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ARTICLE
Received 20 Feb 2014 |Accepted 9 Jul 2014 |Published 18 Aug 2014
Quantitative super-resolution imaging of
Bruchpilot distinguishes active zone states
Nadine Ehmann1,*, Sebastian van de Linde2,*, Amit Alon3, Dmitrij Ljaschenko1, Xi Zhen Keung1,
Thorge Holm2, Annika Rings4,5, Aaron DiAntonio6, Stefan Hallermann4,5, Uri Ashery3,7, Manfred Heckmann1,
Markus Sauer2& Robert J. Kittel1
The precise molecular architecture of synaptic active zones (AZs) gives rise to different
structural and functional AZ states that fundamentally shape chemical neurotransmission.
However, elucidating the nanoscopic protein arrangement at AZs is impeded by the
diffraction-limited resolution of conventional light microscopy. Here we introduce new
approaches to quantify endogenous protein organization at single-molecule resolution in situ
with super-resolution imaging by direct stochastic optical reconstruction microscopy
(dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ
cytomatrix (CAZ) is composed of units containing B137 Bruchpilot (Brp) proteins, three
quarters of which are organized into about 15 heptameric clusters. We test for a quantitative
relationship between CAZ ultrastructure and neurotransmitter release properties by engaging
Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic
organization of Brp distinguishes different physiological AZ states and link functional
diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.
DOI: 10.1038/ncomms5650 OPEN
1Department of Neurophysiology, Institute of Physiology, University of Wu
¨rzburg, 97070 Wu
¨rzburg, Germany. 2Department of Biotechnology and
Biophysics, University of Wu
¨rzburg, 97074 Wu
¨rzburg, Germany. 3Department of Neurobiology, Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv 69978, Israel. 4European Neuroscience Institute, University Medical Center Go¨ttingen, 37077 Go¨ttingen, Germany. 5Carl-Ludwig-Institute
of Physiology, Medical Faculty, University of Leipzig, 04103 Leipzig, Germany. 6Department of Developmental Biology, Washington University
School of Medicine, St. Louis, Missouri 63110, USA. 7Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel. * These authors contributed
equally to this work. Correspondence and requests for materials should be addressed to M.S. (email: m.sauer@uni-wuerzburg.de) or to R.J.K.
(email: robert.kittel@uni-wuerzburg.de).
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Amajor challenge facing the scientific exploration of brain
function is the accurate interpretation of structure–
function relationships1. The synaptic active zone (AZ) is
a specialization within the presynaptic terminal, which is
functionally defined as the site of neurotransmitter release and
can be morphologically identified by the proteinaceous AZ
cytomatrix (CAZ)2. At AZs, complex molecular interactions
deliver the speed, precision and plasticity unique to neuro-
transmission3. The CAZ is believed to provide a structural
platform for these interactions. In electron microscopy (EM), the
CAZ is revealed as an electron-dense structure, whose morpho-
logy varies between different types of neurons and between
individual synapses formed by the same partner cells.
Importantly, activity-dependent structural variations of the
CAZ can occur at individual synapses in a dynamic manner
and appear to correlate with functional properties of transmitter
release4–7. The mechanistic coupling of molecular composition,
CAZ structure and neurotransmission, however, remains largely
elusive.
Despite a gradually emerging comprehensive protein catalogue,
we still lack basic information describing how the nanoscopic
organization of proteins gives rise to synaptic function. In
essence, this is because of the diffraction-limited resolution of
light microscopy that has hindered access to the spatial
nanodomain in a physiologically relevant context. In recent
years, super-resolution imaging methods have emerged that
enable far-field fluorescence microscopy with spatial resolutions
approaching virtually EM8–13. Here, super-resolution imaging by
single-molecule photoactivation or photoswitching and position
determination (localization microscopy) captures an outstanding
position because it reconstructs the super-resolved image from
single-molecule localization events. Thus, it can deliver
information about molecular distributions, even giving absolute
number of proteins present in subcellular compartments,
provided that each molecule is labelled with an intact
fluorophore, detected above a certain photon threshold, and
that the number of localizations measured per individual
fluorophore is accessible14. This provides insight into biological
systems at a molecular level.
Neurotransmitter release is controlled by a multi-step process
of vesicle delivery and molecular maturation at the AZ to
generate fusion-competent synaptic vesicles. The availability
of such readily releasable vesicles (RRVs) and their calcium-
dependent probability of fusion fundamentally determine
synaptic performance15. The precise spatial organization of AZ
constituents shapes such basic elements of presynaptic function
and contributes to use-dependent synaptic plasticity. Here, we set
out to test whether a quantitative analysis of the CAZ
ultrastructure could provide information on these functional
properties.
To this end, we focused specifically on Bruchpilot (Brp), a
major structural and functional component of the CAZ in
Drosophila. Brp performs a dual function of clustering
Ca2þchannels and concentrating synaptic vesicles at neuro-
muscular AZs16,17. By promoting excitation–secretion coupling,
Brp supports efficient transmitter release, shapes synaptic
plasticity5,18 and participates in certain forms of learning19.
Using STED (stimulated emission depletion) microscopy,
previous work has provided an ultrastructural description of the
orientation of Brp in the CAZ16,20,21. Building upon this
basic understanding, we sought to extract quantitative data on
the number and precise spatial arrangement of Brp molecules
within AZs.
The present study puts forward a novel approach to extract
protein counts from macromolecular assemblies at single-
molecule resolution. We demonstrate this procedure by
estimating endogenous Brp protein copies in their native
environment by direct stochastic optical reconstruction micro-
scopy (dSTORM)11,22.Drosophila mutants were used to analyse
different AZ states, and electrophysiology was applied to
functionally calibrate super-resolution images. Our results
demonstrate that functional information on neurotransmission
is provided by the nanoscopic organization of Brp.
Results
Localization microscopy of the CAZ nanostructure. To obtain
detailed structural information on the CAZ in situ, we used
dSTORM at the glutamatergic larval Drosophila neuromuscular
junction (NMJ). The CAZ was recognized with a monoclonal
antibody (mAb BrpNc82) directed against a C-terminal epitope of
Brp20,23. To optimize structural resolution, we used a secondary
F(ab0)
2
fragment labelled on average with 1.3 Cy5 fluorophores.
With an IgG size of 8–10 and B4 nm for the F(ab0)
2
fragment,
we estimate B13 nm for the spatial dimensions of the antibody–
fluorophore complex24,25. Localization microscopy relies on
temporally separating fluorescence emission from single
fluorophores within a diffraction-limited area. The position of
single-molecule fluorescence signals can then be precisely
determined (localized) by fitting of a two-dimensional (2D)
Gaussian function to the point-spread function. The localizations
of all emitters are finally used to reconstruct a super-resolution
image10–13. Owing to the high brightness of Cy5, a localization
precision of 6–7 nm was achieved (s.d., in lateral direction)
using either localizations of individual, isolated fluorophore-
labelled antibodies in the sample or nearest neighbour
analysis26 (Fig. 1a,b and Supplementary Fig. 1; see Methods
section).
