Synaptic Vesicle Cycle

Transcription

1 Synaptic Vesicle Cycle proteins SNAREs SNARE complex formation calcium-regulated fusion maintenance lipids (peptidergic vesicles)

2 vesicle budding and fusion specificity? regulation by calcium? --usually constitutive

3 SV Purification SV separation by density (equilibrium sedimentation) and by size (velocity sedimentation)--svs unique

4 differential centrifugation to get synaptosomes lysis in hypotonic buffer to release SVs density gradient controlled pore glass (size exclusion chromatography) --for organelles (rather than proteins)!

5 proteins excised from gel, sequenced: all different kinds: peripheral membrane proteins (synapsin, synuclein) lipid anchored (rab3, cysteine string protein) single TMD (type 2 synaptobrevin, 1 synaptotagmin) polytopic (synaptophysin, SV2) H+-ATPase (soluble and membrane sectors) --quantified in Takamori et al (2006)

6 Trafficking Assay (Rothman)

7 assay relies on addition of sugars to VSV-G protein requires transit from cis-golgi of VSV-G-expressing mutant CHO cells (defective in glycosylation) to later Golgi stack of WT CHO cells without VSV-G activity requires cell extract as well as membranes many proteins required, how to identify them? --inactivate extract with NEM (-SH reagent) --add back protein from untreated extract --if one protein, can be purified: NEM-sensitive factor NSF is an ATPase--used to find associated proteins

8 binding in non-hydrolyzable ATP elute with ATP--releases only those bound dependent on ATP --soluble NSF attachment receptors (SNAREs) but how do we know they are functional? clostridial toxins block NT release are Zn-dependent proteases specifically cleave SNAREs --toxins have less effect on spontaneous than evoked release

9 SNARE complex

10 revised model zippering mechanism N-termini of v- and t-snares together energy for fusion provided by binding SNARE complex very stable --dissociates only by boiling in SDS! OR addition to ATP to NSF

11 SNARE: spatial specificity? BUT SNARE complex formation is promiscuous! so what regulates SNAREs? rab proteins? (small GTPases)

12 SNARE complex formation SM proteins n-sec1 (munc-18): both pos and neg roles in neurotransmission?

13 syntaxin has an auto-inhibitory domain --must be removed to form SNARE complex GTP rab3 munc-13/munc-18 syntaxin sequential protein-protein interactions result in formation of SNARE complex

14 tomosyn inhibits SNARE complex tomosyn has a SNARE motif ~synaptobrevin forms inhibitory complex with t-snares mutant shows increased RRP (McEwen et al, 2006) tomo KO, open syntaxin (downstream) --rescue munc13 KO

15 Calcium-regulated fusion: C2 domains C2 mediates Ca ++ -dependent phospholipid binding could mediate Ca ++ -evoked release does calcium regulates other presynaptic events?

16 munc13 (another SM protein) (Rosenmund et al, 2002) double KO = no release rescue with different isoforms alone confers different forms of short-term plasticity how does munc13 work?

17 synaptotagmin (Drosophila) single C2 domain (Littleton and Bellen, 1994) Hill coefficient reduced from ~4 to ~2 WT interpreted as dimer (4 C2 domains)

18 synaptotagmin 1 (mouse KO) wt k-o (Geppert et al, 1994) k-o reduces synchronous release no effect on asynchronous increases spontaneous release complexin k-o has same phenotype multiple synaptotagmin isoforms

19 complexin binds to SNARE complex (Pabst et al, 2000) not to individual SNAREs

20 (Chen et al, 2002) over-expression inhibits release complexin KO ~synaptotagmin KO

21 flipped SNARE assay (Rothman) SNAREs flipped to outside of cell (Hu et al, 2003) blue nuclei appear in red cytoplasm: fusion

22 regulation by synapse formation black widow spider venom (α-ltx) binds to neurexin but depends on Ca ++ and α-ltx action does not calcium-independent receptor for α-ltx is a GPCR neurexin binds to neuroligin important for specification of excitatory and inhibitory synapses

23 maintenance of the nerve terminal csp a chaperone (DnaJ domain) linked to membrane by extensive palmitoylation originally thought to cluster Ca ++ channels

24 9-11 days --no phenotype early days (Fernandez-Chacon et al, 2004) csp k-o exhibits degeneration maintenance factor?

25 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

26 Lipids PC12 permeabilized cell assay (Martin) ATP-dependent priming Ca ++ -dependent triggering

27 sequential lipid modification 1) PI phosphorylation (3 proteins required): PI transfer protein PI4K PIP5K 2) CAPS (Ca++-activated protein for secretion) ~munc-13

28 what do the lipids do in exocytosis? need cone-shaped at neck of fusion pore may influence SNARE zippering --direct roles in fusion as well as regulation

29 References Discussion paper Giraudo, C.G., Eng, W.S., Melia, T.J. and Rothman, J.E A clamping mechanism Involved in SNARE-dependent exocytosis. Science 313: Reviews Synapses by Cowan, Sudhof and Stevens Johns Hopkins Press. Jahn, R. Lang, Sudhof, TC Membrane fusion. Cell 112: Chen, X., Tomchick, D.R., Kovrigin, E., Arac, D., Machius, M., Sudhof, T.C., and Rizo, J. (2002). Three-dimensional structure of the complexin/snare complex. Neuron 33, Fernandez-Chacon, R., Wolfel, M., Nishimune, H., Tabares, L., Schmitz, F., Castellano-Munoz, M., Rosenmund, C., Montesinos, M.L., Sanes, J.R., Schneggenburger, R. et al. (2004). The synaptic vesicle protein CSP alpha prevents presynaptic degeneration. Neuron 42, Geppert, M., Goda, Y., Hammer, R.E., Li, C., Rosahl, T.W., Stevens, C.F., and Sudhof, T.C. (1994). Synaptotagmin I: a major Ca++ sensor for transmitter release at a central synapse. Cell 79, Hay, J.C., and Martin, T.F.J. (1992). Resolution of regulated secretion into sequential MgATPdependent and calcium-dependent stages mediated by distinct cytosolic proteins. J. Cell Biol. 119, Littleton, J.T., Stern, M., Perin, M., and Bellen, H.J. (1994). Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants. Proc. Natl. Acad. Sci. USA 91, McEwen, J.M., Madison, J.M., Dybbs, M., and Kaplan, J.M. (2006). Antagonistic regulation of synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51, McNew, J.A., Parlati, F., Fukuda, R., Johnston, R.J., Paz, K., Paumet, F., Sollner, T.H., and Rothman, J.E. (2000). Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, Pabst, S., Hazzard, J.W., Antonin, W., Sudhof, T.C., Jahn, R., Rizo, J., and Fasshauer, D. (2000). Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions. J Biol Chem 275, Rosenmund, C., Sigler, A., Augustin, I., Reim, K., Brose, N., and Rhee, J.S. (2002). Differential control of vesicle priming and short-term plasticity by Munc13 isoforms. Neuron 33, Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B.R., and Montecucco, C. (1992). Tetanus and botulinum-b neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, Sollner, T., et al SNAP receptors implicated in vesicle targeting and fusion. Nature 362, Sutton, R.B., Fasshauer, D., Jahn, R., and Brunger, A.T. (1998). Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395, Takamori, S. et al Molecular anatomy of a trafficking organelle. Cell 127, Walent, J.H., Porter, B.W., and Martin, T.F. (1992). A novel 145 kd brain cytosolic protein reconstitutes Ca++-regulated secretion in permeable neuroendocrine cells. Cell 70,