T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $15.00
The Journal of Cell Biology, Vol. 177, No. 5, June 04, 2007 843–855
Synapses are highly specialized and asymmetric intercellular
junctions organized into morphologically, biochemically, and
physiologically distinct subdomains. At the presynaptic terminal
membrane, active zones mediate Ca2+-dependent synaptic vesi-
cle fusion, whereas the surrounding periactive zones are essen-
tial for synaptic vesicle endocytosis and the control of synaptic
terminal growth (Sone et al., 2000; Zhai and Bellen, 2004).
Definition of distinct synaptic subdomains is not restricted
to the plasma membrane but is also clearly visible within the
presynaptic terminal cytoplasm. Notably, synaptic vesicles are
clustered at the cell cortex, in the vicinity of active zones. In
addition, they seem organized into functional subpools display-
ing distinct release and recycling properties (Rizzoli and Betz,
2005). Such an organization requires the precise traffi cking and
targeting of vesicles to their appropriate location and the spe-
cifi c recruitment and release of subsets of vesicles, depending
on the stimulation conditions. One of the main challenges syn-
apses have to face is maintaining such a highly organized struc-
ture while constantly adapting their morphology and strength in
response to developmental programs and/or external stimuli.
Indeed, synaptic terminals can adjust their size; the number,
size, and composition of their pre- and postsynaptic membrane
specializations; and the availability and release competence of
cytoplasmic synaptic vesicles. These dynamic changes require
the maintenance of precise physical and functional connections
between pre- and postsynaptic compartments, as well as be-
tween cytoplasmic and plasma membrane subdomains.
To date, the mechanisms allowing such a dynamic re-
organization are still poorly understood. However, using the
Drosophila melanogaster neuromuscular junction (NMJ) as a
genetic model, different components of periactive zones, in-
cluding transmembrane proteins and adaptor molecules, have
been implicated in the control of terminal outgrowth (Schuster
et al., 1996b; Beumer et al., 1999; Sone et al., 2000; Koh et al.,
2004; Marie et al., 2004). Cell adhesion molecules (CAMs) of
the Ig superfamily seem particularly important in maintaining
The Ig cell adhesion molecule Basigin controls
compartmentalization and vesicle release
at Drosophila melanogaster synapses
Florence Besse,1 Sara Mertel,2,3 Robert J. Kittel,2,3 Carolin Wichmann,2,3 Tobias M. Rasse,2,4 Stephan J. Sigrist,2,3
and Anne Ephrussi1
1Developmental Biology Unit, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
2European Neuroscience Institute Göttingen, D-37077 Göttingen, Germany
3Institut für Klinische Neurobiologie und Rudolf-Virchow-Zentrum, Universität Würzburg, D-97078 Würzburg, Germany
4Hertie-Institute for Clinical Brain Research, University of Tübingen, D-72076 Tübingen, Germany
property of neuronal cells requires the coordinated re-
arrangement of synaptic membranes and their associated
cytoskeleton, yet remarkably little is known of how this
coupling is achieved. In a GFP exon-trap screen, we
identifi ed Drosophila melanogaster Basigin (Bsg) as an
immunoglobulin domain-containing transmembrane pro-
tein accumulating at periactive zones of neuromuscular
junctions. Bsg is required pre- and postsynaptically to
ynapses can undergo rapid changes in size as well
as in their vesicle release function during both plas-
ticity processes and development. This fundamental
restrict synaptic bouton size, its juxtamembrane cyto-
plasmic residues being important for that function. Bsg
controls different aspects of synaptic structure, including
distribution of synaptic vesicles and organization of the pre-
synaptic cortical actin cytoskeleton. Strikingly, bsg function
is also required specifi cally within the presynaptic terminal
to inhibit nonsynchronized evoked vesicle release. We thus
propose that Bsg is part of a transsynaptic complex regulat-
ing synaptic compartmentalization and strength, and co-
ordinating plasma membrane and cortical organization.
Correspondence to Stephan J. Sigrist: firstname.lastname@example.org.
de; or Anne Ephrussi: email@example.com
Abbreviations used in this paper: BRP, Bruchpilot; CAM, cell adhesion molecule;
CSP, cysteine string protein; eEJC, nerve-evoked excitatory junctional current; NMJ,
neuromuscular junction; PSD, postsynaptic density; SSR, subsynaptic reticulum.
The online version of this article contains supplemental material.
