Module 5 - Fusion Flashcards
(112 cards)
1
Q
what drives fusion?
A
SNARE proteins
2
Q
super briefly what is the mechanism underlying fusion?
A
SNAREs form tight coiled-coils bringing membranes together
3
Q
what do SNARE required to form coiled-coil domains correctly?
A
template proteins
4
Q
what are the 3 issues to think about fusion?
A
- specificity: can’t fuse with any membrane
- Overcoming energy barriers: lipid bilayer surrounds vesicles
- Regulation: ex regulated secretion, regulation of receptor expression
5
Q
what is T-SNARE?
A
target SNARE; where v-SNARE binds (SNAP-25 and Syntaxin)
6
Q
what was the first fusion assay that was done?
A
mixing golgi from a VSVG+ cell line lacking glycolytic enzyme with golgi from a cell line that has the glycolytic enzyme. measure glycosylation = measure fusion, because golgi needed to fuse to have VSVG and glycolytic enzyme together
7
Q
why was VSVG protein used in the assay?
A
it was well known and it gets glycosylated in the golgi with that glycolytic enzyme
8
Q
what did the VSVG+ glycolysis- cell line mixed with glycolysis+ cell line assay show?
A
no fusion (glycosylation) occurred without adding cytosol and ATP
9
Q
what was the glycolytic enzyme used in the assay?
A
GlcNAc Transferase
10
Q
before, forward transport through the golgiwas thought to be only vectorial. What do we know now?
A
forward transport happens via cisternal maturation, and vesicular trafficking is for backward transport
11
Q
backward vesicular transport in the golgi is mediated by what?
A
COPI
12
Q
knowing that the golgi vesicle go backward change what in the results of the glycolysis vsvg assay?
A
not much; just that probably the important cargo was the glycolytic enzyme and not VSVG
13
Q
what is NEM? what did it do when added to the cytosol (to try and find what part of cytosol is important for fusion)?
A
n-ethyl maleimide: modifies cysteine residues.
It inactivated fusion
14
Q
how did they restore fusion after NEM addition?
A
by purifying the proteins (had been modified by NEM)
15
Q
finding NEM impact on fusion led to the identification of the first factor for fusion which is?
A
NSF (NEM sensitive factor)
16
Q
what did they find after NSF? what was its role?
A
SNAP: Soluble NSF Attachment Protein; needed for NSF binding to golgi membrane
17
Q
what did NSF turned out to be?
A
an ATPase!
18
Q
what did they find after SNAP? what was its role?
A
3 SNAP receptors: SNARE proteins!
19
Q
how did they identify SNARE proteins?
A
Bound NSF and SNAPs to a column; Incubate with brain (lots of synaptic vesicles); Release receptors with ATP
20
Q
what is the most regulated fusion and perhaps most common in any cell?
A
synaptic vesicles release
21
Q
what are the 3 SNARE proteins and their origins?
A
- VAMP/synaptobrevin: (v-SNARE) isolated as a synaptic vesicle specific protein
- SNAP-25: synaptosomalassociated protein of 25 kD
- Syntaxin: isolated using screens for proteins that bound to synaptotagmin
22
Q
what is synaptotagmin?
A
a calcium sensor
23
Q
what do tetanus and botulinum toxin do? what did another lab randomly found their targets to be?
A
toxins that block fusion event for neurotransmitter release; target were VAMP, SNAP25, Syntaxin
24
Q
how did Schekman find SNARE proteins at the same time?
A
in a yeast screen for mutants that block secretion (membranes accumulate inside, increasing density of that mutant, increased density isolated by centrifugation)
25
how was tetanus-toxin insensitive VAMP discovered?
after a lab claimed that SNARE-independent apical fusion existed (was not blocked by tetanus which target SNAREs)
26
are SNAREs sufficient to drive fusion and its specificity?
no, it required 2 additional partners.
SNAREs showed some specificity but not complete.
27
what was the function of the additional 2 partners required for fusion ?
both template the SNARE binding to avoid improper coil coils.
one is a tethering protein important for specificity
28
how is the SNARE coiled-coil structure formed?
1 v-SNARE + 3 t-SNAREs
29
how many coils are in coiled-coil structure in the fusion events?
four coils (very strong) provided by 4 separate proteins, except SNAP-25 which provides 2 coils
30
how are coiled-coil SNARE structures formed?
v-SNARE and t-SNAREs interaction on both lipid bilayer is favored and spontaneously fuse
31
where does the energy come to overcome the barrier to fusion?
from the formation of the four-fold coiled-coil
32
what differs between V- and t-SNAREs that stabilizes their interaction?
v-SNARE has an arginine residue in the middle of the coiled-coil that interactions with t-SNAREs glutamines
33
what are the V- and T-SNARE in transmitter release?
V-SNARE is VAMP/Synaptobrevin;
T-SNAREs are SNAP-25 (two coiled coils) and Syntaxin(one coil)
34
how tight is the coiled-coil SNARE structure?
really tight; is protease and heat and detergent resistant!!
