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ANAT365 > Module 5 - Fusion > Flashcards

Module 5 - Fusion Flashcards

1
Q

what drives fusion?

A

SNARE proteins

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2
Q

super briefly what is the mechanism underlying fusion?

A

SNAREs form tight coiled-coils bringing membranes together

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3
Q

what do SNARE required to form coiled-coil domains correctly?

A

template proteins

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4
Q

what are the 3 issues to think about fusion?

A
  1. specificity: can’t fuse with any membrane
  2. Overcoming energy barriers: lipid bilayer surrounds vesicles
  3. Regulation: ex regulated secretion, regulation of receptor expression
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5
Q

what is T-SNARE?

A

target SNARE; where v-SNARE binds (SNAP-25 and Syntaxin)

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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

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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

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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

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9
Q

what was the glycolytic enzyme used in the assay?

A

GlcNAc Transferase

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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

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11
Q

backward vesicular transport in the golgi is mediated by what?

A

COPI

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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

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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

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14
Q

how did they restore fusion after NEM addition?

A

by purifying the proteins (had been modified by NEM)

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15
Q

finding NEM impact on fusion led to the identification of the first factor for fusion which is?

A

NSF (NEM sensitive factor)

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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

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17
Q

what did NSF turned out to be?

A

an ATPase!

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18
Q

what did they find after SNAP? what was its role?

A

3 SNAP receptors: SNARE proteins!

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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

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20
Q

what is the most regulated fusion and perhaps most common in any cell?

A

synaptic vesicles release

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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
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22
Q

what is synaptotagmin?

A

a calcium sensor

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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

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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)

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25
Q

how was tetanus-toxin insensitive VAMP discovered?

A

after a lab claimed that SNARE-independent apical fusion existed (was not blocked by tetanus which target SNAREs)

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26
Q

are SNAREs sufficient to drive fusion and its specificity?

A

no, it required 2 additional partners.
SNAREs showed some specificity but not complete.

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27
Q

what was the function of the additional 2 partners required for fusion ?

A

both template the SNARE binding to avoid improper coil coils.
one is a tethering protein important for specificity

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28
Q

how is the SNARE coiled-coil structure formed?

A

1 v-SNARE + 3 t-SNAREs

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29
Q

how many coils are in coiled-coil structure in the fusion events?

A

four coils (very strong) provided by 4 separate proteins, except SNAP-25 which provides 2 coils

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30
Q

how are coiled-coil SNARE structures formed?

A

v-SNARE and t-SNAREs interaction on both lipid bilayer is favored and spontaneously fuse

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31
Q

where does the energy come to overcome the barrier to fusion?

A

from the formation of the four-fold coiled-coil

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32
Q

what differs between V- and t-SNAREs that stabilizes their interaction?

A

v-SNARE has an arginine residue in the middle of the coiled-coil that interactions with t-SNAREs glutamines

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33
Q

what are the V- and T-SNARE in transmitter release?

A

V-SNARE is VAMP/Synaptobrevin;
T-SNAREs are SNAP-25 (two coiled coils) and Syntaxin(one coil)

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34
Q

how tight is the coiled-coil SNARE structure?

A

really tight; is protease and heat and detergent resistant!!

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35
Q

what is the actual role of NSF?

A

break up coiled-coil structure to recucled SNARE proteins! (not involved in the fusion reaction)

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36
Q

what happens to v- and t-SNARE respectively after fusion event?

A

v-SNARE is recycled to vesicle, t-SNARE is recycled at the target membrane

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37
Q

what are the 2 addition partners and their specific role?

A
  • Unc13: tether (physically connect vesicle to its target membrane)
  • Unc18: organizer. acts with Unc13 as a template to coiled-coil
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38
Q

what happens if you remove Unc13 or Unc18?

A

you get zero synaptic fusion

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39
Q

what kind of regulate fusion is specially required for transmitter release?

A

FAST regulated fusion

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40
Q

what is the calcium sensory for fast transmitter release?

A

synaptotagmin

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41
Q

what does complexin do in colaboration with synaptotagmin for fast fusion?

A

they trap the SNAREs in a pre-fusion complex

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42
Q

what releases the pre-fusion SNARE complex?

A

calcium

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43
Q

what is Unc13 role in SNARE pre-fusion complex?

A

it regulates the number of pre-fusion complex and the localization of calcium channels

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44
Q

Why study exocytosis by studying transmitter release?

A

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

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45
Q

what are all the event that happens in a synaptic release? in what timeframe does this happen?