JCB • VOLUME 177 • NUMBER 5 • 2007 844
the integrity of synaptic terminals but also in transmitting sig-
nals to the cell interior, thereby promoting differentiation of
pre- and postsynaptic specializations and regulating synaptic
structure and function (Schuster et al., 1996a; Stewart et al.,
1996; Sone et al., 2000; Polo-Parada et al., 2001; Rougon and
Hobert, 2003; Yamagata et al., 2003). Moreover, the actin-rich
presynaptic cytoskeleton is important for rearranging synaptic
domains and for controlling synaptic vesicle distribution and
release ability (Dillon and Goda, 2005). How the linkage between
cortical cytoskeleton, cytoplasmic vesicle pools, and specialized
membrane domains is mediated and, more generally, how plasma
membrane and cytoplasmic membranes are spatially and func-
tionally connected largely remain to be elucidated.
Here, we identify the transmembrane Ig CAM Basigin
(Bsg) as a new component of periactive zones at D. melanogaster
NMJ synapses. Bsg is the only D. melanogaster member of the
Basigin/Embigin/Neuroplastin family of glycoproteins, of which
mammalian Bsg has been shown to have multiple functions, in-
cluding in tumor progression (Nabeshima et al., 2006). It seems
to regulate cell architecture and cell–cell recognition (Fadool and
Linser, 1993; Curtin et al., 2005), act in signaling (Guo et al.,
1997; Tang et al., 2006), and act as a chaperone for transmem-
brane proteins (Kirk et al., 2000; Zhou et al., 2005). By analogy to
other mammalian cell surface glycoproteins, and in particular to
the CD44 transmembrane protein family (Ponta et al., 2003), Bsg
may be essential for establishment of transmembrane complexes
and for organization of cell structure and signal transduction cas-
cades. Interestingly, mammalian Bsg and Neuroplastin have been
suggested to play a role in memory functions and long-term
potentiation, respectively, although their precise function has not
been determined (Naruhashi et al., 1997; Smalla et al., 2000).
Our in vivo study shows that D. melanogaster Bsg is re-
quired in both pre- and postsynaptic compartments to control
formation and growth of synaptic varicosities (or boutons) at
larval NMJs. We also show that Bsg is a bona fide Ig CAM
because (1) it can promote cell–cell adhesion and (2) its trans-
membrane and/or juxtamembrane cytoplasmic domains are
critical for its function in vivo. Furthermore, down-regulation of
bsg affects the size of postsynaptic receptor fi elds, as well as the
distribution of synaptic vesicles within neuronal terminals.
These defects are associated with alterations of the actin/Spec-
trin network, suggesting that Bsg accumulation at the plasma
membrane regulates synaptic compartmentalization and archi-
tecture. Strikingly, we found that Bsg function is also essential
within the presynaptic compartment for the restriction of neuro-
transmitter release. Based on our in vivo data, we propose that
Bsg may be part of a transsynaptic complex surrounding active
zones and involved in the coordinated development of pre- and
postsynaptic membranes, as well as in the functional coupling
of plasma membrane and cortical subdomains.
at D. melanogaster NMJs
To identify new proteins controlling synapse development, we
searched for proteins specifi cally accumulating at developing
NMJs of D. melanogaster larvae. We performed a protein-trap
screen in which GFP fusion proteins expressed from their en-
dogenous promoters are randomly generated (Morin et al., 2001)
and screened the expression pattern of ?350 GFP+ lines (see
Materials and methods). Thereby, we identifi ed 10 lines exhibit-
ing GFP expression at the larval NMJ and focused on three inde-
pendent lines showing strong GFP accumulation at larval NMJs,
but only low GFP levels along the motoneuron axons, and at the
surface of muscle fi bers (Fig. 1, A and A′). In these lines, a strong
GFP signal is also observed in different neuropil structures of
the larval brain (Fig. 1 B).
Using inverse PCR, we found that in each of these three
lines the protein-trap cassette was inserted in the gene CG31605,
encoding the D. melanogaster homologue of the mammalian
protein CD147/EMMPRIN/Basigin, Basigin (Bsg; Curtin et al.,
2005). According to predictions, the artifi cial GFP exon should
be incorporated upon splicing into mature transcripts whose
transcription starts upstream of the insertion, resulting in the
in-frame incorporation of GFP. We confi rmed this by RT-PCR
Figure 1. GFP expression pattern in bsg protein-trap lines. (A and A′)
Third instar larva heterozygous for the protein-trap insertion, stained with
anti-GFP antibodies (A and A′, green), anti-HRP antibodies (A′, red), and
phalloidin (A′, blue). (B) GFP-Bsg accumulation in heterozygous larval
brain. (C) Western blot of total extracts from w larvae (left) or larvae het-
erozygous (protein-trap/+; middle) or homozygous (protein-trap; right) for
a protein-trap insertion, probed with anti-Bsg and anti-GFP antibodies.
