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Transcript of Synaptic Vesicle Cycle: Exocytosis membrane fusion tethering docking priming fusion regulation brake...
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Synaptic Vesicle Cycle: Exocytosis
membrane fusion tethering docking priming fusion
regulation brake calcium sensor location
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general mechanism:vesicular transport
usually constitutivevesicles do not accumulatecannot isolate key intermediateunless process regulated
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SV
SV Purification
synaptic vesicles the smallest biological membranesvery homogeneous in size, shapeseparate by density (equilibrium sedimentation) and size (velocity sedimentation):
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synaptosomes: sheared-off nerve terminals
lysis in hypotonic buffer lysed pellet 1 (LP1): synaptic plasma membrane lysed pellet 2 (LP2): synaptic vesicles
gradient fractionation equilibrium (density) gradient velocity (size) gradient: SVs unique
size exclusion chromatography: controlled pore glass for organelles (rather than proteins):
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proteins excised from gel, sequenced:
differ in membrane association:peripheral membrane proteins (synapsin, synuclein)lipid-anchored (rab3, cysteine string protein)single TMD (type 2 synaptobrevin, 1 synaptotagmin)polytopic (synaptophysin, SV2)--quantified in Takamori et al (2006)
the function of many remain unknown
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trafficking assay (Rothman)
assay for glycosylation of VSV-G proteinrequires transport between stacks from different cells
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activity requires many proteins identified by functional complementation
inactivate extract with NEM (-SH reagent)add back protein from untreated extractNEM-sensitive factor (NSF)
NSF is an ATPase--used to find associated proteins:
binding in non-hydrolyzable ATPelute with ATP--releases only those bound
dependent on ATP--soluble NSF attachment receptors (SNAREs)
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specifically cleave SNAREs --less effect on spontaneous than evoked release
but how do we know they are functional?clostridial toxins block NT release --Zn-dependent proteases
(A. Brunger)
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revised model
zippering mechanismN-termini of v- and t-SNAREs togetherenergy for fusion provided by binding SNARE complex very stable --dissociates only by boiling in SDS! OR addition of ATP to NSF (before endocytosis)
?biochemical correlates of docking and priming?
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SNARE distribution
BUT SNARE complex formation is promiscuousAND only some complexes produce fusion
what regulates the SNAREs?
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SM proteins essential for fusionBUT munc18 (n-sec1) stabilizes closed state?!munc18 also binds to assembled SNARE complex --positive regulator as well as negative? switch to open?
?rab munc18 syntaxinGTP
regulation at t-SNARE itself: syntaxin has an auto-inhibitory domain --must be removed to form SNARE complex
(Sudhof and Rothman, 2009)
docking and priming: SM proteins
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(Rosenmund et al, 2002)
munc13
double KO = no releaserescue with different isoforms alone confers different forms
of short-term plasticity (Pr)mechanism?
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tomosyn has a SNARE motif ~synaptobrevin forms inhibitory complex with t-SNAREs
mutant shows increased readily releasable pool:
(McEwen et al, 2006)
sucrose
tomo KO, open syntaxin both rescue unc13 KO but only priming —not evoked release
EPSCs
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Ca++-dependent triggering
C2 mediates Ca++-dependent phospholipid binding could mediate Ca++-evoked release
dimer contains 4 C2 domains: does this confer the sensitivity to [Ca++]4? --delete one of the two C2 domains
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(Littleton and Bellen, 1994)
single C2 domain
Hill coefficient reduced from ~4 to ~2WT interpreted as dimer (4 C2 domains)
Drosophilaloss of syt eliminates evoked releasedelete one C2 domain:
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syt 1 KO (mouse)
wt KO
KO reduces synchronous release no effect on asynchronousincreased spontaneous release in Drosophila --not in mammals
multiple synaptotagmin isoforms vary in calcium sensitivity
complexin KO has similar phenotype
(Geppert et al, 1994)
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complexin binds to SNARE complex
(Pabst et al, 2000)
not to individual SNAREs
over-expression inhibits releasecomplexin KO ~synaptotagmin KO
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(Giraudo et al., 2009)
central helix binds to SNARE complex specific mutations block binding
increase spontaneous releasereduce evoked release~KO
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N-terminal accessory helix required for clamping
Ca++, synaptotagmin displace complexin allow completion of SNARE complex fusion
(Maximov et al, 2009)
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but accessory helix clamps adjacent SNARE complex!!
spontaneous unclamping
zigzag
(Krishnakumar et al., 2011)
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active zone
how are SVs coupled to Ca channels? nano-domain coupling: insensitive to EGTA (low affinity buffer)
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peptidergic vesicles(large dense core vesicles)
different calcium requirements LDCVs require more stimulation BUT have higher intrinsic Ca++ sensitivity --further from Ca++ entry sites
also involves SNAREs, synaptotagmins ?complexins as brake?
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lipids
PC12 permeabilized cell assay (Martin)
ATP-dependent primingCa++-dependent triggering
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1) PI phosphorylation (3 proteins required): PI transfer protein PI4K PIP5K
2) CAPS (Ca++-activated protein for secretion) ~munc13
--probably not unique to LDCVs
sequential lipid modification
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black widow spider venom (-LTX) triggers release independent of Ca++
how?
-LTX binds to neurexinneurexin binds to neuroligin important for specification of excitatory and inhibitory synapsesBUT binding depends on Ca++ ?!
calcium-independent receptor for -LTX --GPCR
neither neurexin or CIRL required for -LTX effect
regulation of synapse formation
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Synaptic Vesicle Pools
(Rizzoli and Betz, 2005)
extreme functional heterogeneitybut do they differ biochemically?
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stimulation disperses synapsin and other SV proteinsnot blocked by clostridial toxintriggered by Ca++ entry, phosphorylation
vesicle mobilization: synapsins
synapsins are peripheral membrane proteins
(Chi et al, 2001)
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styryl dyes
image loss of dye with stimulation (exocytosis only)
blocking phos-phorylation slows exocytosis
dispersion correlateswith destaining--synapsin dispersionrequired for release
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(Gitler et al., 2008)
synapsins increase recycling pool
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Conclusions
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total internal reflection fluorescence (TIRF) microscopy
individual exocytic eventsretinal bipolar cells
residents fuse fasterthan newcomers
(Zenisek and Almers, 2000)
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References
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