Rhodamine B

Eyes were fixed in 4% PFA in PBS at 4°C overnight and washed in PBS. Retinas were dissected, permeabilized in PBS, 1% BSA, and 0.5% Triton X-100 at 4°C overnight, rinsed in PBS, washed twice in PBlec (PBS, pH 6.8, 1% Triton-X100, 0.1 mM CaCl₂, 0.1 mM MgCl₂, 0.1 mM MnCl₂), and incubated in biotinylated isolectin B4 (Bandeiraea simplicifolia; L-2140; Sigma-Aldrich) 20 μg/ml in PBlec at 4°C overnight. After five washes in PBS, samples were incubated with streptavidin conjugates (Alexa 488, 568, or 633; Molecular Probes) diluted 1:100 in PBS, 0.5% BSA, and 0.25% Triton X-100 at 4°C for 6 h. TO-PRO 3 (1:1,000; Molecular Probes) served for nuclear staining. After washing and a brief postfixation in PFA, the retinas were either flat mounted using Mowiol/DABCO (Sigma-Aldrich) or processed for multiple labeling. The following antibodies were used: GFAP (1:75; Z 0334; Dako), VEGFR2 (1:50; 555307; BD PharMingen), VEGFR2 (1:50; AF644; R&D Systems), F4/80 (1:100; MCAP497; Serotec), fibronectin (1:200; A 0245; Dako), VE-cadherin (1:1, culture supernatant provided by Dietmar Vestweber, University of Muenster, Muenster, Germany), and Ki67 (1:200; NCL-Ki67p; Novo Castra). Alexa-488, 568, or 633 conjugated secondary antibodies (Molecular Probes). Rhodamine-phalloidin served for actin staining (1:40; Molecular Probes). VEGFR2 signal was amplified using the TSA^TM Fluorescein System (NEL701; NEN) according to instructions. Flat mounted retinas were analyzed by fluorescence microscopy using a Nikon E1000 microscope equipped with a digital camera (Nikon Coolpix 990) and by confocal laser scanning microscopy using a Leica LCS NT. Images were processed using Adobe Photoshop^®.
For visualization of vascular lumina, FITC-conjugated Dextran (FD-2000S; Sigma-Aldrich) was warmed to 37°C and perfused through the heart of deeply anaesthetized mice (Avertin, intraperitoneally 10 μl/g body weight).
Mouse VEGF-A, PDGF-B, VEGFR1, and VEGFR2 cDNA fragments were used for whole mount in situ hybridization as described (Fruttiger, 2002 (link)). For double labeling, immunolabeling was performed after a 10-min postfixation in 4% PFA.
BrdU labeling was achieved by a 2-h BrdU pulse before fixation (100 μg Brd U/g body weight, intraperitoneally). For double labeling, isolectin labeling was followed by a 30 min 4% PFA fixation, three washes in PBS, a 1-h incubation in 6 M HCl and 0.1% Triton X-100, six washes in PBT, blocking, and anti-BrdU antibody (1:50, 347580; BD PharMingen) incubation.

Plasmid Constructs—Plasmids for mouse synaptobrevin 2 and
the t-SNARE heterodimer (mouse SNAP-25B and rat syntaxin 1A), were kindly
provided by J. E. Rothman, Columbia University, New York. C2A, C2B, and C2AB
were expressed in pGEX-2T or -4T as described previously
(38 (link)). Syb and the t-SNARE
complex were expressed as previously described
(18 (link),
19 (link)). The overlapping primer
method was used to generate site-directed point mutations in a modified
pTrcHisA construct containing the syt 1 C2AB domain (amino acids
95–421). This modified pTrcHisA vector has the gene 10-leader, Xpress
Epitope, and enterokinase recognition and cleavage site removed.