At roughly 200 kDa, Brp is a large coiled-coil domain protein
that adopts an elongated and polarized orientation perpendicular
to the AZ membrane. Its membrane-proximal N terminus helps
to cluster Ca2þchannels at the AZ, whereas the C-terminal
region of Brp reaches into the cytoplasm to tether synaptic
vesicles16,17,20. Thus, localizations defined by mAb BrpNc82
correspond to the distal region of macromolecular CAZ-
filaments observed in electron micrographs of high-pressure
frozen NMJs20,27. The increase in spatial resolution by dSTORM
uncovered detailed information of the CAZ, concealed in
diffraction-limited conventional fluorescence microscopy
(Fig. 1c,d). In the present study, we define an individual AZ via
its CAZ, which is an interconnected region of Brp
immunoreactivity. Within one such Brp assembly, localization
microscopy resolved a substructural arrangement of Brp into
modules (Fig. 1d). Hereafter, this further level of organization is
termed a CAZ-unit. An AZ could contain single, individual
(Fig. 1d1) or multiple, closely grouped CAZ-units (Fig. 1d2). The
same organizational principle was also observed with Alexa Fluor
700 (A700) and Alexa Fluor 532 (A532) fluorophores, although at
lower spatial resolution than obtained with Cy5 (Supplementary
Fig. 1). For the following analyses, we therefore used Cy5 and
considered only single, clearly distinguishable CAZ-units
(Fig. 1d1).
A wild-type (wt) CAZ-unit (en face view, that is, optical axis
perpendicular to the membrane) contains on average 1,021±43
(s.e.m.) localizations and, considering its size (0.095±0.003 mm2
s.e.m., n¼144 CAZ-units), is likely analogous to a CAZ structure
as defined by STED (termed donut)16 and EM (termed T-bar)28.
Several T-bars may reside at one synapse, and this is reflected by
groups of narrowly spaced CAZ-units (for example, triple T-bar
AZ in Fig. 1d2). As an AZ consists of 1,257±89 localizations
(s.e.m.), it will contain on average 1.2 CAZ-units. This calculation
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is consistent with the number of T-bars detected per synapse in
electron micrographs28,29.
Moving on to the next level of organization, we studied the
substructure of CAZ-units. EM tomography at the Drosophila
NMJ has reported macromolecular CAZ-filaments with a
diameter of B10 nm (ref. 27). How many Brp proteins
contribute to one filament is, however, unknown. In order to
describe the spatial arrangement of Brp molecules in quantitative
terms, we analysed the distribution of localizations using a
clustering algorithm that was modified from Bar-On et al.30 and
took into account the filament diameter in electron micrographs
(Fig. 2 and Supplementary Movie 1; see Methods section). The
density-based analysis calculated that an average CAZ-unit
contains 14.5±0.4 multiprotein clusters (s.e.m.) with an elliptic
shape (29.8±0.2 nm s.e.m. long radius 19.9±0.1 nm s.e.m.
short radius) and an average size of 1,568±21 nm2(s.e.m.).
Considering the dimensions of the antibody complex (B13 nm)
and our localization precision (6–7 nm; Fig. 1a,b), the average
Brp cluster size indeed very closely matches the diameter of
CAZ-filaments (see Discussion section).
Quantifying the substructural organization of Brp in the CAZ.
To attain further quantitative information on the molecular AZ
architecture, we developed an approach to count Brp molecules in
their native environment. Owing to the single-molecule sensi-
tivity of dSTORM, fluorophore localizations can be used not only
to extract information about the distribution of individual
molecules30, but crucially, also to approximate the absolute
number of protein copies. Since the number of localizations
measured for a fluorophore-labelled antibody is influenced by its
nano-environment, reference experiments using different
antibody concentrations had to be conducted at the CAZ to
unequivocally correlate localization counts with the number of
underlying Brp protein epitopes (Fig. 3; see Methods section).
First, titrations were performed with secondary antibodies to
unravel the number of localizations detected for a single Cy5-
labelled secondary antibody (2ndary Ab-Cy5) bound to the
primary antibody within the CAZ (Fig. 3b). In order to reliably
identify the CAZ at low 2ndary Ab-Cy5 concentrations (high
dilutions), mAb BrpNc82 was co-stained with Alexa Fluor 488
(2ndary Ab-A488). Next, the concentration of the primary
antibody was titrated to estimate saturation of Brp epitopes
(Fig. 3c). And finally, the number of secondary antibodies bound
to each primary antibody was estimated. To obtain this
information, a low concentration of mAb BrpNc82 was combined
with a normal concentration of 2ndary Ab-Cy5. Comparing the
number of Cy5 localizations per putative Brp epitope with those
of a single 2ndary Ab-Cy5 (Fig. 3b) provided an approximation of
1.59 2ndary Ab-Cy5 per mAb BrpNc82. To visualize the CAZ in
this experiment, co-staining was performed with an antibody
directed against an N-terminal Brp epitope (rabbit-BrpN-term plus
2ndary Ab-A488 anti-rabbit)20.
Taking these considerations into account allows for quantita-
tive image analysis and delivers an estimate of 137±29 (s.e.m.)
Brp molecules per CAZ-unit (conversion factor of molecules per
localization: 0.134±0.028 s.e.m.; see Methods section). Corre-
spondingly, B7 Brp molecules are recognized per multiprotein
cluster (52.2±0.7 localizations s.e.m., n¼2,102 clusters). Accord-
ing to this calculation, Brp proteins would assemble as polarized
rod-like heptamers via coiled-coils to form a multiprotein
filament. The plausibility of this stoichiometry can be appreciated
by comparison with other filamentous protein structures, for
example, seven subfibrils form intermediate filaments of B10 nm
diameter31.
Interestingly, the image analysis described that B26% of Brp
localizations in CAZ-units are not clearly grouped into clusters
(Fig. 2d, black triangles). Taking the image background into
account (only 78±7 localizations per mm2s.e.m.), we estimate
that o1% of localizations within CAZ-units (B104localizations
per mm2) are caused by unspecific labelling. This indicates that a
substantial fraction of Brp molecules are not part of macro-
molecular filaments.
In localization microscopy, labelling density, in addition to
localization precision, critically determines the ability to resolve
spatial features of a given structure32. Thus, the high density of
Brp molecules at the CAZ and the strong affinity of the antibodies
form the basis for the high spatial resolution in dSTORM
experiments. While epitope shielding may well introduce an error
to our approximation of Brp protein and cluster numbers, the
tight correlation with EM data lends strong support to our
quantitative approach.
Ultrastructural analysis of different AZ states. Brp reorganiza-
tions are involved in synaptic plasticity operating on time
scales ranging from milliseconds to days5,6,17,33. Such plastic
rearrangements could, in principle, involve changes in Brp
40
a
c
b
d
9,000
6,000
3,000
0
40 40
80
80
0
40
20
20
y (nm)
Number of localizations
y (nm)
x (nm)
x (nm)
–20
–20
–40
–40
0
0
1
22
1
12 12
Figure 1 | dSTORM resolves substructural information on the CAZ.
(a) 2D localization pattern of a single, unspecifically bound Cy5 F(ab0)
2
fragment. (b) Aligned distribution of 209,537 localizations from 21,436
unspecifically bound antibodies. A 2D Gaussian fit gives a localization
precision (s.d.) of 7.16±0.02 nm. (c,d)dSTORM (right panels) uncovers a
substructural organization of the CAZ, disguised in epifluorescence images
(left panels) of wt NMJs stained against Brp. Thereby, assemblies of Brp
into clusters, termed CAZ-units, could be identified. Panel d1 shows two
separate AZs (arrows), viewed en face, each containing one CAZ-unit, and
panel d2 shows a single AZ composed of three CAZ-units (arrowheads).
Lower panels display magnification of boxed regions. Scale bars, 2mm(c,d)
and 500 nm (c1,c2,d1,d2).
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protein number per CAZ34 or the spatial orientation of Brp
within the CAZ17. To test our quantitative imaging approach, we
analysed different AZ states by using two previously investigated
Drosophila mutants with altered Brp organization.