(D) Alignment of D. melanogaster and human Bsg proteins. The green
triangle indicates the location of GFP insertion.
BSG IN SYNAPTIC VESICLE DISTRIBUTION AND RELEASE • BESSE ET AL.845
(unpublished data) and Western blot analysis using anti-Bsg anti-
bodies raised against the D. melanogaster protein (Fig. 1 C).
D. melanogaster Bsg is a small transmembrane protein
composed of two extracellular Ig-like domains, a highly con-
served transmembrane domain, and a short cytoplasmic tail
(Fig. 1 D). Mammalian Basigin has been described as a multi-
functional protein regulating different processes, including tu-
mor invasion, reproduction, and sensory and memory functions
(Muramatsu and Miyauchi, 2003; Toole, 2003). Interestingly,
bsg is highly expressed in the mouse nervous system (Fan et al.,
1998), and Bsg protein is present in purifi ed postsynaptic den-
sities (PSDs) of mouse central nervous system synapses (Collins
et al., 2006). In D. melanogaster, Bsg has been proposed to regulate
cellular architecture during eye morphogenesis (Curtin et al.,
2005). The cellular mechanisms underlying its functions, how-
ever, are still poorly understood. Bsg is the only D. melanogaster
member of a mammalian protein family including Basigin,
Embigin, and Neuroplastin/gp65/gp55. All the members of this
family have been suggested to regulate cell–substratum adhe-
sion and/or cell–cell adhesion, and are therefore proposed to
belong to the Ig CAM family (Fadool and Linser, 1993; Huang
et al., 1993; Kasinrerk et al., 1999; Smalla et al., 2000).
Basigin accumulates at periactive zones
To check if the distribution of tagged Bsg refl ects that of the
endogenous protein, we stained wild-type larvae with anti-Bsg
antibodies. Endogenous Bsg shows a localization pattern identi-
cal to that of the GFP fusion (Fig. 2 A), and both precisely co-
localize with Discs large (Dlg), a transmembrane protein present
both pre- and postsynaptically, but mainly accumulating in
stacks of postsynaptic membranes named subsynaptic reticu-
lum (SSR; Fig. 2, A″ and B″; Lahey et al., 1994). Like Dlg, Bsg
accumulates to higher levels at type Ib than at smaller type Is
boutons (Fig. 2, A and B; and not depicted). To exclusively
visualize the presynaptic expression of Bsg, we next expressed
a GFP-tagged Bsg fusion specifi cally in the presynaptic com-
partment (Fig. 2 C) and observed a robust GFP signal at NMJs.
Consistent with an accumulation of Bsg at the presynaptic
membrane, the inner aspect of both endogenous Bsg and GFP-
Bsg protein-trap fusion labels partially overlap with the pre-
synaptic membrane marker HRP (Fig. 2, D and E).
Further analysis revealed that Bsg is not homogenously
distributed at the membrane but is excluded from active zones
labeled with anti-Bruchpilot (BRP) NC82 antibodies (Fig. 2 F;
Wagh et al., 2006). Therefore, similar to other transmembrane
proteins involved in the structural control of synaptic terminals,
such as Dlg or Fasciclin II (Fas II), Bsg localizes to periactive
zones (Sone et al., 2000).
Identifi cation of basigin mutants
According to data from the D. melanogaster genome project,
bsg encodes nine distinct putative transcripts, of which eight
encode the same protein (Fig. 3 A). One of the predicted tran-
scripts, RG, encodes a slightly different protein. However, this
transcript is barely detected after quantitative RT-PCR on larval
fi llet extracts (unpublished data).
To address the in vivo requirement for Bsg at the larval
NMJ, we searched for P element insertions near the transcrip-
tion starts of the longer transcripts and found five belonging
to the same lethal complementation group. Three of these
(lk13638, lk14308, and NP3198), when placed in trans
over a defi ciency covering the locus (Df[2L]Exel7034, hereafter
referred to as Df), cause an embryonic/early larval lethality that
can be rescued by ubiquitous expression of a bsg transgene (Fig.
3 B), and represent strong hypomorphic alleles (Curtin et al.,
2005; unpublished data). Two other insertions, NP6293 and
l(2)SH1217, behave genetically as weaker hypomorphic alleles,
as, respectively, 30 and 50% of the corresponding hemizygotes
Figure 2. Bsg accumulation at the NMJ.