Protein Expression and Purification—To generate His-tagged
syb and t-SNARE heterodimers, Escherichia coli were grown at 37
°C to an A₆₀₀ of 0.8, and protein expression was
induced with 0.4 mm isopropyl
1-thio-β-d-galactopyranoside. Four hours after induction the
bacteria were collected by centrifugation, and the pellet was resuspended in
resuspension buffer (25 mm HEPES-KOH, 400 mm KCl, 20
mm imidazole, and 5 mm 2-mercaptoethanol). Resuspended
bacteria were subjected to sonication (2 × 45 s, 50% duty cycle). Triton
X-100 (2%), protease inhibitors (1 mg of aprotinin, pepstatin, and leupeptin;
0.5 mm phenylmethylsulfonyl fluoride), and 0.1 mg/ml RNase and
DNase were added to the sonicated material, and the mixture was incubated for
2–3 h with rotation at 4 °C. Insoluble material was removed by
centrifugation (Beckman JA17 rotor, 17K rpm), and the supernatant was applied
to a Ni²⁺ column using an AktaFPLC™ (GE-Amersham
Biosciences). The column was washed extensively with resuspension buffer
containing 1% Triton X-100 and then 1% n-octylglucoside wash buffer
(25 mm HEPES-KOH, 400 mm KCl, 50 mm imidazole, 10% glycerol, 5 mm 2-mercaptoethanol, 1%
n-octylglucoside). The bound protein was eluted using
n-octylglucoside wash buffer with 500 mm imidazole.
For the C2AB point mutants, E. coli were grown as above, however,
following addition of isopropyl 1-thio-β-d-galactopyranoside
bacteria were grown for an additional 4 h at 30 °C. Bacteria were
collected by centrifugation, resuspended in His₆ buffer (25
mm HEPES-KOH, 500 mm NaCl, 20 mm imidazole),
and sonicated as mentioned above. Samples were incubated with 1% Triton X-100
and protease inhibitors for 1 h followed by centrifugation to remove the
insoluble material. The supernatant was incubated with
Ni²⁺-Sepharose HP beads overnight. The following day, the
Ni²⁺ beads were washed with 20 volumes of His₆ buffer
containing 1 m NaCl, 20 volumes His₆ buffer supplemented
with 0.1 mg/ml RNase and DNase, and eluted with 1.5 volumes of elution buffer
(25 mm HEPES-KOH, 500 mm NaCl, 500 mm imidazole). Eluted protein was dialyzed against 50 mm HEPES-KOH,
150 mm NaCl, and 10% glycerol.
For glutathione S-transferase-tagged proteins, E. coli were grown as described above for the point mutants. However, they were
resuspended in phosphate buffered-saline containing 10% glycerol and 1
mm dithiothreitol. Bacteria were sonicated, treated with Triton
X-100, and protease inhibitors. Insoluble material was removed as described.
The supernatant was collected, and incubated overnight at 4 °C with
glutathione-Sepharose beads with rotation. Beads were washed extensively with
phosphate buffered-saline (10% glycerol, 1 mm dithiothreitol)
containing 0.1 mg/ml RNase and DNase, and the protein was removed by thrombin
cleavage (50 units/ml of bead slurry for 2 h at 20 °C). The supernatant
was collected and treated with phenylmethylsulfonyl fluoride to inactivate the
thrombin.
All proteins were analyzed by SDS-PAGE and stained with Coomassie Blue to
determine the purity and concentration against a bovine serum albumin standard
curve. The concentration of C2A, C2B, and C2AB was verified using a BCA
protein assay kit compatible with reducing agents.
Protein Reconstitution—Vesicles were prepared as described
previously (19 (link)). Briefly,
lipids supplied in chloroform were combined in various molar ratios (t-SNARE
vesicles: 15% PS, 30% PE, 55% PC; v-SNARE vesicles: 15% PS, 27% PE, 55% PC,
1.5% NBD-PE, 1.5% Rhodamine-PE), dried under a stream of nitrogen, and
subjected to vacuum for >1 h. Proteins to be reconstituted were diluted in
elution buffer to yield ∼100 copies per vesicle. Syb was diluted to 0.19
mg/ml and the syntaxin·SNAP-25 complex was diluted to 0.8 mg/ml in
elution buffer. The dried lipid film was solubilized using these respective
protein mixes and subsequently diluted with reconstitution buffer (25
mm HEPES-KOH, 100 mm KCl, 10% glycerol, 1 mm dithiothreitol). Protein-free vesicles were prepared as described above;
however, the protein was omitted.
