Synaptic Vesicle Cycle: Exocytosis

membrane fusion tethering docking priming fusion

regulation brake calcium sensor location

general mechanism:vesicular transport

usually constitutivevesicles do not accumulatecannot isolate key intermediateunless process regulated

SV

SV Purification

synaptic vesicles the smallest biological membranesvery homogeneous in size, shapeseparate by density (equilibrium sedimentation) and size (velocity sedimentation):

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):

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

trafficking assay (Rothman)

assay for glycosylation of VSV-G proteinrequires transport between stacks from different cells

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)

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)

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?

SNARE distribution

BUT SNARE complex formation is promiscuousAND only some complexes produce fusion

what regulates the SNAREs?

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

(Rosenmund et al, 2002)

munc13

double KO = no releaserescue with different isoforms alone confers different forms

of short-term plasticity (Pr)mechanism?

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

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

(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:

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)

complexin binds to SNARE complex

(Pabst et al, 2000)

not to individual SNAREs

over-expression inhibits releasecomplexin KO ~synaptotagmin KO

(Giraudo et al., 2009)

central helix binds to SNARE complex specific mutations block binding

increase spontaneous releasereduce evoked release~KO

N-terminal accessory helix required for clamping

Ca++, synaptotagmin displace complexin allow completion of SNARE complex fusion

(Maximov et al, 2009)

but accessory helix clamps adjacent SNARE complex!!

spontaneous unclamping

zigzag

(Krishnakumar et al., 2011)

active zone

how are SVs coupled to Ca channels? nano-domain coupling: insensitive to EGTA (low affinity buffer)

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?

lipids

PC12 permeabilized cell assay (Martin)

ATP-dependent primingCa++-dependent triggering

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

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

Synaptic Vesicle Pools

(Rizzoli and Betz, 2005)

extreme functional heterogeneitybut do they differ biochemically?

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)

styryl dyes

image loss of dye with stimulation (exocytosis only)

blocking phos-phorylation slows exocytosis

dispersion correlateswith destaining--synapsin dispersionrequired for release

(Gitler et al., 2008)

synapsins increase recycling pool

Conclusions

total internal reflection fluorescence (TIRF) microscopy

individual exocytic eventsretinal bipolar cells

residents fuse fasterthan newcomers

(Zenisek and Almers, 2000)

References

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