Behrends, Jan C.

Steinbrenner, Daniel

Elsevier - Publisher Connector 

Tuesday, March 8, 2011 407a[1] Aure´lien Roux, Katherine Uyhazi, Adam Frost, and Pietro De Camilli. Na-
ture 2006.
[2] Martin Lenz, Jacques Prost, and Jean-Francois Joanny. Phys. Rev. E 2008.
[3] Sandrine Morlot, Martin Lenz, Jacques Prost, Jean-Francois Joanny, Aure´-
lien Roux. submitted
2201-Pos Board B187
GED-Zippering Induced by PH Domain-Membrane Insertion Elicits Dy-
namin Assembly-Dependent GTPase Activation
Rajesh Ramachandran.
The multi-domain GTPase dynamin catalyzes the scission of deeply invagi-
nated clathrin-coated pits (CCPs) from the plasma membrane employing do-
main-specific protein-membrane and protein-protein interactions. By
monitoring the dynamics of pleckstrin homology (PH) domain-mediated dyna-
min-membrane, and GTPase effector domain (GED)-mediated dynamin-dyna-
min interactions in real time, the requirement for allosteric conformational
coupling in dynamin GTPase activation is revealed. Membrane insertion-defec-
tive dynamin-1 mutant, Dyn1 I533A uncouples intermolecular GED-GED in-
teractions from PH domain-membrane interactions, and is therefore rendered
incompetent in eliciting assembly-dependent GTPase activation. Therefore,
stable PH domain-membrane interactions allosterically trigger intermolecular
GED-zippering to effect dynamin assembly-dependent GTPase activation dur-
ing clathrin-mediated endocytosis.2202-Pos Board B188
Serotonin Release and Matrix Decondensation Precede Dissipation of the
Proton Gradient in Secretory Granule Exocytosis
Daniel Steinbrenner, Jan C. Behrends.
Exocytosis is a rapid but complex reaction involving multiple steps from the
initial protein interactions to formation of the fusion pore and release of vesicle
content. The latter is generally viewed as the last step in the sequence. For se-
cretory granules of mast cells, it has been proposed that dissipation of the gran-
ular proton gradient is an early event, permitting decondensation (swelling) of
the granule matrix and subsequent re-
lease of small molecule cargo, such as
serotonin (Willams and Webb 2000,
J.Cell Sci. 113, 3839). Here, we have ex-
amined this issue using simultaneous ca-
pacitance recording and amperometry as
well as optical (fluorescence and trans-
mitted light) recording. Using RBL-1
and RBL-2H3 cells loaded with seroto-
nin and transfected with synapth-
pHluorin as a reporter of intragranular pH, we find that, in contrast
alkalinization of the granules is the last event in the series, lagging by several
100 ms both serotonin release and matrix decondensation, which start immedi-
ately after fusion pore expansion. Our finding is in line with the idea that gran-
ular pH change is greatly delayed by the strong buffering capacity of both
serotonin and the proteoglycan matrix.2203-Pos Board B189
The Conformational Change of SNAP25 during the Exocytosis
Ying Zhao, Qinghua Fang, Khajak Berberian, Kassandra Kisler,
Wolfhard Almers, Manfred Lindau.
It is widely assumed that a conformational change in the SNARE complex
induces fusion pore opening and transmitter release. The SNAP-25 FRET
construct SCORE (SNARE complex reporter) has CFP as FRET donor in-
serted at the N-terminal end of SN1 and the fluorescent protein Venus
(YFP) as FRET acceptor inserted at the N-terminus of SN2. It was previ-
ously shown that SCORE that reports a FRET change in response to stimu-
lation that is calcium dependent but is not abolished by tetanus toxin
treatment, which abolishes fusion (An and Almers 2004 Science
306:1042). To determine if the conformational change reported by SCORE
is directly associated with fusion we monitored SCORE fluorescence in chro-
maffin cells in TIRF mode while exocytotic events at the cell bottom were
spatially and temporally monitored by a microfabricated electrochemical de-
tector (ECD) array with four Pt electrodes patterned on a glass coverslip
(Hafez et al. PNAS 2005 102:13879). After the SCORE-expressing cells
were placed on the top of the ECD array, the amperometric currents and
fluorescence images were simultaneously recorded. Based on the charge ra-
tios of oxidation currents recorded by the four electrodes, the localization ofsingle exocytotic events was determined. The time course of CFP and YFP
fluorescence in the 4 camera pixels at the release site was extracted for 10 s
time intervals before and after the amperometric spike. Averaging >300
events revealed a transient increase of YFP and a transient decrease of
CFP fluorescence by ~2.5% at the time of amperometric events, The in-
creased FRET level returned to the pre-exocytotic level within ~5s. This re-