35
what is the actual role of NSF?
break up coiled-coil structure to recucled SNARE proteins! (not involved in the fusion reaction)
36
what happens to v- and t-SNARE respectively after fusion event?
v-SNARE is recycled to vesicle, t-SNARE is recycled at the target membrane
37
what are the 2 addition partners and their specific role?
- Unc13: tether (physically connect vesicle to its target membrane)
- Unc18: organizer. acts with Unc13 as a template to coiled-coil
38
what happens if you remove Unc13 or Unc18?
you get zero synaptic fusion
39
what kind of regulate fusion is specially required for transmitter release?
FAST regulated fusion
40
what is the calcium sensory for fast transmitter release?
synaptotagmin
41
what does complexin do in colaboration with synaptotagmin for fast fusion?
they trap the SNAREs in a pre-fusion complex
42
what releases the pre-fusion SNARE complex?
calcium
43
what is Unc13 role in SNARE pre-fusion complex?
it regulates the number of pre-fusion complex and the localization of calcium channels
44
Why study exocytosis by studying transmitter release?
the post-synaptic ligand gated ion channels allows for exquisite electrical measurement of release;
- it is a really sensitive (can measure one vesicle, milliseconds)
- allows to look at pre-fusion events
45
what are all the event that happens in a synaptic release? in what timeframe does this happen?
1. presynaptic AP
2. Ca2+ current
3. exocytosis (release rate)
4. evoked postsynaptic surrent
5. postsynaptic AP
Happens in only a few milliseconds!
46
describe synaptotagmin structure
family of proteins that have two C2 domains
47
how many syts (Synaptotagmin) are there in the mammalian genome? how many syts isoforms can support fast transmitter release?
17 Syts.
3 isoforms.
48
what can mutations in calcium binding region cause?
alteration of the calcium dependency of vesicle release
49
what happens when you get rid of Syts?
does not block transmitter release (unlike Unc13 or 18), but blocks FAST transmitter release
50
what 2 syt isoforms are we interested in?
SytI and II for fast transmitter release
51
what is the relationship of neurotransmitter release & calcium concentration? what explains this?
exponential to 4th or 5th power! syts can bind 2-3 calcium in a cooperative way
52
how is Syt's C2 domain affinity for calcium? what does this mean?
low affinity, which means faster but requires a high concentration of calcium
53
how do synapses deal with the high concentration of calcium requirement?
the synaptic vesicles are localized right next to the calcium channels
54
what is the Calyx of Held and why is it used to study synapses?
auditory synapse with a very large presynaptic terminus that allows to directly record from
55
what is the best way of actually measuring the calcium dependence of release?
light-induced release of calcium
56
at what calcium concentration does neurotransmitter release peak?
10 uM intracellular calcium. narrow range.
57
what Syt is expressed at calyx of held?
Syt 2
58
what is an osmotic/hyptotonic shock and what does it trigger at the calyx of held of Syt2 KO?
changing solution osmolarity with unsoluble Sucrose; it triggers the release of PRIMED vesicles only
59
what stays the same/differs when Syt2 is KO at the calyx of held? (remember, only syt of calyx is Syt2)
same number of primed vesicles, no effect at low calcium conc.;
loss of the majority of evoked release from large calcium concentration
60
what syt mediates asynchronous transmitter release?
syt7. syt7 can not support evoked release
61
what does it mean that sytKO makes no difference in EPSP(Transmitter release) at low calcium concentrations?
Syt2 is not active at low Calcium concentration because it has low affinity for calcium
62
what do C2A vs C2B domain of Syts bind to?
C2A binds 3 calcium.
C2B binds to the SNARE complex and 2 calcium.
63
C2A and B domains bind calcium best when ?
there are phospholypids
64
what happens if you get rid of C2A domain?
no more synaptic release
65
how many of the 3 Syts involved in fast transmitter release do you need?
all 3 are important
66
C2A may be important for coordinating
what?
multiple SNAREs to act together
67
can you overcome the energy barrier of fusion with one SNARE complex?
no you need more than one. they can be coordinated via C2A.
68
how many binding sites exist for SYTs in the SNARE complex?
2
69
what is complexin?
coiled-coil protein important for transmitter release, but not required.
It is involved in Syt1 and 7 binding to SNARE
70
syt 1, 2, and 9 have a specific ____ ______ that defines the syt 1 family
crystal structure
71
what triggers the final zippering of fusion?
Calcium binding to C2B makes it insert in the lipid bilayer, which induces a conformational change, completes the coiled-coil, membranes curve and fuse
72
what prevents the complete zippering up before calcium binding?
VAMP is partially occupied by complexin or Syt and is not completely in the coiled-coil
73
what is Syt7? where is it not found?
plasma membrane syt protein important for asynchronous release. it is not found in synaptic vesicles
74
unc18 is necessary for fusion, but it can also ...?