A
  1. presynaptic AP
  2. Ca2+ current
  3. exocytosis (release rate)
  4. evoked postsynaptic surrent
  5. postsynaptic AP
    Happens in only a few milliseconds!
46
Q

describe synaptotagmin structure

A

family of proteins that have two C2 domains

47
Q

how many syts (Synaptotagmin) are there in the mammalian genome? how many syts isoforms can support fast transmitter release?

A

17 Syts.
3 isoforms.

48
Q

what can mutations in calcium binding region cause?

A

alteration of the calcium dependency of vesicle release

49
Q

what happens when you get rid of Syts?

A

does not block transmitter release (unlike Unc13 or 18), but blocks FAST transmitter release

50
Q

what 2 syt isoforms are we interested in?

A

SytI and II for fast transmitter release

51
Q

what is the relationship of neurotransmitter release & calcium concentration? what explains this?

A

exponential to 4th or 5th power! syts can bind 2-3 calcium in a cooperative way

52
Q

how is Syt’s C2 domain affinity for calcium? what does this mean?

A

low affinity, which means faster but requires a high concentration of calcium

53
Q

how do synapses deal with the high concentration of calcium requirement?

A

the synaptic vesicles are localized right next to the calcium channels

54
Q

what is the Calyx of Held and why is it used to study synapses?

A

auditory synapse with a very large presynaptic terminus that allows to directly record from

55
Q

what is the best way of actually measuring the calcium dependence of release?

A

light-induced release of calcium

56
Q

at what calcium concentration does neurotransmitter release peak?

A

10 uM intracellular calcium. narrow range.

57
Q

what Syt is expressed at calyx of held?

A

Syt 2

58
Q

what is an osmotic/hyptotonic shock and what does it trigger at the calyx of held of Syt2 KO?

A

changing solution osmolarity with unsoluble Sucrose; it triggers the release of PRIMED vesicles only

59
Q

what stays the same/differs when Syt2 is KO at the calyx of held? (remember, only syt of calyx is Syt2)

A

same number of primed vesicles, no effect at low calcium conc.;
loss of the majority of evoked release from large calcium concentration

60
Q

what syt mediates asynchronous transmitter release?

A

syt7. syt7 can not support evoked release

61
Q

what does it mean that sytKO makes no difference in EPSP(Transmitter release) at low calcium concentrations?

A

Syt2 is not active at low Calcium concentration because it has low affinity for calcium

62
Q

what do C2A vs C2B domain of Syts bind to?

A

C2A binds 3 calcium.
C2B binds to the SNARE complex and 2 calcium.

63
Q

C2A and B domains bind calcium best when ?

A

there are phospholypids

64
Q

what happens if you get rid of C2A domain?

A

no more synaptic release

65
Q

how many of the 3 Syts involved in fast transmitter release do you need?

A

all 3 are important

66
Q

C2A may be important for coordinating
what?

A

multiple SNAREs to act together

67
Q

can you overcome the energy barrier of fusion with one SNARE complex?

A

no you need more than one. they can be coordinated via C2A.

68
Q

how many binding sites exist for SYTs in the SNARE complex?

A

2

69
Q

what is complexin?

A

coiled-coil protein important for transmitter release, but not required.
It is involved in Syt1 and 7 binding to SNARE

70
Q

syt 1, 2, and 9 have a specific ____ ______ that defines the syt 1 family

A

crystal structure

71
Q

what triggers the final zippering of fusion?

A

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
Q

what prevents the complete zippering up before calcium binding?

A

VAMP is partially occupied by complexin or Syt and is not completely in the coiled-coil

73
Q

what is Syt7? where is it not found?

A

plasma membrane syt protein important for asynchronous release. it is not found in synaptic vesicles

74
Q

unc18 is necessary for fusion, but it can also …?

A

INHIBIT fusion by sequesterin Syntaxin in a closed conformation

75
Q

how is priming related to unc18 function of inhibition of fusion?

A

priming works to relieve the Unc18 inhibition (releasing syntaxin)

76
Q

what is RRP?

A

readily releasable pool (primed vesicle)

77
Q

osmotic shock can release …? how?

A

release the RRP, because the two membranes (vesicular and synaptic) are already very close

78
Q

what is UNC13’s role?

A

(it’s a tether but also) determines the size of the RRP and releases syntaxin from Unc18.
important for modulation of release.

79
Q

what is post-tetanic potentiation?