sult directly indicates that the SNAP25 conformational change is directly
associated with fusion. Supported by NIH grant R01GM085808.
2204-Pos Board B190
On-Chip Electroporation Results in Calcium- and Chloride- Stimulated
Exocytosis Assayed with Planar Electrochemical Microelectrodes
Jaya Ghosh, Xin Liu, Kevin D. Gillis.
We are developing microfabricated devices consisting of arrays of electro-
chemical electrodes in order to increase the throughput of single-cell mea-
surements of quantal exocytosis from neuroendocrine cells and to develop
technology that allows simultaneous electrochemical detection and fluores-
cence imaging of single fusion events. One component of this effort is to
develop on-chip methods for stimulating exocytosis from select cell popula-
tion on the chip. We have demonstrated that the same electrochemical mi-
croelectrode can be used to electroporate an adjacent cell and then measure
quantal exocytosis using amperometry. Trains of voltage pulses, 5-7 V in
amplitude and 0.1-0.2 ms in duration, can reliably trigger exocytosis in
Ca2þ-containing bath solutions using gold and diamond-like-carbon elec-
trodes. Electrodes fabricated from Indium-Tin-Oxide were less effective at
eliciting exocytosis via electroporation. Higher rates of exocytosis were ob-
served with bath solution containing higher [Ca2þ]e, and experiments with
semi-transparent electrodes and the Ca2þ indicator fura-4F demonstrate
a rise in [Ca2þ]i upon electrical stimulation. Little or no exocytosis was ob-
served in Ca2þ-free (5 mM EGTA) solutions with glutamatic acid as the
major anion. Surprisingly, exocytosis could be elicited upon electroporation
in 0 Ca / 5 mM EGTA solutions if the bath contained high Cl- concentra-
tions (140 mM NaCl or KCl). This is surprising since the same solution
does not induce exocytosis when loaded into the cell through a patch pipette
during whole-cell recording. We conclude that electric fields sensitize gran-
ules to undergo exocytosis in response to Cl-. Supported by NIH R01
NS048826.
2205-Pos Board B191
Functional Characterization of Putative Synaptotagmin-Binding Inter-
faces in SNAP-25
Ralf Mohrmann, Heidi De Wit, Emma Connell, Bazbek Davletov,
Matthijs Verhage, Jakob B. Sørensen.
SNARE complexes possess multiple negative surface charges that probably
mediate association with regulatory factors during exocytosis. Several dis-
tinct groups of negatively charged residues in SNAP-25 have been identified
as essential for binding synaptotagmin-1 (syt-1). One group exists at the C-
terminal end (D172/D179/D186/D193), while another site extends around
Layer 0 (D51/E52/E55). A third potential group may consist of D166/
E170, as we previously found that E170 is essential for fast release. To in-
vestigate the nature of the binding interface, we introduced alanine-substitu-
tions at the three putative binding sites and characterized the resulting
mutant variants after expression in SNAP-25 -/- chromaffin cells. Secretion
was induced by photolysis of caged-Ca2þ and assayed by capacitance mea-
surements and amperometric recordings. We found substantial differences in
the phenotypes of these mutant variants, which excludes that all sites coop-
erate in the same mode of syt-1 interaction. Though no variant fully phe-
nocopied the changes seen in syt-1 -/- cells, SNAP-25A D51A/E52A/
E55A caused a slowdown of secretion reminiscent of the syt-1 -/- phenotype.
SNAP-25A D166A/E170A decreased total release substantially and abol-
ished the fast burst component, while SNAP-25A D172A/D179A/D186A/
D193A had little effect on secretion. Pull-down assays demonstrated that
mutation of the two groups around Layer 0 indeed restricted syt-1 binding,
while mutation of the C-terminal site did not decrease affinity towards syt-1.
By electron microscopy we found that all mutations significantly affected
docking of vesicles, which depends on both SNAP-25 and syt-1 (De Wit
et al., 2009, Cell 138:935-946). This implies that SNAP-25/Syt-1 assisted
docking might rely on another mode of interaction between SNAP-25 and
syt-1 than fusion triggering. Our data thus suggest that the interaction be-
tween SNAREs and syt-1 might involve multiple interaction modes, and
that negative charges around Layer 0 most likely function as a syt-1 binding
interface during exocytosis triggering.


Serotonin Release and Matrix Decondensation Precede Dissipation of the Proton Gradient in Secretory Granule Exocytosis 

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Serotonin Release and Matrix Decondensation Precede Dissipation of the Proton Gradient in Secretory Granule Exocytosis

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