for fusion, but isn’t directly, its required for disassembly of SNAREs which enables the recycling.

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45
Q

NSF yeast version? found by who?

A

Sec18 by Shekman.

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46
Q

Rothman (1993) used what tissue?

A

Bovine brain synapses

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47
Q

What is the middle binding partner between SNAREs and NSF?

A

SNAPs

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48
Q

What is ATP-Gamma-S?

A

Non-hydrolisable version, Stablilising in an activate state.

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49
Q

SNAREs coil from which end first?

A

Amino to carboxyl.

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50
Q

Chemicals that evidence SNAREs ?

A

Clostridial neurotoxins e.g. and Tetanus toxin- clevaes SNARE proteins.

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51
Q

Clostridial neurotoxin structures?

A

Have a light and a heavy chain. The heavy chain has a receptor binding domain needed for getting into the neuron.
The light chain cleaves proteins, its an endopeptidase

52
Q

Clostridial neurotoxins; there is one type of ……, which targets ….., but 7 types of……., which targets ….,…… and….

A

Tetanus- targets synaptobrevin (1,2)

Botulinum- A-G types, 4 also target synaptobrevin, 2 (A and E) targets SNAP25 and C targets both synaxin and Snap25.

53
Q

What does Botox do? work how?

A

Pico amounts of Botulinum- blocks NT across NMJ by cleaving SNARES.

  • Binds to receptors on the pre-synaptic terminal.
  • Internalised into an acidic endosomal environment
  • this allows the dissociation of the LC.
  • Heavy chain Hn domain forms pore allowing LC to pass out of the vesicle into cytosol
  • cleave SNARE proteins so vesicles cant fuse with the membrane and release across the synapse.
54
Q

Most lethal Botnulinum?

A

C- targets both syntaxin and SNAP25, neurons retract and die.

55
Q

Botulinum and Tetanus are examples of which toxins?

A

Clostridial neurotoxins

56
Q

Clostridial toxins HC exerts its action how?

A

100kd- has a receptor binding domain to receptors on the pre-synaptic neuronal terminal membrane, required for internalization.

57
Q

Clostridial toxins LC exerts its action how?

A

50Kd-contains metallo-endopeptidase domain for cleaving the SNARE proteins

58
Q

Botox can be a Medicine for?

A

medicine for Squints, spasms, migraine and even incontinence.

59
Q

Number of mammalian SNARES? simialr?

A

38- common domains- all have a SNARE domain, some TMD, and Ha Hb Hc doman.

60
Q

Which botulinum is used in botox?

A

A-cleaves SNAP25 removing 9 AA from the c terminus only.

61
Q

What is the result of Botulinum A on the SNARE it targets?

A

Botulinum A-cleaves SNAP25 removing 9 AA from the c terminus only.results in unproductive syntaxin–SNAP-25 dimers

62
Q

Experimental evidence about botulinum and SNAREs?

A

Davletov 2004
The cleaved SNAP25 by botnulinum E (-26AA), had v poor binding to syntaxin which was GST linked. Fluorescent tagged rabbit (to SNAP) and mouse (to synatxin) antibodies to Snares allowed visuallisation and wihtout the toxin the SNAP25 like syntaxin was found at the plasma membrane, but with E SNAP25 coexpression was not seen.

63
Q

Experimental example of redundancy in SNAREs?

A

Erwin Neher- 2003
mutated different SNAREs found that SNAP23 can rescue the function of SNAP-25 knockout mice, albeit not to full function- vesicle fusion is slow. Using electrode can measure capacitance of membrane to work out amount of fusion. Around 10 vesicles fused vs WT 300, SNAP-23= 80 vesicles. (all same Ca rise).

Amperomter trace confirms is adrenaline vesicles.

64
Q

Characteristics of SNAP23?

A

insensitive to toxins, involved in constitutive secretion and immune cells.

65
Q

SNAP27 role in..?

A

long term potentiation?

66
Q

SNARE specificity comes from?

A

-physical arrangement of SNAREs (ie. location)- different SNAREs found on different membranes.
–organization/proximity of organelles
–inherent ability of cognate snares to form complexes
–Rab proteins and other regulators

67
Q

What are SM proteins?

A

Sec/Munc18 like proteins- essential for membrane fusion as they stop the formation of deadends which don’t allow synaptobrevin to the acceptor complex of SNAP25 and syntaxin.

68
Q

Vesicle Fusion energy from?

A

The energy built up in the coiling of the SNARE proteins, which enables the energy to overcome the negative repelling membranes.

69
Q

Why may there be different SNAREs on different compartments?

A

Give an address sequence.

