JC2 paper

Question 1

How is neurotransmitter release by Ca2+ triggered eocytosis regulated?

Answer 1

• regulated by core fusion machinary - e.g. SNAREs , MUNC proteins

Question 2

What are roles of Munc 18?

Answer 2

• Munc 18 locks syntaxin in a closed conformation - prevents forming unproductive SNARE complex and holds syntaxin so it is available to form productive SNARE complex
• Munc -18 also binds to N terminus on syntaxin (when in SNARE complex) to hold in open configuration in order to form a productive SNARE complex

CATALYSE MEMBRANE FUSION

Question 3

What region of UNC13munc13s is essential for it’s function?

Answer 3

MUN domain (C terminus)
-required for vesicle PRIMING function of UNC13-Munc13s

Question 4

what happens in neurons lacking UNC13-munc13s proteins?

Answer 4

-vesicle release is abolished

Question 5

What does the syntaxin LE mutaitons do and what does it show?

Answer 5

• syntaxin-1 LE mutation - opens syntaxin in the absence of Unc13 and rescues vesicle release
• this suggest unc-13 has some role in the opening of syntaxin -1

Question 6

what sequence in the MUN domain hydrophobic pocket is essential ofr vesicle priming

Answer 6

-the NF sequence

asparagine and phenylalanine

Question 7

what is an angstrom?

Answer 7

unit of length used in measuring wavelengths of light (10 -10)

Question 8

what does FRET stand for?

Answer 8

-fluorescence resonance energy transfer

Question 9

Give the principles of FRET used in this experiment

Answer 9

• Is a fluorescence donor on SNAP-25
• Fluorescence/rhodamine acceptor on syntaxin 1
• Transfer of fluorescence occurs when donor and acceptor sites (SNAP-25 and syntaxin-1 are close - so in a SNARE complex)

-FRET sensitive to distance - efficacy of energy transfer is inversely proportional to the distance between donor and acceptor sites

Question 10

what is a proteoliposome?

Answer 10

• liposome hwere one or more proteins have been added artificially

Question 11

how is lipid mixing/fusion measured?

Answer 11

By fluorescence dequenching methods

- when fusion occurs fluorescence is quenched

Question 12

what happens in NFAA mutation?

Answer 12

liposome fusion is abolished

• shows importance of NF sequence in hydrophobic pocket of MUN mun13 domain
• NF sequence crucial for SYNAPTIC VESICLE PRIMING

Question 13

what happens when UNC13L or S is expressed in unc-13 mutant worms?

Answer 13

• locomotion is rescued and neurotransmission defects are also rescued
• rescue vesicle exocytosis

Question 14

Give the model for the mechanism which shows activity of MUN

Answer 14

-syntaxin 1a is in closed conformation with mun-18
(munc18-syntaxin complex)
- MUN domain causes syntaxin1a to open (munc18 still bound) (intermediate 1)
- Addition of SNAP 25 - binds to syntaxin to form T-SNARE complex (intermediate 2)
- Addition of synaptobrevin forms SNARE complex

-After dissociation of synaptobrevin the syntaxin1a-SNAP25 complex undergoes addition of MUN (intermediate 2)

Question 15

what domains, e.g. ABCD have highest activity in MUN domain?

Answer 15

• domains B and C
• A and D have additional roles but cannot stimulate transition from munc18-syntaxin1 to SNARE complex without both B and C

Question 16

what is the point in using hypertonic sucrose solution?

Answer 16

• induce synaptic vesicle exocytosis in a Ca2+ independent manner
• This measures readily releasable pool of vesicles
• Shows the changing in SNARE complex formation is due to SNAREs not forming properly (due to absence of WT NF sequence) not due to Ca2+ dependent reasons.
• shows NF sequence has role in both the spontaneous and evoked release of vesicles (role in vesicle PRIMING)