The small vesicle-associated GTPase Rab3 regulates the
enrichment of Brp at AZs. At Rab3 mutant (rab3rup) larval
NMJs, altered synaptic transmission is accompanied by fewer
Brp-positive synapses, although these display greatly enlarged Brp
aggregates34. The hypomorphic mutant, brpnude, lacks merely the
last 17 C-terminal amino acids of Brp (that is, B1% of the entire
protein; Fig. 3a). While the overall CAZ structure is left intact,
brpnude T-bars display a strikingly reduced vesicle tethering
capacity that leads to slowed vesicle recruitment and short-term
depression of neurotransmitter release17.
In line with previous work, we used confocal microscopy to
confirm that rab3rup NMJs contain fewer Brp-positive AZs
nm
400
ab
cd
300
200
100
0
400
300
200
100
0100 200 300
400
300
200
100
0100 200 300
nm
100 200 300
100
80
60
40
20
Density
400
300
200
100
0100 200 300
Figure 2 | Density-based analysis reveals that CAZ-units are built from multiple Brp clusters. (a) Spatial distribution of localizations in a specific
CAZ-unit (shown as inset). (b) Density distribution of localizations in a 20-nm search radius (number of localizations within Eps-environment colour
coded). Difference between elevation lines, 2 nm. (c) Centres of mass, that is, local density maxima as discovered by algorithm. (d) Final clusters as defined
by algorithm. Each colour represents a different cluster. Parameters used for algorithm: 20nm search radius (Eps) and 16 neighbours threshold (k).
MergeBrpNc82-Cy5BrpNc82-A488
1
2
10 10
2
1
10–5 10–4
2ndary Ab-Cy5 dilution
2ndary Ab-Cy5 dilution
2ndary Ab-Cy5 Nc82
brpnude
mAb BrpNc82
2ndary Ab-A488
C-term
Brp
N-term CAZ-unit (side view)
High Low LowHigh
10–3 10–2 10–1 110–4
mAb BrpNc82 dilution
mAb BrpNc82 dilution
10–3 10–2 10–1
100
100
Localization/CAZ
Localization/CAZ
1,000
CAZ-filament
a
bc
Figure 3 | Quantification of Brp molecules in the CAZ. (a) Schematic description of the experiments. A filamentous CAZ-unit is shown in grey and the
polarized orientation of Brp is illustrated. The blue shaded area indicates the approximate region of the mAb BrpNc82 epitope, the red arrow marks the
C-terminal (C-term) truncation in brpnude.(b) Cy5 and A488 labelled 2ndary F(ab0)
2
fragments were diluted at a constant overall concentration of
5.2 10 8M (for example, 1: 100% Cy5 to 0% A488 and 103: 0.1% Cy5 to 99.9% A488) and a fixed mAb BrpNc82 dilution (1/2,000; 0.5 10 3)to
estimate the number of localizations corresponding to a single Cy5-labelled antibody attached to Brp via mAb BrpNc82. Individual Brp proteins are indicated
by filled circles (grey). Binding of single Cy5 antibodies (magenta) to the CAZ was verified through comparison with the A488 epifluorescence
signal (green; see Methods section). Images depict examples of several antibody dilutions (indicated by 1 and 2 in the graph). (c) Titration of mAb BrpNc82
at fixed Cy5- and A488 antibody concentration (5.2 10 8M; 1% Cy5, 99% A488) provides information on epitope saturation by the primary
antibody. Data in (b)(n¼8–10 NMJs per dilution) and (c)(n¼4–5 NMJs per dilution, error bars show s.e.m.) were fit to a logistic function (see Methods
section). Arrowheads denote antibody concentrations used in experiments (Figs 1, 2, 4 and 7). Scale bar, 200 nm. N-term, N terminus.
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(B35% of controls; control: 472±43 s.e.m., n¼14 NMJs;
rab3rup: 164±15, n¼14, rank sum test Po0.001; Fig. 4e and
Table 1; ref. 34) and then applied dSTORM to study the
nanoscopic organization of Brp at individual AZs. In rab3rup
mutants, the CAZ was significantly enlarged (control: 0.120±
0.006 mm2s.e.m., n¼16 NMJs; rab3rup: 0.212±0.01 mm2,n¼11,
rank sum test Po0.001; Fig. 4f and Table 1) and contained more
Brp molecules (control: 1,257±89 localizations s.e.m., n¼16
NMJs; rab3rup: 1,999±98, n¼11, rank sum test Po0.001; Fig. 4f
and Table 1). Electron micrographs have described a high
concentration of T-bars at a subpopulation of rab3rup AZs, which
likely correspond to those AZs that are Brp-positive34.In
addition, dSTORM resolved a complex organization of the
large rab3rup CAZ, often lacking a clearly distinguishable modular
composition (Fig. 4, enlarged boxed regions, lower panel).
CAZ-units could therefore not be unequivocally identified at
rab3rup AZs.
In contrast, brpnude NMJs contained normal numbers of Brp-
positive AZs (brpnude: 397±25, n¼13, rank sum test P¼0.254
versus control; Fig. 4e and Table 1; ref. 17) and displayed a
modular arrangement of Brp into units within the CAZ (Fig. 4,
enlarged boxed regions upper panel). Interestingly, dSTORM
revealed a decrease in the area of the CAZ at brpnude AZs
(0.097±0.005 mm2s.e.m., n¼13 NMJs, rank sum test P¼0.005
versus control; Fig. 4f and Table 1). This structural property had
heretofore not been recognized using high-resolution imaging via
STED or EM17. Despite this decrease in size, the average brpnude
CAZ contained normal numbers of Brp molecules (1,129±104
localizations s.e.m., n¼13 NMJs, rank sum test P¼0.28 versus
control; Fig. 4f and Table 1). This prompted us to investigate the
brpnude
rab3rup
1
1
2
2
1
1
2
2
3,000
1,500
0
0.15
0.3
0
300
AZs/NMJ
CAZ area (µm2)
600
0
*
*
*
*
*
*
Control
brpnude
rab3rup
Control
brpnude
rab3rup
Control
brpnude
rab3rup
Localizations/CAZ
0.02
0.01
0
0 100
Radial distance (nm)
200 300
CAZ-unit density
(localizations per nm2)
*
*
*
*
*
Figure 4 | Variable nano-organization of Brp in the CAZ. (a,b) Overview of brpnude and (c,d)rab3rup NMJs stained against Brp. Enlarged boxed
regions (left panels epifluorescence and right panels dSTORM) demonstrate the ordered arrangement of brpnude CAZs, with Brp immunoreactivity largely
confined to the CAZ-unit margins, and the disordered nanoscopic organization of greatly enlarged rab3rup CAZs. (e–g) Quantification of imaging data
acquired with confocal (e, rank sum test versus controls (n¼14 NMJs); brpnude (n¼13) P¼0.254; rab3rup (n¼14) Po0.001) and localization microscopy
(f, rank sum test versus controls (n¼16 NMJs): brpnude (n¼13) P¼0.005, rab3rup (n¼11) Po0.001; g, rank sum test versus controls (n¼16 NMJs):
brpnude (n¼13) P¼0.28, rab3rup (n¼11) Po0.001). (g)En face views of individual CAZ-units were aligned according to their centres of mass and
the radial density distributions of Brp localizations were plotted (dark lines: average and shaded area: s.e.m.). Compared with controls (black), the Brp
epitope was distributed more narrowly in brpnude (grey) CAZ-units. Scale bars, 2 mm(a–d) and 500 nm (enlarged boxed regions).
Table 1 | Structure of AZs.
Confocal dSTORM
AZ/NMJ Area (lm2) Localizations Number of Brp molecules
Control CAZ 472±43 (n¼14 NMJs) 0.120±0.006 (n¼16 NMJs) 1,257±89 168±34
brpnudeCAZ 397±25 (n¼13 NMJs) 0.097±0.005 (n¼13 NMJs) 1,129±104 151±35
rab3rupCAZ 164±15 (n¼14 NMJs) 0.212±0.01 (n¼11 NMJs) 1,999±98 268±58
AZ, active zone; Brp, Bruchpilot; CAZ, active zone cytomatrix; dSTORM, direct stochastic optical reconstruction microscopy; NMJ, neuromuscular junction.
