Un double rôle de la VH - Club Exocytose

Lundi 26 mai - Session 1 – 16h10/18h50
Transmitter release at the mouse calyx of Held synapse with
and without cysteine string protein
R. Schneggenburger*, M. Wölfel*, R. Fernández-Chacon#@, T.C. Südhof#.
*
Max-Planck Institut für biophysikalische Chemie, Abt. Membranbiophysik, AG Synaptische
Dynamik und Modulation, Göttingen, Germany # Center for Basic Neuroscience, U.T.
Southwestern Medical Center, Dallas, Tx USA @ Dept. of Physiology and Biophysics, University
of Sevilla School of Medicine, Sevilla, Spain.
T
he calyx of Held forms an excitatory, glutamatergic synapse onto principal neurons
of the medial nucleus of the trapezoid body, a relay nucleus in the auditory
brainstem. Due to its unusually large size (about 10 - 15 µm), patch-clamp recordings
can be made directly at the calyx of Held, thus offering unique possibilities for studying
Ca2+-secretion coupling at a CNS nerve terminal (see e.g. Schneggenburger et al. TINS
2002 for a review). Here, we investigate the role of the presynaptic vesicle protein
cysteine string protein (CSP) in Ca2+-secretion coupling at the calyx of Held, by using
CSP knock-out mice (-/-) and their wild-type littermates (+/+) as controls.
Knock-out mice were viable at birth, but they showed early lethality at 3-4 weeks
of age. In slices from P9 – P13 mice, the presynaptic voltage-gated Ca2+-currents
(ICa,pre) were 1.1 ± 0.37 nA (n=18) and 1.26 ± 0.33 nA (n=13) for CSP -/- and +/+ mice,
and thus, not significantly different. The voltage-dependence of ICa,pre, as well as its
sensitivity to ω-Aga-IVa (200 nM) were unchanged (92 ± 4% and 93 ± 7% block for CSP
-/- and +/+). Thus, in both genotypes P/Q type Ca2+ channels contributed a large
fraction of ICa,pre. Also, transmitter release, assessed by recording AMPA-receptor
mediated excitatory postsynaptic currents (EPSCs) after various types of presynaptic
stimuli was not changed in P9 - P13 CSP -/- mice, showing that the lack of CSP does
not lead to an immediate loss of function in Ca2+-secretion-coupling.
Given the finding of an early lethality of CSP-/-mice (by P25, ≈ 50% of CSP -/mice died), we next analyzed synaptic transmission at the calyx of Held at a later
developmental stage (P20 - P23). In many, but not all synapses of CSP -/- mice, we
found a largely reduced EPSC amplitude, and the remaining EPSC was generated by
more asynchronous transmitter release. EPSC amplitudes were 5.6 ± 5.4 nA and 13.3 ±
3.1 nA for CSP -/- and +/+ (n = 5 each). The delayed onset of a synaptic phenotype in
CSP -/- mice might be caused by a loss of the proposed chaperone function of CSP in
presynaptic terminals (Tobaben et al. 2001, Neuron) and a subsequent, delayed
accumulation of misfolded presynaptic proteins of various molecular identity.
Alternatively, a protein-protein interaction involving CSP and other presynaptic
protein(s) might become essential for synaptic function at a late stage in postnatal
development.
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Lundi 26 mai - Session 1 – 16h10/18h50
Release and retention of proteins after exocytosis by single
chromaffin granules
David Perrais!,2, Justin W. Taraska1, Ingo Kleppe3 & Wolfhard Almers1
1
Vollum Institute, Oregon Health & Science University, Portland, OR, USA. 2 present address,
Physiologie Cellulaire de la Synapse, CNRS and Université Bordeaux 2, Bordeaux, France. 3
Physiological laboratory, University of Cambridge, Cambridge, UK.