Vesicles were dialyzed against reconstitution buffer overnight, changing
the buffer once. The dialyzed vesicles were collected, mixed with 80%
Accudenz, and transferred into ultra clear centrifuge tubes. A step gradient
was prepared by addition of 30 and 0% Accudenz layers onto the vesicle layer.
The samples were centrifuged at 41,000 rpm for 5 h (SW-41 rotor) or 55,000 rpm
for 1.75 h (SW-55 rotor). Vesicles were collected from the 0–30%
interface and analyzed by SDS-PAGE to verify protein incorporation.
Fusion Assays and Data Analysis—Fusion assays were carried
out in white-bottom 96-well plates with total reaction volumes of 75 μl.
Each reaction contained 45 μl of t-SNARE vesicles or protein-free vesicles,
5 μl of NBD-Rhodamine-labeled v-SNARE vesicles, and 1.5 μl of 10
mm EGTA. C2AB, C2A, or C2B were added to each reaction as indicated
in the figures. Samples were preincubated at 37 °C for 20 min followed by
injection of 5 μl of 18 mm Ca²⁺ to give a final
concentration of 1 mm free Ca²⁺. Following
Ca²⁺-injection fluorescence intensity was monitored for 60 min at
37 °C using a BioTek Synergy HT plate reader equipped with 460/40
excitation and 530/25 emission filters. The maximum fluorescence signal was
obtained by addition of 25 μlof n-dodecyl
β-d-maltoside to each reaction well; samples were monitored
for an additional 30 min until a stable baseline was obtained.
The fusion data were normalized by setting the initial time point to 0% and
the maximal fluorescence signal in detergent to 100%. All graphs and plots
were generated and analyzed using Prism 4.0 software (GraphPad, Inc.).
Co-flotation and Assembly Assays—100-μl reactions were
prepared containing 50 μm C2A or C2B or 10 μm C2AB, 45 μl of either t-SNARE heterodimer, syntaxin alone, or protein-free
vesicles, 2 μl of 10 mm EGTA, and reconstitution buffer in the
presence or absence of 1 mm free Ca²⁺. Components were
incubated at room temperature for 30 min with shaking. Following incubation,
the vesicles were mixed with 100 μl of 80% Accudenz (with or without
Ca²⁺), transferred to ultra clear centrifuge tubes, and layered
with 35%, 30%, and 0% Accudenz (with or without Ca²⁺) to form a
step gradient. Gradients were centrifuged in a SW-55 rotor (55,000 rpm, 1.75
h), and 40 μl of vesicles was collected at the 0–30% interface and
analyzed by SD-SPAGE and Coomassie staining or immunoblotting. Samples to be
immunoblotted were transferred to nitrocellulose by the semi-dry method,
nonspecific sites were blocked with 3% nonfat dry milk, and proteins were
probed for with the indicated primary antibodies (diluted 1:1,000 in 1% nonfat
dry milk) and a goat-anti mouse horseradish peroxidase-linked secondary
antibody (diluted 1:20,000 in 1% nonfat dry milk). Blots were incubated with
enhanced chemiluminescent substrate and exposed to film.
PS Binding Assays—Mutant syt 1 C2AB-glutathione
S-transferase fusion proteins were expressed and purified as
described above using glutathione-Sepharose beads, however, the protein was
not eluted by thrombin cleavage. Sepharose beads containing 10 μg of bound
protein were incubated with protein-free liposomes (15% PS, 29.25% PE, 55% PC,
and 0.75% Rhod-aminePE) in the presence or absence of Ca²⁺ for 15
min with gentle agitation. All buffers contained 0.2 mm EGTA and
Ca²⁺ concentrations were prepared from a 100 mm stock
solution (Thermo Electron Corp, Beverly, MD) using WebMaxC
(www.stanford.edu/~cpatton/webmaxcS.htm).