Confocal microscopy was used to approximate the number of AZs per NMJ (via their Brp-positive CAZ), and super-resolution imaging by dSTORM was engaged to quantify ultrastructural properties of
the CAZ and to estimate Brp protein copies. Data are presented as mean±s.e.m.
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spatial organization of Brp in more detail. Because the
arrangement of Brp within individual brpnude CAZ-units
appeared very ordered, we analysed its radial distribution. We
found that the Brp epitope is more confined to the perimeter at
brpnude CAZ-units and more uniformly distributed in controls
(Fig. 4g).
Functional properties of AZ states. Next, we performed elec-
trophysiological recordings to obtain mechanistic descriptions of
AZ function in both mutants. During low levels of activity,
synaptic transmission is fairly normal at both rab3rup and brpnude
NMJs (Supplementary Figs 2 and 3)17,34. While both paired-pulse
stimulation (Fig. 5a) and high-frequency trains of activity
(Fig. 5b) provoked similarly pronounced short-term depression
of evoked excitatory postsynaptic currents (eEPSCs) in both
mutants, closer inspection revealed subtle differences in short-
term plasticity (STP). Whereas rab3rup NMJs showed normal
biphasic recovery kinetics, brpnude synapses displayed a slow
initial phase of recovery, characteristic of slow vesicle recruitment
(Fig. 5b)17. These dissimilar properties of recovery indicate
different origins of depression at brpnude and rab3rup synapses.
To describe synaptic depression in quantitative terms of the
number of RRVs (N), their release probability (p
vr
) and the rate of
vesicle reloading at release sites (k
þ1
), an established modelling
approach was used (Fig. 6; see Methods section)5,18. Two
constrained STP models were used to reproduce individual
high-frequency trains with ensuing biphasic recovery. For brpnude
NMJs, both models ascribed short-term depression to (i) a
decreased rate of replenishing (ii) a regular number of RRVs (iii)
possessing a normal average release probability (Fig. 6b and
Supplementary Table 1). This is related to (i) impaired vesicle
tethering to the CAZ, and agrees with (ii) a normal
electrophysiologically measured RRV pool size and (iii) regular
calcium channel clusters at brpnude AZs17.
Consistent with previous interpretations34,35, both models
predicted few RRVs possessing a high p
vr
at rab3rup NMJs (Fig. 6b
and Supplementary Table 1). Despite normal vesicle reloading
rates, this setting is susceptible to short-term depression owing to
the depletion of a large RRV fraction at stimulus onset.
In principle, short-term depression may be provoked by a
range of pre- and postsynaptic factors36–38. However, the
established information on these two specific mutants17,34,35
and a previous analysis of postsynaptic properties18 allowed us
to focus on the recruitment of RRVs to the AZ and the availability
of release sites as rate-limiting factors of synaptic transmission.
Dissecting structure–function relationships. Building upon the
mutant analyses of different AZ states, we sought to link quan-
titative information on the CAZ ultrastructure with functional
properties of neurotransmitter release. In view of the modelling
results, the number of Brp-positive AZs per NMJ (B35% of
controls in rab3rup; Fig. 4e) scales roughly with the number of
RRVs (Fig. 6b). Such a calculation predicts on average B3 RRVs
per AZ in all three genotypes (wt B2.5, brpnude B3.2, rab3rup
B3.1; see Methods section). Hence, in terms of RRVs, the almost
twofold larger rab3rup CAZ cannot compensate for reduced AZ
numbers.
Since Brp contributes to clustering Ca2þchannels at the AZ16,
the increased recruitment of Brp proteins to the rab3rup CAZ
(Fig. 4f) will likely also increase the recruitment of Ca2þchannels
and may tighten the spatial coupling to RRVs. This will elevate
p
vr
, consistent with the modelling results (Fig. 6b) and the larger
Ca2þchannel clusters reported at rab3rup AZs34.
To further test this structure–function interpretation, which
was reached by studying mutant AZ states, we turned to an
intrinsic physiological property of the Drosophila NMJ. Two
glutamatergic motorneurons, containing either small (type Is) or
big (type Ib) boutons, innervate larval muscles28. Previous studies
have described a functional gradient along the type Ib
motorneuron, with larger Ca2þsignals and correspondingly
higher p
vr
at the terminal bouton despite a constant density of
AZs35,39. A structural correlate of this gradient has, however,
not been identified. Hence, we used dSTORM to study the
ultrastructural organization of Brp at wt motorneurons. Our data
Control
40
20
0.0
Time (s)
0
0
1
*
**
*
*
50
100
0
0
50
50
100
100
Paired-pulse ratio
eEPSC amplitude
(–nA)
Last 10 eEPSCs
0
Control
rab3rup
20
40
10010
0.4
0.8
1.2 **
**
*
**
**
*
**
**
*
1,000
Inter-stimulus interval (ms)
0.5
Time post train (s) Time post train (s)
brpnude
brpnude
rab3 rup
1 = 126 ms
1 = 215 ms
1 = 110 ms
2 = 6.0 s
2 = 5.4 s
2 = 8.6 s
Figure 5 | Electrophysiological characterization of different AZ states. (a) Representative traces (normalized amplitude of the first eEPSC) and
average data of two-electrode voltage clamp recordings from larval NMJs show similar depression of brpnude (grey) and rab3rup (pink) eEPSCs during
paired-pulse stimulation (rank sum test versus controls: brpnude for 10 ms P¼0.014, for all other intervals Pr0.01; rab3rup for 10 ms P¼0.016, for
30 and 100ms Pr0.01) and (b) 60 Hz trains (eEPSC 91–100 mean; control: 34.3±1.5 nA s.e.m., n¼10 NMJs; brpnude:23.9±1.3nA, n¼10, rank sum
test Po0.001 versus control; rab3rup:27.3±1.4 nA, n¼11, P¼0.004 versus control). The fast phase of recovery after stimulation is selectively slowed in
brpnude (centre panel), while all three genotypes show a normal slow recovery phase (right). Scale bars, (a) 30 nA, 30 ms. Data are presented as
mean±s.e.m.
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resolve a clear gradient of both CAZ size and the number of Brp
molecules per CAZ along the Ib neuron (Fig. 7). With the largest
values at terminal boutons, this structural diversity closely
matches the functional gradient35,39.
Discussion
Here, we introduce a novel procedure to determine endogenous
protein numbers in tissue by localization microscopy using
standard labelling with primary antibodies and secondary F(ab0)
2
fragments. Correlating the nanoscopic organization of a single
large protein with electrophysiological recordings enabled us to
link the filamentous CAZ ultrastructure to neurotransmitter
release properties.
Our data are in line with the observation that p
vr
scales with
AZ size40. Studies at Drosophila NMJ and hippocampal synapses
have also reported that larger AZs provide for more RRVs5,40,41.