C
hromaffin cell dense core granules package numerous proteins, but the
characteristics of their release following exocytosis is largely unknown. We have
imaged single exocytic events with evanescent field microscopy in chromaffin cells
transfected with dense core proteins fused with GFP. We have used three different
proteins, neuropeptide Y, tissue plasminogen activator, which are both secreted, and a
membrane protein, VAMP2. We imaged the cells at high time resolution (20 images / s)
while stimulating them with voltage steps and recording the membrane capacitance with
a patch pipette. We observed two classes of protein behavior following exocytosis
caused by depolarization, either fast complete release, or incomplete release after the
opening and closing of the fusion pore. Retained protein was used as a marker to trace
fusion pore open time, which varied from less than a second to several minutes. The
proportion of each behavior differed between proteins observed and the extracellular
calcium concentration, demonstrating a “molecular sieve” mechanism where granule
components are selectively released from individual granules during exocytosis.
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Lundi 26 mai - Session 1 – 16h10/18h50
Transfert Ca2+/calmoduline-dépendant du domaine
juxtamembranaire de la VAMP 2 de la bicouche cis vers la
bicouche trans ?
Luc De Haro1, Géraldine Ferracci2, Sandrine Opi3, Cécile Iborra1, Christian Lévêque1,2,
Stéphanie Quetglas1, Raymond Miquelis2, Michael Seagar1.
1- Laboratoire Neurobiologie des Canaux Ioniques, INSERM U464, 13916 Marseille.
2- Unité méthodologique des Interactions Moléculaires, IFR Jean Roche, 13916 Marseille.
3- Laboratoire d’Ingénierie des Systèmes Macromoléculaires, CNRS U9027, 13402 Marseille.
L
a synaptobrévine (VAMP2), protéine membranaire des vésicules synaptiques, est
impliquée dans la formation des complexes SNARE, processus indispensable à
l’exocytose régulée. Le domaine entre les acides aminés 77-90 de la VAMP fixe des
phospholipides chargés ou le complexe Ca2+/calmoduline (Cam). Ces interactions
sont mutuellement exclusives : la Cam inhibe la fixation de 3H-liposomes (DPPC 75%
DOPS 25%) sur la GST-VAMP. Par analyse SPR Biacore*, la Cam peut décrocher la
GST-VAMP 1-96 fixée à des liposomes immobilisés sur ship hydrophobe. Par ailleurs,
les expériences de mutagénèse montrent que le domaine 77-90 joue un rôle essentiel
dans l’exocytose induite dans les cellules PC12 (1,2).
L’analyse, à l’aide de peptides synthétiques, de la fluorescence des 2 Trp en
positions 89 et 90 montre que le domaine 77-90 se lie à des liposomes DOPS 25% en 2
étapes : une adsorption avec augmentation du signal, puis une insertion avec
modification du λ du pic d’émission. L’insertion n’est observée que si la couche lipidique
est en phase liquide.
A priori la VAMP pourrait interagir avec des lipides vésiculaires (cis) ou de la
membrane plasmique (trans). L’hypothèse cis est confirmée par l’inaccessibilité des
épitopes de la VAMP 77-90 lorsque la protéine entière est incorporée dans un
protéoliposome DOPS 25%. De plus l’interaction cis inhibe la liaison des liposomes en
trans. Par contre, l’incorporation dans un proteoliposome 100% PC (phopspholipide non
chargé qui n’induit pas de liaison en cis) permet la fixation de 3H-liposomes en trans.
Quel est le rôle de la Cam dans ce système ? Nos résultats montrent que la Cam
déplace les interactions lipidiques en cis ce qui déclenche la liaison irréversible des
lipides en trans. Ainsi, pendant l’assemblage du complexe SNARE, la Cam transférerait
le domaine juxtamembranaire de la VAMP d’une adsorption sur la membrane
vésiculaire vers une insertion dans la membrane plasmique. La conséquence d’un tel
processus Ca2+- dépendant serait la mise en quasi contact des deux bicouches.