Next, beads were washed three times with reconstitution buffer with the
corresponding Ca²⁺ concentration. Bound liposomes were solubilized
by addition of reconstitution buffer with 1% Triton X-100. The fluorescence
intensity was measured using a BioTek Synergy HT plate reader equipped with
530/25 excitation and 590/35 emission filters. The resulting fluorescence was
normalized to the maximum intensity as determined by nonlinear regression. The
[Ca²⁺]_½ and Hill slope for each mutant were
determined by fitting the normalized data with sigmoidal dose-response
curves.
Dynamic Light Scattering—Phospholipid vesicles (15% PS, 30%
PE, 55% PC) were prepared by drying the phospholipids under a stream of
nitrogen, subjected to vacuum for >2 h, and suspended in HEPES-buffered
saline (50 mm HEPES, pH 7.4, 0.1 m NaCl, 10% glycerol, 1
mm dithiothreitol). Small unilamellar liposomes were prepared using
a mini-extruder (Avanti Polar Lipids) with a 50 nm pore size membrane
(Whatman). Dynamic light scattering experiments were performed on an N4 plus
Submicron Particle Size Analyzer (Beckman Coulter, Inc.), with a scattering
angle of 90°. Data were analyzed with PCS software. Liposomes (0.05
mm phospholipids) were mixed with either 4 μm or 10
μm protein in HEPES-buffered saline. Samples were loaded into a
cuvette, and all of the experiments were thermostatically controlled at 22
°C. To determine the kinetics of vesicle aggregation, the particle size in
the lipid-protein mixture was estimated at 305, 535, 765, 995, 1225, and 1455
s after the addition of 1 mm Ca²⁺ or 0.2 mm EGTA. Particle size was measured again after addition of 5 mm EGTA.
FIGURE 1.

The isolated C2B domain of syt 1 is sufficient to regulate
Ca²⁺-triggered membrane fusion. A, shown is a
schematic diagram depicting the components of the in vitro fusion
assay. B, fusion assays were carried out using donor v-SNARE
vesicles, t-SNARE acceptor vesicles, and 10 μm of the C2AB, C2A,
or C2B domain of syt 1. Components were incubated together for 20 min at 37
°C in the presence of 0.2 mm EGTA, followed by the addition of
Ca²⁺ (arrow) to give a final free concentration of 1
mm. Fluorescence intensity was measured every minute for 60 min and
normalized as described under “Experimental Procedures.”
Reconstituted v- and t-SNARE vesicles were composed of either: 100% PC,
15%PS/85%PC, 30%PE/70%PC, or 15%PS/30%PE/55%PC. Shown are representative
traces from three independent experiments. C, binding of each domain
to t-SNARE vesicles was monitored using a co-flotation assay; t-SNARE vesicles
used in the fusion assays were incubated with 10 μm C2AB, 30
μm C2A, or 30 μm C2B in the presence or absence of
1 mm Ca²⁺. Bound material co-floated through a density
gradient and was analyzed by SDS-PAGE and Coomassie Blue staining. Shown is a
representative gel of three independent experiments. Note: the line between the PS/PC and PS/PC/PE samples indicates the data were obtained from
two different gels.

Materials—Lipids were purchased from Avanti Polar Lipids
(Alabaster, AL). Immunoblotting substrate and reducing agent compatible BCA
kits were from Pierce-Thermo Scientific (Rockford, IL). Affinity media
(Ni²⁺-Sepharose HP and glutathione-Sepharose beads were from
GE-Amersham Biosciences, Pittsburgh, PA). Accudenz was from Accurate Chemical
& Scientific Corp. (Westbury, NY). All other general laboratory chemicals
and supplies were from Sigma or Fisher Scientific.