Surprisingly, our analysis of the grossly enlarged rab3rup CAZ
does not support this notion. A possible explanation could be
provided by the disordered accumulation of Brp at the rab3rup
CAZ (Fig. 4, enlarged boxed regions, lower panel) that suffices to
increase p
vr
via calcium channel clustering but fails to provide
k–0
k–1 k+1
Pvr
N
Model 1
Model 1
Control Control brpnude rab3 ru
p
brpnude
100 *
0.5
0.0
0.5
0.0
k+1
500
0
NPvr
0
100
0
100
0
0 1 10 100
Time (s)
EPSC amplitude (nA)
Model 2
Model 2
k+0
N0
k–0
k–1 k+1
k+0
N0
N1
k2
Pvr1 Pvr2
N2
**
*
*
*
*
*
*
*
**
*
*
rab3 rup
Figure 6 | Mechanistic interpretation of AZ function. (a) Model 1 (blue; encompassing one pool of RRVs refilled from a finite supply pool) and model 2
(green; containing two pools of RRVs with different release probabilities) were used to describe the experimental data (example fits to average
data in right panel). (b) The release parameters, obtained by fitting individual trains plus recovery experiments (control: n¼10, brpnude:n¼10 and rab3rup:
n¼11), distinguish between brpnude and rab3rup phenotypes. Statistics used Kruskal–Wallis with Dunn’s multiple comparison tests. Plots show mean±s.e.m.
1
2
3
4
5
6
#
Is
Ib
*
*
#
Epifluorescence dSTORM
0
1,000
Localizations/CAZ
2,000
0123
**
456
12
Ib bouton
number from end
3456
0.05
CAZ area (µm2)
0.10
0.15 ***
Anti-HRP-A488
BrpNc82-Cy5
Figure 7 | Neuronal gradient of the CAZ ultrastructure. (a,b) Epifluorescence dual-channel images.(a) Staining against horseradish peroxidase
(anti-HRP, grey) displays type Is and type Ib motorneurons (indicated by arrowheads) at a wt NMJ. Ib boutons are numbered beginning at the distal end (in
the lower left corner a neurite passes by the NMJ). (b) Corresponding Brp signal, which could be clearly allotted to a specific bouton. Enlarged boxed
regions show examples of CAZs imaged with dSTORM. (c) Quantification of the CAZ ultrastructure uncovers gradients in CAZ size (Ib distal:
0.136±0.006 mm2s.e.m., n¼269 CAZs; Ib proximal: 0.099±0.008 mm2s.e.m., n¼105, rank sum test Po0.001) and Brp localizations per CAZ (Ib distal:
1,583±77 localizations s.e.m., n¼269 CAZs; Ib proximal: 1,200±110 localizations s.e.m., n¼105, rank sum test P¼0.003) along type Ib motorneurons,
which closely match the functional gradient35,39. Scale bars, 5 mm(a,b) and 200 nm (enlarged boxed regions). Data are presented as mean±s.e.m.
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additional vesicle release sites. However, keeping in mind that
release persists and Nappears unaltered in brp-null mutants,
Brp itself is unlikely the primary determinant of a release site16,18.
In any case, it will be of great interest to clarify why the wt
CAZ is subdivided into Brp-positive CAZ-units, and to
investigate whether this modular arrangement is matched by a
complementary substructural organization of the opposing
postsynaptic density.
Our results demonstrate that counting Brp proteins and
quantifying their spatial distribution provides more detailed and
precise information than merely measuring the CAZ area. This is
exemplified by the small brpnude CAZ that contains a normal
number of Brp localizations and gives rise to an unchanged
p
vr
(Figs 4f and 6b and Table 1). Instead, Brp localizations are
more confined to the perimeter of brpnude CAZ-units (Fig. 4g).
Altered post-translational modification of Brp can promote
vesicle tethering to the CAZ and provokes spreading out of
AZ-filaments42. Conversely, the small brpnude CAZ correlates
with deficient vesicle tethering and slow vesicle recruitment,
leading to short-term depression (Figs 5 and 6)17. Hence, vesicle
tethering may contribute to the shape of the CAZ by contorting
proteinaceous filaments or, alternatively, the precise CAZ
conformation affects its tethering ability. While this cannot be
specified at present, either way the spatial organization of Brp
provides information on vesicle reloading kinetics.
By engaging dSTORM, we discovered a heretofore unrecog-
nized gradient of the CAZ ultrastructure along a glutamatergic
neuron, concealed by the limited resolution of confocal micro-
scopy35. Importantly, this finding provides a mechanistic basis for
the functional diversification of AZs35,39. A functional gradient
has not been described for type Is motorneurons. Moreover, AZs
of Is neurons reportedly possess a higher p
vr
than their Ib
counterparts, although this functional feature is not matched by a
larger number of T-bars per AZ28,29,43. Similarly, we detected
comparable numbers of Brp localizations at type Is and Ib
CAZs (the Is CAZ is in fact slightly smaller) and found no
ultrastructural gradient in the Is neuron (Supplementary Fig. 4).
This consideration highlights that Brp is not the sole determinant
of p
vr
and motivates the study of further AZ protein constituents.
Intriguingly, for example, synaptic vesicle size differs between
these two different glutamatergic neurons44.
The present investigation emphasizes how fundamentally
different AZ states may be disguised by giving rise to similar
facets of short-term depression (Fig. 5a). For descriptions of
synaptic function, the degree of paired-pulse depression is
routinely interpreted to reflect the magnitude of release prob-
ability. In light of these results and recent data from different
synapses reporting fast vesicle reloading45, transient fusion46 and
release site clearance47, analysing the CAZ nanostructure to test
alternative interpretations of depression may well provide new
insights into the molecular control of neurotransmission.
In order to estimate the number of Brp molecules in the CAZ,
several hurdles had to be overcome. In general, localization
microscopy in combination with photoactivatable fluorescent
proteins10,12 appears to represent the method of choice for
quantification purposes48 because fluorescent proteins offer the
distinct advantage of specific stoichiometric labelling of target
molecules. On the other hand, we were interested in endogenous
protein levels. Therefore, overexpression of fusion proteins could
not be deployed, and substitution of native proteins by transgenic
variants that display wt expression and function remains
challenging. In addition, misfolded fluorescent proteins and
those that cannot be photoactivated or photobleached already
after only a few excitation/emission cycles, and thus emit an
insufficient number of photons, withdraw themselves from
detection and localization14,49.
Besides localization microscopy, stepwise stochastic photo-
bleaching of fluorophores upon illumination with light can be
used to determine protein numbers50. Stepwise photobleaching is,
however, limited to low protein numbers because the likelihood
of missed events increases exponentially with the number of
molecules51.
As an alternative approach, we evaluated the use of standard
immunocytochemistry, that is, organic fluorophores and anti-
bodies for quantification of endogenous protein levels. Here,
challenges to be accepted include epitope accessibility, antibody
affinity and multiple localizations owing to on/off switching on
expanded time scales. Brp proteins appear to oligomerize as
coiled-coils to form elongated, polarized filaments20. Therefore, it
is conceivable that such a structural organization leads to a
separation of epitopes along the filament circumference,
promoting antibody accessibility despite a high density of Brp
proteins (Supplementary Fig. 5). That said, differences in steric
hindrance may exist at the level of individual AZs and in different
genotypes. Furthermore, since epitopes can be shielded or lost
during fixation, the determined number of Brp proteins might
well represent a lower estimate.
Organic fluorophores exhibit certain advantageous character-
istics, such as higher brightness and photostability than
fluorescent proteins. Thus, in combination with the fact that
each fluorophore can be localized multiple times, a higher
percentage of accurately localized fluorophores can potentially be
achieved. Nonetheless, the difficulty of extracting reliable
information on how often a fluorophore-labelled antibody is
localized remains, especially because the photoswitching perfor-
mance of fluorophores is sensitively influenced by their local
environment52. As a consequence, isolated fluorophores located
outside of the investigated cellular compartment cannot be used
as reference. Therefore, we developed an elaborate but secure
two-colour method for identifying individual fluorophore-
labelled antibodies in the structure of interest in order to
determine the typical number of localizations. However, certain
photophysical effects that depend, for example, on local
fluorophore densities and photoswitching characteristics can
never be completely ruled out in quantitative localization
microscopy experiments. Nevertheless, by titrating primary and
secondary antibodies, the described procedure delivers reliable
estimates for the number of accessible protein epitopes per CAZ.