A

the fact that RRP increases when there is a lot of release (probably homeostatic response to loss of vesicles)

80
Q

other than PTP what precisely increases the size of the RRP?

A

second messengers like diacylglycerol produced by activation of Gq

81
Q

describe the unc13 domain and what they bind

A
  • 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
Q

what Unc13 domains serve to activate it?

A

the domain in the PM: calmodulin binding, C1, and C2B binding domains;
normally inhibit Unc13. when they bind, they release the inhibition.

83
Q

what happens if you mutate calmodulin binding site in Unc13?

A

everything is normal except that post-tetanic potentiation is reduced, because the is no calcium-induced increase in RRP

84
Q

what is presumed to be calcium’s effect on Unc13’s function?

A

it accelerates Unc13’s ability to remove Unc18’s inhibitory effect on syntaxin

85
Q

what happened when you removed DAG binding domain (C1) on Unc13?

A

similar effect as calmodulin binding site removal: reduction of PTP

86
Q

what is RIM?

A

protein that links synaptic vesicles to calcium channel via Unc13

87
Q

how does RIM binding domain (C2A) on Unc13 allows for specific timing of activation?

A

RIM binding compete with Unc13 homodimerization that inactivates Unc13, to activate Unc13 only when it is at the synaptic terminal

88
Q

what happens if you delete RIM binding domain (C2A)?

A

Unc13 can still work

89
Q

do every species have a C2A domain in Unc13? what might that do?

A

no; they may have less localization of SNARE to calcium channels

90
Q

Can replace most active zone scaffold protein with just

A

Unc13 coupled to a subunit of the calcium channel important for release (?)

91
Q

BASICALLY what is Unc13’s role?

A

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
Q

what did they try to prove about syt2 localization in the paper?

A

already know it is postsynaptic, they proves that syt3 is also presynaptic

93
Q

what were the presynaptic markers?

A

vglut1 and synaptophysin

94
Q

what was the postsynaptic marker?

A

PSD95

95
Q

what did Syt3 align the best with?

A

bassoon: active pre synaptic zone

96
Q

did it vesicular markers colocalize with syt3? what can we conclude?

A

no: syt3 is not in vesicles

97
Q

they had synaptosomes with only vglut1, only psd95, or both. which ones colocalized with syt3? what does that mean?

A

synaptosomes with vglut1 AND PSD95, and synaptosomes with only vglut1.
this indicated presynaptic enrichment.

98
Q

did western blot show Syt3 in vesicles?

A

no

99
Q

what happened to spontaneous EPSCs, evoked EPSCs, and pre-synaptic calcium current in Syt3KO neurons at the calix of held? why?

A

nothing different because Syt3 doesn’t affect basal release properties

100
Q

what happened when they stimulate the presynaptic axon at low vs high frequencies in Syt3KO neurons?

A

no difference at low frequencies.
>10Hz: less synaptic release and slower recovery of pre-synaptic vesicles.

101
Q

what are the differences between BAPTA and EGTA calcium buffers?

A

BAPTA is very fast and can compete with synaptotagmin.
EGTA is slower and can’t compete.

102
Q

if EGTA is slow, why do they use it?

A

it can bind slow, higher affinity calcium

103
Q

what is the effect of EGTA on WT and Syt3KO neurons?

A

WT: EGTA reduces the recovery of transmission to the level of Syt3KO recovery.
Syt3KO: EGTA has no effect because the recovery is already

104
Q

what kind of calcium is buffered by EGTA?

A

residual calcium

105
Q

so what kind of calcium mediates Syt3 recovery actions if EGTA has no effect on Syt3KO?

A

Residual calcium

106
Q

were they able to rescue the recovery rates in SYT3KO by adding a modified Syt3 that doesn’t bind calcium?

A

no

107
Q

what is the major difference between Syt3 and Syt7?

A

Syt7 binds calcium slower than Syt3

108
Q

what are PPF vs Recovery thought to be mediated by?

A

PPF mediated by increased release probability.
Recovery mediated by accelerated vesicle replenishment. (Syt3)

109
Q

what were Syt7KO effect on neurons in calix of held and climbing fibers?

A

no effect on the recovery of transmission

110
Q

what was Syt3KO’s effect on PPF (pair pulse facilitation)?

A

lost of PPF

111
Q

what was Syt7KO’s effect on PPF (pair pulse facilitation)?

A

also reduced PPF

112
Q

what new model did they come up with at the end of the paper?

A

Syt3 promotes loose to tight docking of vesicles

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