INHIBIT fusion by sequesterin Syntaxin in a closed conformation
75
how is priming related to unc18 function of inhibition of fusion?
priming works to relieve the Unc18 inhibition (releasing syntaxin)
76
what is RRP?
readily releasable pool (primed vesicle)
77
osmotic shock can release ...? how?
release the RRP, because the two membranes (vesicular and synaptic) are already very close
78
what is UNC13's role?
(it's a tether but also) determines the size of the RRP and releases syntaxin from Unc18.
important for modulation of release.
79
what is post-tetanic potentiation?
the fact that RRP increases when there is a lot of release (probably homeostatic response to loss of vesicles)
80
other than PTP what precisely increases the size of the RRP?
second messengers like diacylglycerol produced by activation of Gq
81
describe the unc13 domain and what they bind
- C2A domain: binds RIM
- calmodulin binding binding domain: binds calcium with high affinity at low conc
- C1 domain: binds DAG
- C2B domain: binds lipids and calcium
- MUN domain: binds SNAREs and Unc18
- C2C end domain: give synaptic vesicle specificity
82
what Unc13 domains serve to activate it?
the domain in the PM: calmodulin binding, C1, and C2B binding domains;
normally inhibit Unc13. when they bind, they release the inhibition.
83
what happens if you mutate calmodulin binding site in Unc13?
everything is normal except that post-tetanic potentiation is reduced, because the is no calcium-induced increase in RRP
84
what is presumed to be calcium's effect on Unc13's function?
it accelerates Unc13's ability to remove Unc18's inhibitory effect on syntaxin
85
what happened when you removed DAG binding domain (C1) on Unc13?
similar effect as calmodulin binding site removal: reduction of PTP
86
what is RIM?
protein that links synaptic vesicles to calcium channel via Unc13
87
how does RIM binding domain (C2A) on Unc13 allows for specific timing of activation?
RIM binding compete with Unc13 homodimerization that inactivates Unc13, to activate Unc13 only when it is at the synaptic terminal
88
what happens if you delete RIM binding domain (C2A)?
Unc13 can still work
89
do every species have a C2A domain in Unc13? what might that do?
no; they may have less localization of SNARE to calcium channels
90
Can replace most active zone scaffold protein with just
Unc13 coupled to a subunit of the calcium channel important for release (?)
91
BASICALLY what is Unc13's role?
acts as a hub to regulate how many SNAREs are in a pre-fusion complex (via calmodulin binding) and to regulate localization to calcium channels (via RIM)
92
what did they try to prove about syt2 localization in the paper?
already know it is postsynaptic, they proves that syt3 is also presynaptic
93
what were the presynaptic markers?
vglut1 and synaptophysin
94
what was the postsynaptic marker?
PSD95
95
what did Syt3 align the best with?
bassoon: active pre synaptic zone
96
did it vesicular markers colocalize with syt3? what can we conclude?
no: syt3 is not in vesicles
97
they had synaptosomes with only vglut1, only psd95, or both. which ones colocalized with syt3? what does that mean?
synaptosomes with vglut1 AND PSD95, and synaptosomes with only vglut1.
this indicated presynaptic enrichment.
98
did western blot show Syt3 in vesicles?
no
99
what happened to spontaneous EPSCs, evoked EPSCs, and pre-synaptic calcium current in Syt3KO neurons at the calix of held? why?
nothing different because Syt3 doesn't affect basal release properties
100
what happened when they stimulate the presynaptic axon at low vs high frequencies in Syt3KO neurons?
no difference at low frequencies.
>10Hz: less synaptic release and slower recovery of pre-synaptic vesicles.
101
what are the differences between BAPTA and EGTA calcium buffers?
BAPTA is very fast and can compete with synaptotagmin.
EGTA is slower and can't compete.
102
if EGTA is slow, why do they use it?
it can bind slow, higher affinity calcium
103
what is the effect of EGTA on WT and Syt3KO neurons?
WT: EGTA reduces the recovery of transmission to the level of Syt3KO recovery.
Syt3KO: EGTA has no effect because the recovery is already
104
what kind of calcium is buffered by EGTA?
residual calcium
105
so what kind of calcium mediates Syt3 recovery actions if EGTA has no effect on Syt3KO?
Residual calcium
106
were they able to rescue the recovery rates in SYT3KO by adding a modified Syt3 that doesn't bind calcium?
no
107
what is the major difference between Syt3 and Syt7?
Syt7 binds calcium slower than Syt3
108
what are PPF vs Recovery thought to be mediated by?
PPF mediated by increased release probability.
Recovery mediated by accelerated vesicle replenishment. (Syt3)
109
what were Syt7KO effect on neurons in calix of held and climbing fibers?
no effect on the recovery of transmission
110
what was Syt3KO's effect on PPF (pair pulse facilitation)?
lost of PPF
111
what was Syt7KO's effect on PPF (pair pulse facilitation)?
also reduced PPF
112
what new model did they come up with at the end of the paper?
Syt3 promotes loose to tight docking of vesicles