A major challenge facing the field of super-resolution
microscopy is the development of analytical tools to quantify
data sets and to help provide biological interpretations30. The
implementation of clustering algorithms provided an objective
description of the distribution pattern of Brp molecules
within the CAZ and revealed the organization of Brp into
supramolecular clusters. Considering their structural properties,
these clusters likely correspond to the multiprotein CAZ-
filaments observed in EM27 (STED microscopy displays B9
‘dots’ per AZ41). Why are the clusters elliptical? We speculate
that an answer can be provided by the arrangement of
fluorophores around CAZ-filaments in space. When CAZ-units
are viewed en face (optical axis perpendicular to AZ membrane),
filaments bent outwards could be viewed at a right angle to
their long axis at the level of the mAb BrpNc82 epitope (Fig. 3a
and Supplementary Fig. 5). In the images, the filament diameter
(B10 nm) only contributes to the separation of encircling fluoro-
phores in x and y. Hence, the largest separation will be seen
for Cy5 molecules located at opposite sides of the filament
and aligned with the CAZ-unit circumference (Supplementary
Fig. 5).
Intriguingly, the quantitative analysis described a substantial
population of un-clustered Brp proteins in the CAZ. It will be of
great interest to investigate the biological significance of this
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observation and to test whether, for example, the fraction of ‘free’
Brp proteins changes during synaptic activity.
The complex nanoscopic organization and highly dynamic
interactions of AZ proteins pose a formidable challenge to
deciphering structure–function relationships of neurotransmis-
sion. A synergy of sophisticated biochemistry53,54 with super-
resolution microscopy55 holds great promise for assembling a
comprehensive molecular blueprint of the AZ.
Methods
Fly stocks.The following genotypes were used in this study: w1or w1118 (con-
trols), brpnude(5.38)/df(2R)BSC29 (brpnude)17 and rab3rup/df(2R)ED2076 (rab3rup)34.
Data were obtained from male third instar Drosophila larvae raised at 25°C.
Electrophysiology.In brief, two-electrode voltage clamp recordings (Axoclamp
900A amplifier, Molecular Devices) of eEPSCs (V
holding
60 mV, stimulation
artefact removed for clarity in figures) and minis (V
holding
80 mV, 90 s recording)
were made from muscle 6 (segments A2 and A3) at room temperature with
intracellular electrodes (resistances of 10–20 MO, filled with 3 M KCl) essentially as
previously reported18. The composition of the extracellular haemolymph-like
solution (HL-3)56 was (in mM): NaCl 70, KCl 5, MgCl
2
20, NaHCO
3
10, trehalose
5, sucrose 115, HEPES 5 and CaCl
2
1.5, pH adjusted to 7.2. Muscle cells with an
initial membrane potential between 50 and 70 mV, and input resistances of
Z4MOwere accepted for analysis. Signals were sampled at 10 kHz, low-pass
filtered at 1 kHz and analysed with Clampfit 10.2 (Molecular Devices). EPSCs were
evoked by stimulating the innervating nerve (300 ms pulses typically at 10 V) via a
suction electrode.
Ten eEPSCs were averaged per cell for each paired-pulse interval and for low-
frequency stimulation. Paired-pulse recordings were made at 0.2 Hz with inter-
stimulus intervals of (in ms): 10, 30, 100, 300 and 1,000. Ten seconds of rest were
afforded to the cell in between recordings. The amplitude of the second response in
10 ms inter-pulse recordings was measured from the peak to the point of
interception with the extrapolated first response. High-frequency stimulation
followed an established protocol18,57 consisting of 100 pulses applied at 60 Hz. The
recovery was monitored by stimulating at increasing intervals following the train
(in ms): 25, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000 and
100,000.
Modelling.STP modelling of 60 Hz trains and of the recovery thereafter was
performed as previously described5,18. Two constrained STP models were used.
First, a model with one pool of release-ready vesicles refilled from a finite supply
pool was used (model 1). Model 1 is characterized by the following parameters: g,
the Ca2þdependence of facilitation; a, that defines the release probability; N, the
number of RRVs; N
0
, the number of vesicles in the supply pool from which the
readily releasable pool is refilled; and k
þ1
,k
-1
,k
þ0
and k
-0
, the refilling rates of N
and N
0
, respectively. In addition, we also used a model with two pools of release-
ready vesicles and heterogeneous release probabilities (model 2). In model 2, a
small pool of release-ready vesicles (N
2
) with a high release probability (p
vr2
)is
refilled with a rate k
2
from a larger pool (N
1
), which has a lower vesicular release
probability (p
vr1
), and which, in turn, is refilled from a supply pool (N
0
). The
refilling rates of these pools (k
þ1
,k
1
,k
þ0
and k
0
) are defined as in model 1.
The release probabilities (p
vr
for model 1, and p
vr1
and p
vr2
for model 2) were
defined according to a biophysical Ca2þ-dependent model of facilitation with one
single free parameter (a)18,58. Both models had two additional free parameters:
Nfor model 1, N
1
for model 2 and k
þ1
for both models, resulting in three free
parameters for each model. The remaining parameters were constrained to values
previously estimated at the Drosophila NMJ18 with the facilitation parameter (g)58
adjusted to 0.4 mm1to reproduce the initial facilitation observed here with an
extracellular Ca2þconcentration of 1.5 mM.
Individual experiments including depression during 60 Hz train stimulation and
the recovery from depression were fitted with either models 1 or 2 as previously
described18. The best-fit parameters for both models are shown for each individual
experiment of the different genotypes as mean and s.e.m. in Fig. 6b.
Confocal imaging.Larvae were dissected in ice-cold HL-3, fixed for 10 min using
4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) and blocked for
30 min in PBT (PBS with 0.05% Triton X-100, Sigma) containing 5% normal goat
serum (Jackson ImmunoResearch). Preparations were incubated with primary
antibodies at 4 °C overnight. After one short and three 20 min washing steps, the
filets were incubated with secondary antibodies for 2 h followed by another three
washing steps. The samples were mounted in Vectashield (Vector Laboratories) for
confocal imaging or kept in PBT for dSTORM measurements. Primary antibodies
were used in the following dilutions: monoclonal antibody (mAb) BrpNc82 (1:250,
provided by E. Buchner) and rabbit-GluRIID (1:1,000, provided by S.J. Sigrist).
Alexa Fluor 488-conjugated mouse (Invitrogen) and Cy3-conjugated rabbit (Dia-
nova) antibodies were used at 1:250. Images were acquired with a Zeiss LSM5
Pascal confocal system (objective: 63 , numerical aperture 1.25, oil). For each set
of experiments, all genotypes were stained in the same vial and imaged in one
session. To estimate synapse numbers laser power was adjusted individually for
each NMJ.
Brp punctae and GluRIID clusters (NMJ 6/7, segments A2, A3) were examined
using ImageJ software (National Institutes of Health) in principle as previously
described6. After background subtraction, a Gaussian blur (0.9 px s.d.) was applied
to maximum z-projections of confocal stacks and masks were generated (threshold
mean grey value of 25 for Brp and 30 for GluRIID). After superimposing the binary
mask on the original blurred image, spot detection and segmentation via the ‘Find
Maxima’ operation was performed to extract particle numbers.
To estimate the number of release sites (N) per AZ, the modelling prediction of
N(average value of both models) was divided by the number of AZs on muscle 6
identified in confocal images, that is, half of NMJ 6/7 (ref. 28).