70
Q

Who invented the patch clamp technique?

A

Erwin Neher

Electrophysiology experiment on Chromaffin cells with vesicles in adrenaline.

71
Q

What was the technique used to measure fusion by Erwin Neher for fusion events? (1st)

A

He invented patch clamp, and used this to measure storage of charge of a membrane (capacitance)
membrane electrical charge directly proportional to the surface area of the membrane. So if a vesicle fuses the surface area is increased by the size of the vesicle. Endocytosis decreases. This allows us to measure fusion events in Chromaffin cells etc

72
Q

2nd technique Erwin Neher developed?

A

Cage compounds- Ca in cages and the UV light flash breaks the chemical cage open and Ca inside is released and can bind to Ca binding proteins- trigger fusion.

73
Q

Advantages to the Cage compounds Experimental technique?

A

Erwin Neher.
allows fusion without needing neuronal transmission and upstream ion channels, Ca released by UV light, so useful for KO and mutant proteins etc, don’t need to worry about ion channels upstream just final stages.

74
Q

Why was it important to include an amperometer trace in Erwin Nehers experiment in chromaffin cells?

A

If increase calcium levels may force fusion of other vesicles and the capacitance technique only shows non-specific fusion, so wanted to check it was all the adrenaline vesicles that were testing.

75
Q

Which experiment proved SNAREs are not only needed for fusion but docking also?

A

Imig 2014
Cryo-EM on synapses isolated from KO mice of every SNARE.
GFP tagged vesicles docked at an active site.
SNAP25 KO- no vesicles docked. In absence of any SNARES this happened. but could see vesicles in the cell, this showed SNAREs necessary for docking as well as fusion.
Earlier experiments before cryo-EM harder to distinguish docking and fusion.

76
Q

Experimental evidence about Rab proteins?

A

Rab 5 and APPL1 on endosomes.

77
Q

Munc vs Unc?

A

mammalian UNC

78
Q

Later experiment in Rothman Lab Shen at al (2007) procedure?

A

Made artificial liposomes and add back SNARE components (reconstitute). FRET signal if the target and vesicle liposomes fuse.
Preincubate one at 4=degrees and not the other before adding SNAREs at 37degrees.

79
Q

results of Rothman Lab Shen at al (2007) procedure?

A

If preincubate liposomes and add munc18 fusion increases- this was suprising as munc18 was thought to be a negative regulator.
Explaination: Can bind to the N peptide (open) as well as the Habc domain (closed- Habc domain covers Snare domain).

80
Q

Experiment to prove Munc18 necessary for fusion?

A

2006 KO Munc18- No change in Capacitance after UV flash (increases Ca intracellularly), so no fusion. If reconstitute restore fusion.

81
Q

Experiment to prove Munc18 necessary for docking?

A

Munc18 KO chromaffin cells, and reintroduce Munc18 GFP fusion protein by viral vector. Count docked vesicles at membrane and reduced in KO and reconstition saved. Total vesicle number the same.

82
Q

Munc18 creates ….. which help with docking?

A

Microdomains, colocalisation of vesicles to Munc 18 hotspot clusters at the membrane.

83
Q

Munc18 KO rescued by?

A

De Wit
SNAP25 overexpression rescues docking but not fusion.
Why? Munc18 binds to syntaxin to prevent two binding to SNAP25, but if overexpress Snap25 still bind 1:1 so synaptobrevin can still bind allowing tethering.

84
Q

Procedure to experiment Munc13 KO?

A

Augustin 1999 Mouse KO- embryonic lethal but can isolate the embryo brain and the neurons synapse with themselves and enable studing. If stick an electode in can stimulate an AP, NT release to activate its own receptors and keep going.

85
Q

Result of Munc13 KO?

A

Augustin 1999.
Reduction in fusion so current of EPSC.
Proved is a fusion defect, by artifically inserting Ca and only the WT responds.
The readily releasable pool of vesicles is reduced. By putting a high sucrose conc outside the neuron (Jam-artifically brings membranes together as osmosis shrinks neurons) and still no EPSC- so no readily releasable pool to release. NOT PRIMED

86
Q

Munc 13 acts after …. but before …

A

after docking, before fusion.

87
Q

Munc13 binding partners?

A

Yeast 2 hybridscreen to show that syntaxin binds to Munc13 direct interaction, and forms a 7s complex.

88
Q

Worm study? Procedure?

A

Janet Richmond- C-elegans, stimulate pre- neuro and measure current in the post. Did this with various KOs.

89
Q

Worm study result?