1- Quetglas S, Iborra C, Sasakawa N, de Haro L, Kumkura K, Sato K, Lévêque C, Seagar M. Ca2+dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding
domain of synaptobrevin. PNAS 2000 ; 97(17) : 9695-700.
2- Quetglas S, Lévêque C, Miquelis R, Sato K, Seagar M. Calmodulin and lipid binding to synaptobrevin
regulates calcium-dependent exocytosis. EMBO J 2002 ; 21(15) : 3970-9.
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Lundi 26 mai - Session 1 – 16h10/18h50
Genetic evidence suggesting that the V0 domain of VATPase is required for exocytosis independently of its role
as a proton pump
S. Liégeois, A. Janoshazi, J.M. Garnier, M. Labouesse
IGBMC, BP10142, F-67400 ILLKIRCH
C
urrent models of exocytosis are based on genetic data in yeast and biochemical
data in neurons. According to the SNARE hypothesis, the final sequence of events
in exocytosis would involve the pairing of a v-SNARE protein on the vesicle membrane
with a t-SNARE protein on the target membrane, followed by a complete fusion of the
two membranes. Whether the SNARE hypothesis is valid for all cargo proteins during
exocytosis in epithelial cells, particularly at the apical membrane, is unclear. We
previously showed that the gene che-14 is required for secretion in the epidermis. We
performed a genetic screen to uncover additional mutations with a che-14-like
phenotype and cloned two of them, rdy-1 and rdy-2 (rdy, Rod-like larval lethality and
DYe-filling defect).
rdy-1 encodes an a subunit of V-ATPase, a proton pump present in all cells. The
V-ATPase comprises two domains, a cytoplasmic domain responsible for ATP
hydrolysis called V1, and a transmembrane domain responsible for proton transport
called V0 (the a subunit belongs to the latter). RDY-1 is localized at the apical
membrane of excretory, support and epidermal cells. As shown by mosaic experiments
rdy-1 activity in the excretory cell is essential for survival, while its activity in the
epidermis is required for secretion. Preliminary studies indicate that RDY-1 may be
required for secretion of specific but not all proteins.
Recent biochemical analysis in yeast indicates that the V0 domain might have a
function in membrane fusion. To find out if the rdy-1 mutant phenotypes are associated
with a membrane fusion function or with a proton pump activity, we performed sitedirected mutagenesis in order to separate both activities. We altered conserved
residues on a transgene and introduced them in a rdy-1 null animal to assay survival
(as a probable measure of proton pump activity) and cuticle secretion. Of 18 mutations
tested so far, E830Q allows almost normal secretion but makes the animal sick and
vacuolated, while L786S impairs secretion but does not seem to affect the excretory
system. These results support the notion that the V-ATPase might act not only as a
proton pump, but also as part of a fusion pore during secretion at the apical membrane
in epithelial cells.
The rdy-2 gene encodes a tetra-spanning membrane protein without any
homology in protein databases. RDY-2 is also localized at the apical membrane of
some ectodermal epithelial cells, and preliminary evidence using FRET analysis
indicates that it interacts with RDY-1. Moreover, it is interesting to note that the first
protein suspected to be required for apical sorting in vertebrates (VIP/MAL) is also a
tetraspanin.
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Lundi 26 mai - Session 1 – 16h10/18h50
Un double rôle de la V-H+ATPase lors de la libération de
neurotransmetteur?
Nicolas Morel
Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, UPR 9040, 91198 Gif sur Yvette,
France.
U
ne isoforme particulière de l’ATPase à protons est présente dans les terminaisons
nerveuses. Associée aux vésicules synaptiques, elle est nécessaire à
l’accumulation du neurotransmetteur dans son organelle de stockage. Associée à la
membrane présynaptique, elle interagit avec les complexes SNAREs. Le domaine
membranaire de la V-ATPase pourrait participer à la constitution d’un pore de fusion
permettant la libération calcium-dépendante du contenu vésiculaire.
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