Super-resolution imaging.The mAb BrpNc82 was used at a dilution of 1/2,000 to
identify AZs. Goat anti mouse F(ab0)
2
fragments (A10534, Invitrogen) were
labelled with Cy5-NHS (PA15101, GE Healthcare) according to standard coupling
protocols given by the supplier. Purification of the conjugates was performed by
use of gel filtration columns (Sephadex G-25, GE Healthcare). The degree of
labelling was determined by absorption spectroscopy (Jasco) as 1.3 for studies of
the CAZ ultrastructure and 1.3–1.5 for dilution experiments. Samples were stored
in 0.2% sodium azide in PBS and for the experiments, Cy5-labelled secondary
antibody was used at a concentration of 5.2 10 8M.
For dSTORM imaging with Cy5, the sample was embedded in photoswitching
buffer, that is, 100 mM mercaptoethylamine, pH 8.0, enzymatic oxygen scavenger
system (5% (wt/vol) glucose, 5 U ml 1glucose oxidase and 100 U ml 1catalase59)
and mounted on an inverted microscope (Olympus IX-71) equipped with an oil-
immersion objective (60 , numerical aperture 1.45, Olympus) and a nosepiece
stage (IX2-NPS, Olympus)22. For excitation of Cy5, a 641-nm diode laser (Cube
640–100C, Coherent) was used. Telescope lenses and mirror were arranged on a
translation stage to allow for switching between wide-field, low-angle/highly
inclined thin illumination and total internal reflection fluorescence imaging22,60,61.
Fluorescence light from Cy5 was filtered by a dichroic mirror (650, Semrock)
and a band- and long-pass filter (BrightLine 697/75, RazorEdge 647, Semrock), and
imaged on an electron-multiplying CCD camera (EMCCD; Ixon DU897, Andor
Technology). Additional lenses were used to generate a final camera pixel size of
107 nm. Fifteen thousand frames were recorded with a frame rate of 100 Hz at an
irradiation intensity of B5kWcm2. For imaging A488, a 488-nm laser (Sapphire
488 LP, Coherent) and a polychromatic dichroic mirror (410/504/582/669,
Semrock) were used. Fluorescence light from A488 was reflected by a dichroic
mirror (630 DCXR, Chroma) and imaged on a second EMCCD camera equipped
with a band-pass filter (HQ535/50, Chroma).
Goat anti mouse IgG labelled with A532 (A11002, Invitrogen) and A700
(A21036, Invitrogen) was used at a concentration of 6.25 10 9M. The degree of
labelling was determined as 2.0 (A700) and 4.5 (A532). Imaging of A532 and A700
by dSTORM was performed in PBS containing 100 mM mercaptoethylamine, pH
8.3. Using appropriate filter sets (dichroic mirrors: 650 or 545/650; band-pass
filters: RazorEdge 647 or BrightLine 582/75, Semrock), the samples were irradiated
at 641 nm (A700) or 532nm (NANO 250-532-100, Linos; A532) at B5kWcm2.
For titrations of A532-labelled secondary antibodies (Supplementary Fig. 6),
fluorescence light from A700 and A532 was separated by a dichroic mirror (630
DCXR, Chroma) and imaged on two EMCCD cameras.
Super-resolution images were reconstructed using the software package
rapidSTORM62,63. Only fluorescence spots containing 41,000 photons were
analysed. Double-spot emission was analysed by a two-kernel analysis as
described64 applying a maximum two-kernel improvement of 0.1. Raw localization
data obtained from rapidSTORM was examined and further processed with
ImageJ. A subpixel binning of 10 nm px 1was applied. Representative images in
Fig. 7 and magnified views in Figs 1 and 4 are shown with 7 nm binning for clarity.
To measure CAZ (defined by BrpNc82) area and localization numbers, masks
were created by applying a Gaussian blur (1 px s.d.) followed by a minimal
threshold (0.15 counts). After a minimum overlay of the original data with the
masks, CAZs were then identified via their area (300 px to infinity). For the
comparison of genotypes, a total of 812 CAZs in controls, 776 in brpnude and 257 in
rab3rup were analysed and data (presented as mean±s.e.m.) were acquired in two
imaging sessions, each containing all three genotypes stained in the same vial.
Images with a background of 42.3 single spots per mm2were excluded from the
comparative analysis. Unspecific background labels exhibited equal localization
counts in all genotypes (average counts control: 12.0±0.2 localizations s.e.m.,
n¼16 NMJs; brpnude: 12.0±0.1, n¼13; rab3rup: 12.4±0.3, n¼11), indicating
comparable imaging settings.
For the investigation of different motorneurons, a total of 963 (type Ib) and 579
(type Is) CAZs (from NMJ 6/7, segments A2 and A3) were analysed to determine
the gradient. Double-stainings included horseradish peroxidase directly conjugated
to A488 (1:250, Jackson ImmunoResearch) for visualization of boutons. In the
representative images, the epifluorescence signals were background subtracted and
normalized.
To specify the localization precision of dSTORM images, localizations of
unspecific background label (n¼21,436) from all three genotypes were analysed
using ImageJ. Masks were created as described above (Gaussian blur with 1 px s.d.,
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&2014 Macmillan Publishers Limited. All rights reserved.
threshold 0.08 counts). Within a dSTORM image, only selections with a size
between 16 and 100 px (10 nm px 1) and an ellipticity Z0.95 were anaylzed. The
coordinates of localizations within a single selection were aligned to their centre of
mass and a 2D histogram of all localizations (209,537 in total) was generated
(binning: 4 nm 4 nm). A 2D Gaussian function was fitted to this histogram
(adjusted R2¼0.995). The s.d. of the Gaussian function (s
x,y
¼(s
x
þs
y
)/2)) was
determined as 7.16±0.02 nm and is stated as localization precision in this work
(Fig. 1a,b). This value is comparable to the localization precision obtained with an
alternative method based on nearest neighbour analysis26 (Supplementary Fig. 1).
For the investigation of CAZ-units, those structures were chosen that were not
grouped together and were viewed en face, that is, with the AZ membrane
perpendicular to the optical axis20. For the manual selection of CAZ-units, the
genotypes were blinded.
To calculate the radial distribution (Fig. 4g), Mathematica 9.0 (Wolfram
Research) was used to automatically calculate the centre of each chosen CAZ-unit
as the centre of mass (that is, the average localization of all pixels of the CAZ-unit
weighted with the pixel value). Subsequently, the distance of each pixel to the
centre of mass was calculated. These distances were then binned, the pixel values
were added to the corresponding bins and the values were normalized by the area
of each radial slice. The resulting distributions were averaged across all chosen
CAZ-units, resulting in mean and s.e.m. values for the radial distributions of each
genotype.
Quantification of Brp protein numbers.To estimate the number of Brp mole-
cules per CAZ-unit, a number of parameters had to be considered. First, the mAb
BrpNc82 specifically recognizes one epitope per Brp molecule. Second, it is unclear
how many Cy5-labelled F(ab0)
2
fragments can bind to the primary mAb BrpNc82
and how many localizations can be expected per Cy5-labelled secondary antibody.
Here, it has to be considered that the number of localizations detected per antibody
can be strongly influenced by its nano-environment. Hence, the number of loca-
lizations expected for an individual labelled antibody should ideally be derived
from measurements performed under identical imaging and buffer conditions in
the same cellular environment, that is, within the CAZ. In order to derive quan-
titative values of Brp molecules from the localization data, we performed antibody
titrations with (i) different dilutions of Cy5-labelled secondary antibody and a
constant concentration of mAb BrpNc82, and (ii) different dilutions of mAb
BrpNc82 and a constant concentration of Cy5-labelled secondary antibody.