A 
Janet richmond- 
Munc 13 KO- no EPSC
synataxin KO- no EPSC
Munc13 KO x Syntaxin KO but re-express mutated syntaxin which is always open and get EPSC. 
Role in opening syntaxin therefore.
90
Q

Rothman 2013 experiment procedure and findings?

A

Reconstitution experiment using an acceptor and donor liposomes to work out the order of events.

91
Q

Why was a lipid mixing experiment not enough? Yang et al.

A

The lipid mixing doesn’t evidence full fusion (hemifusion) only that lipids on the PM can flip and mix when the outerleaflets join, so did content mixing to check that there was mixing of the cargo- full fusion pore opening.

92
Q

Procedure experiment on Munc13 and GPCRs?

A

Seward lab
Phosphorylate G proteins- Gq-PKC-PIP2-DAG produced. This can bind to Munc13 to regulate.
Chromaffin cells- depolarise the cell and simultaneously add the agonist and measure the capacitance.

GFP tagged Munc13 to see localisation.
Then mutate Munc13- disabled part that recognised DAG

93
Q

Procedure experiment on Munc13 and GPCRs?

A

Seward lab
agonist to GPCRs Gq-PKC-PIP2-DAG produced. This can bind to Munc18 to regulate.
Chromaffin cells- depolarise the cell and simultaneously add the agonist (Histamine) and measure the capacitance using patch clamp.

GFP tagged Munc13 to see localisation.
Then mutate Munc13- disabled part that recognised DAG

94
Q

Results of experiment on Munc13 and GPCRs?

A

after Gpcr agonist, cell capacitance x3.
If activate the receptors more Munc13-GFP tagged came to the PM.
But if mutate the DAG recognition site, block potentiation by the agonist.
Long term potentiation!

95
Q

What domain of Munc13 is necessary for function?

A

Yang et al, The MUN domain- ABCD, BC necessary.

96
Q

Yang et al paper aims?

A

The role of the MUN domain in the opening of syntaxin by Munc13 in synaptic vesicle priming.

97
Q

How are Ras proteins recylced after fusion?

A

Disassembly is catalysed by NSF ATPase, using ATP and it’s adapters (SNAPS). After vesicle fusion the Rab proteins are hydrolysed into the GDP form. GDI (GDP dissociation inhibitor) chaperone, recycles Ras in the GDP form back out of the membrane.

98
Q

Where does ATP come into vesicle fusion?

A

NSF requires to break up SNAREs and recyle them to reuse.

99
Q

What experiment found synaptotagmin?

A

Holt 2006
Proteomic studies quantified the proteins in isolated synaptic vesicles. 15 copies of synaptotagmin (per vesicle), a protein known to be regulated by calcium.

100
Q

Along with synaptotagmin what other copies were found in a proteomic study?

A

Holt 2006
Rab protein-10 copies
Snap25 x2 copies (unusual so either contaminated or to recycle)
synaptobrevin (70copies)

101
Q

How many isoforms of synaptotagmin are there? differences?

A

16 with different Ca affinities and vesicle localisations.

102
Q

Synaptotagmin isoform similarities?

A

All have tandem C2 domains for binding calcium. These also interact with phospholipids to associate with the membrane.

103
Q

synaptotagmin not only found in Neurotrasmitter vesicles but also? why?

A

Secretory vesicles, if regulated by calcium.

104
Q

Ca independent part of SNARE steps?

A

Tethering and docking, with complexin forms the fusion clamp preventing full fusion. But requires Ca for full fusion.

105
Q

Synaptotagmins with low calcium affinities? Use?

A

1,2,9- used for rapid transmission, needs high Ca levels- bind to voltage gated Ca channels, so are right at the cleft active zone.

106
Q

How does synaptotagmin work?

A

Ca2+ induced Fusion initiation- Calcium binds to synaptotagmin, causing an interaction with; synaptotagmin, SNARES and negative phospholipids. Synaptotagmin C2a domain pushes complexin arm out of the way to allow fusion? Bend PM up to vesicle, as Ca+ release the clamp to allow full zippering of the SNARES.

107
Q

C2b domain function?

A

C2b domain: Complexin binds to. Binds to SNAP25, inserting into the SNARE complex.

108
Q

………. is a speed determiner for neurons

A

syntaptotagmin

109
Q

snaptotagmin (syt) 2 vs 7

A

2- fast transmission e.g. sound location.

7- slower e.g. peptide release, basal asyncronous release further away from the active site, higher Ca sensitivity

110
Q

Why is it useful for some synaptotagmins to have low Ca sensitivity?