To evaluate the localizations presented by a Cy5-labelled F(ab0)
2
fragment
attached to Brp via mAb BrpNc82, the concentration of mAb BrpNc82 was kept
constant (1/2,000, that is, experimental concentration) and the secondary antibody
was diluted (1, 1/2, 1/10, 1/100, 1/1,000, 1/10,000 and 1/100,000). Preparations
were simultaneously stained with A488 goat anti mouse F(ab0)
2
fragments
(A11017, Invitrogen) to warrant an overall constant secondary antibody
concentration of 5.2 10 8M for epitope saturation (for example, 1: 100% Cy5 to
0% A488 and 1/1,000: 0.1% Cy5 to 99.9% A488) and to enable the unequivocal
identification of the CAZ at low Cy5 antibody concentrations (Fig. 3b). The
epifluorescence signal (A488) was background subtracted, blurred and contrast
enhanced to identify Cy5 localizations within in the CAZ. NMJs (8–10) were
evaluated for each dilution and the localizations per CAZ were histogrammed and
fit to a Poisson model in order to extract the average number of localizations per
CAZ (L
CAZ
) as the mean of the distribution. L
CAZ
as a function of the Cy5 antibody
dilution (d) was then approximated with the logistic function
L
CAZ
¼L
2
þ(L
1
L
2
)(1 þ(d/d
0
)p)1, where the lowest localization value (L
2
)is
equivalent to the number of localizations corresponding to a single F(ab0)
2
fragment attached to Brp via mAb BrpNc82 (L
1
is the maximum localization value,
pis the Hill coefficient, which was fixed to 1; Fig. 3b). Such titrations are not
limited to Cy5 but can, in principle, also be performed with other fluorophore-
labelled antibodies (Supplementary Fig. 6).
The saturation of mAb BrpNc82 was analysed by using a constant concentration
of secondary antibodies (5.2 10 8M) with a fixed ratio of Cy5 and A488 F(ab0)
2
fragments (1% Cy5: 99% A488) and by diluting the mAb BrpNc82 (1/20, 1/50,
1/100, 1/200, 1/500, 1/1,000, 1/2,000, 1/5,000 and 1/10,000). The localization data
was analysed (4–5 NMJs per dilution) as described for Cy5 secondary antibody
dilutions (Fig. 3c; L
1
¼198.9 and L
CAZ
(1/2,000) ¼70.6).
In order to estimate how many Cy5-labelled secondary antibodies bind per
primary mAb BrpNc82 under experimental conditions (5.2 10 8M Cy5, 100%),
NMJs (n¼5) were stained with a very low concentration of mAb BrpNc82
(1/20,000) together with an antibody directed against an N-terminal Brp epitope
(rabbit-BrpN-term 20, provided by S.J. Sigrist, 1/2,000; labelled by Alexa Fluor 488
goat anti rabbit (A11008, Invitrogen), 1/250). Only solitary spots within CAZs
defined via BrpN-term were measured (threshold Z10 px), histogrammed and fit to
a Poisson model to extract the number of localizations corresponding to one mAb
BrpNc82 (L
E
(Nc82)).
Using the localization values (Table 2) to solve the equation
No:of BrpCAZ unit¼LocCAZ unit
L2Cy5ðÞ
L1Cy5ðÞ
LCAZ Cy5 100 %ðÞ
L1Nc82ðÞ
LCAZ Nc82 1=2;000ðÞ
L2Cy5ðÞ
LENc82ðÞ
ð1Þ
gives an estimate of 137±29 Brp molecules s.e.m. at an average CAZ-unit (we
refrained from cancelling L2(Cy5) to improve the traceability of the equation).
Correspondingly, the conversion factor 0.134±0.028 s.e.m. was used to translate
localizations into molecules for all genotypes (Table 1).
Cluster analysis.For the analysis, we used a home-written density-based algo-
rithm. The base of the algorithm comes from the known algorithm, density-based
spatial clustering of applications with noise: this algorithm simply looks for loca-
lizations that reside within the middle of a circle of radius Eps and enclose at least k
other localizations30,65. Since our data contains a large number of localizations, in
between putative clusters, we added more constraints on cluster detection. The
algorithm starts with finding local maxima of density. The density is defined as the
number of localizations within an Eps radius circle around a localization (Eps-
environment). Each local maximum that has a density that is more than kwill be
defined as a cluster centre. For each cluster centre, the localizations contained
within its Eps-environment are examined for holding the condition of k
localizations. When the condition is held, the current localization along with all the
other localizations within the Eps-environment will become members of this
cluster. The algorithm then moves on to another localization that was found within
the circle of radius Eps from the cluster centre and examines if it holds the
conditions. If not, this localization will be a boundary point for the cluster and the
expansion will end. If it does, this localization will be considered a core-object of
the same cluster, and the cluster will keep expanding until it reaches a boundary
point. In addition to the above conditions, each localization added to the cluster
should have a lower density than the localization that discovered it. If the algorithm
detects an increase in density, this localization will not be part of the previous
cluster. This separates adjacent clusters and prevents creating saddle points
between them.
We chose a search Eps of 20 nm that roughly corresponds to the estimated
radius of an EM filament with the antibody complex attached to it. For a chosen
Eps, the density is calculated as the number of localizations within the Eps-
environment of the current localization. The parameter kwas chosen based on the
density distribution that had a peak B12–18, and kwas large enough to separate
from noise but not too large as to find a sparse number of clusters (k¼16).
After defining the clusters and the non-clustered localizations, we set to
examine a set of different parameters that characterize the clusters. We analysed
the following cluster properties: (a) the number of localizations belonging to each
cluster, and (b) cluster shape and area. We found that most clusters did not show
an exact circular shape but were more elliptic. Therefore, a 2D ellipse was tightly
fitted to each cluster. From this ellipse, the parameters: shape (minor radius divided
by major radius), minor and major axes are calculated. Clusters were defined as
comprising a CAZ-unit if the density was at least four clusters within a circle of
radius 200 nm.
Statistics.Statistical tests were used as indicated. In the figures, the level of sig-
nificance is denoted by asterisks: *Pr0.05, **Pr0.01, ***Pr0.001.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5650 ARTICLE
NATURE COMMUNICATIONS | 5:4650 | DOI: 10.1038/ncomms5650 | www.nature.com/naturecommunications 11
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Acknowledgements
We thank T. Langenhan, S. Doose, K. Neuser and J. Eilers for their discussions;
E. Buchner and S.J. Sigrist for reagents; C. Wirth, M. Oppmann and H. Neuweiler for
technical support; and D. Bar-On for assistance with the cluster analysis. This work was
supported by grants from the German Research Foundation (KI 1460/1-1 and SFB
1047/A05 to R.J.K.; HA 6386/2-1 to S.H.; and SFB 581/B27 to M.H.), the National
Institutes of Health (NS043171 to A.D.), a fellowship from the Graduate School of Life
Sciences, University of Wu¨ rzburg (to N.E.), the Biophotonics Initiative of the German
Ministry of Research and Education (BMBF/grant nos. 13N11019 and 13N12507 to
M.S.), and the German-Israeli Foundation (GIF/grant no. 1125-145.1/2010 to M.S. and
U.A.).
Author contributions
A.D., M.H., M.S. and R.J.K. designed; and D.L., N.E., S.v.d.L. T.H. and X.Z.K.
performed the experiments. A.A., A.R., M.S., N.E., S.H., R.J.K., S.v.d.L., T.H., U.A. and
X.Z.K. evaluated the data. R.J.K. and M.S. supervised the project and wrote the manu-
script with N.E. and contributions from A.A., A.D., A.R., M.H., S.H., S.v.d.L. and U.A.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
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How to cite this article: Ehmann, N. et al. Quantitative super-resolution
imaging of Bruchpilot distinguishes active zone states. Nat. Commun. 5:4650
doi: 10.1038/ncomms5650 (2014).
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