A

It is advantageous for them to have low calcium binding as this then means that the vesicles are only released when there is an action potential and only when located at the exit zones at the mouth of the terminal bulb where the calcium signal is the highest (transient microdomain.) It docks right next to calcium channels therefore binding to theSynprint site of ca channel.

111
Q

syt7 vs other syts experiment?

A

Geppert 1994,
KO mouse syt embryonic lethal (except for syt7), so had to isolate neurons.
Stimulate neuron and measure glutamate release.
In KO asynchronous release vs syncronised.
Yet hypertonic sucrose evoked normal release so fusion can still occur.
If KO syt7 as well no asynchronous events either.

112
Q

Experiment to test necessity of synaptotagmin?

A

Sudoss lab 2001, Knockin mouse of synaptotagmin which Ca cannot bind to. 1 AA change in C2a domain. This caused an alteration from a high release probability neuron to a low release, so increased release over AP number.
Altered Ca sensitivity of the neuron.

113
Q

Two types of vesicle release neurons?

A

Low release probability:Has fewer vesicles primed so fewer released. But Ca primes more and more vesicles as it accumulates, making a larger pool for the next AP (response increases over AP number)
High release probability: Initially lots of primed vesicles but the source of vesicles gets depleted faster. (response decreases with AP number)

114
Q

Experiment to test role of Calcium C2AB domains in fusion?

A

rothman et al 2013
donor and acceptor liposomes, fusion dramatically increases when add C2AB domains of synaptotagmin. Fusion increases as Ca conc does (until saturates)

115
Q

Complexin structure with SNAREs? Scientist predicted?

A

clamps SNARES into a zigzag like structure.(Rothman lab) Complexin bridges (inserts into C2b of synaptotagmin and into snares coils) to make a ring to prevent final fusion, but if one of the two arms is flipped upwards the next in the ring is opened etc (dominos by C2A). This enables the SNARES to fuse.

116
Q

disrupt ca binding vs lipid of synaptotagmin?

A

Ca binding- disrupt fusion

lipids- docking defect (synaptotagmin role in docking also as interacts with the phospholipids)

117
Q

SNAREs and synaptotagmin binds to where on the Ca channel?

A

Synprint site on the VGCC’s alpha channel which makes up the pore, forming a complex with the SNAREs.

118
Q

conc of Ca at the mouth of a pre-synaptic neuron?

A

100uM

119
Q

Which proteins/snares regulate each step of fusion?

A
  1. Munc 18 and Munc 13 regulate the ability for the formation of acceptor sites (syntaxin and SNAP25.)
  2. oligomerization of SNARE pins to tether the vesicles. And Rab proteins bind to Rab receptors and RIM aid.
  3. Complexin and synaptotagmin allows priming.
  4. calcium binding to C2b controls fusion.
120
Q

synaptotagmin C2a vs B domain?

A

c2b- Ca binds to, and binds to symprint of VGCC.

c2A- may push complexin arm out of the way and allow fusion. Binds to phospholipids.

121
Q

Function of Rabs?

A

Attach to vesicles, acting as an address system for the vesicles to the target, as they too have specific Rab effectors on the target to bind to. This brings the membranes close so that SNARES can bind.

122
Q

Who established the structure of Munc13?

A

Yang et al, 2015.
crystallised the MUN domain, after remoxing the flexible loop and other amino acids. Hydrophobic pocket between BC. Mixed parallel and anti alpha helices, connected by interhelical flexible loops and interactions.

123
Q

How was the importance of the Munc13 domains tested?

A

Yang et al 2015.
FRET experiment, Fluoresce when liposomes with syntaxin and Synaptobrevin bind. and different combinations of the MUN domains were tested. BC found to be necessary together (form hydrophobic pocket), but the more domains the better.

The NF inbetween AB was necessary for function. NFAA mutation abolished SNARE liposome fusion. Checked both lipid mixing and content mixing. Only 10% of WT fluorescence, same as having no Munc13 or SNAP25.

The NFAA UNC fialed to evoke EPSC. (42hz to less than 1hz). Light sensitive channels evoked neuron depolarisation.

124
Q

Lipid mixing vs contents mixing?

A

Yang et al 2015.
Liposome experiments.
Lipid mixing- only shows docking, where the phospholipids can exchange over membranes.
Content mixing- full fusion, exchange of contents between two vesicles.

125
Q

Function of RIM proteins?

A

Rim binding to vesicular RAB proteins mediates vesicle docking, and binding to Munc13 activates vesicle priming. RIM binding to Ca channels both directly and indirectly through RIM-BP recruits Ca channels on the presynaptic terminal (allowing for exocytosis).

BMS 109 (20 decks)

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