(A and B) NMJs of w (A) or protein-trap/+ (B)
larvae double stained with anti-Bsg (A) or anti-
GFP (B) antibodies and anti-Dlg antibodies
(A′ and B′). (C) elav-Gal4/+; UAS-GFP-bsg/+
larvae stained with anti-GFP (C) and anti-HRP
(C′) antibodies. (D and E) NMJs of w (D) and
protein-trap/+ (E) larvae double stained with
anti-Bsg (D) or anti-GFP (E) and anti-HRP
(D′ and E′) antibodies. A–E correspond to z
projections of serial confocal sections. Bar,
10 μm. (F) Tangential confocal section of a
protein-trap/+ synaptic bouton stained with
anti-GFP and NC82 anti-BRP antibodies and
plot of the intensity profi les of GFP (green) and
NC82 (pink) stainings across the bouton (sec-
tion is indicated by white marks on the over-
lay). A–E are from muscle 6/7 NMJs, and F is
from muscle 4 NMJ. Bar, 5 μm.
JCB • VOLUME 177 • NUMBER 5 • 2007 846
reach third larval instar (Fig. 3 B). This semilethality is reverted
after precise excision of NP6293 (Fig. 3 B). Consistent with
these data, Western blot analysis shows that Bsg expression lev-
els are greatly reduced in Df/NP6293 and Df/l(2)SH1217 mu-
tant larvae but restored to normal levels after precise excision of
NP6293 (Fig. 3 C). The amount of Bsg specifi cally accumulat-
ing at the NMJ is also signifi cantly reduced in Df/NP6293 larval
fi llets, compared with wild type (Fig. 3 D). Together, these re-
sults show that NP6293 and l(2)SH1217 are bsg mutant alleles
suitable for analysis of larval NMJ development and matura-
tion. We therefore renamed them bsg6293 and bsg1217 and used
them for subsequent studies.
Basigin is required in both pre-
and postsynaptic compartments
to restrict bouton size and allow
effi cient NMJ expansion
To determine whether bsg mutants exhibit defects in their mo-
toneuron connection pattern and/or NMJ morphology, we ex-
amined synaptic boutons and motoneuron membranes of both
Df/bsg6293 and Df/bsg1217 third instar larvae. Axonal targeting
is not altered to a visible degree in these animals. However,
the growth of synaptic boutons is strongly altered, as revealed
by the considerable increase in their size (Fig. 4, C–G; and
Table S1, available at http://www.jcb.org/cgi/content/full/
jcb.200701111/DC1). In particular, the proportion of very
large boutons (>12 μm2) is greatly increased in bsg mutants
compared with controls (Fig. 4 G and Table S1; P < 0.001).
The observed increase in bouton size is associated with a con-
comitant reduction of both NMJ branching and bouton number
(Fig. 4, C–F and H; and Table S1), keeping the overall NMJ
size close to normal (muscle 4 NMJ area: 165.33 ± 9 μm2 [n =
18] and 163.97 ± 15 μm2 [n = 12] for Df/bsg6293 and w
larvae, respectively; P > 0.05). Moreover, defects in bouton
size and number are already observed in second instar animals
(Fig. S1) and revert after precise excision of NP6293 (Fig.
4 H and Table S1).
To explicitly determine whether these growth defects
could be rescued and whether they refl ected pre- and/or post-
synaptic function of bsg at the NMJ, we expressed a wild-type
copy of bsg in specifi c compartments of Df/bsg6293 larvae. Ex-
pression of wild-type Bsg solely in muscles (using mhc-Gal4 or
24B-Gal4) or in neurons (using elav-Gal4), partially, but signif-
icantly, rescued both the increase in bouton size and the reduc-
tion of bouton number observed in mutant larvae (Fig. 4, I, J, L,
and M; and not depicted). Near-complete rescue of bouton size
and junction growth was obtained only when expressing wild-
type Bsg both pre- and postsynaptically (Fig. 4, K, L, and M;
and Table S1).
Collectively, we conclude that Bsg is needed for effi cient
outgrowth of larval NMJs and that its function is required in
both pre- and postsynaptic compartments to defi ne boutons of
proper size. Such a dual requirement is documented for the Ig
CAM Fas II and is thought to refl ect the establishment of trans-
synaptic homophilic interactions (Schuster et al., 1996a). We
thus tested whether Bsg might also promote cell–cell adhesion.
As shown in Fig. 4 N, S2 cells transfected with a GFP-Bsg con-
struct strongly adhere to each other, whereas S2 cells transfected
with a control GFP construct do not. Thus, Bsg promotes cell
aggregation, consistent with the idea that Bsg could regulate
the addition and growth of synaptic boutons through modulation
of cell adhesion.
Figure 3. bsg locus and mutants. (A) Genomic organiza-
tion and intron–exon structure of bsg. Untranslated and
coding regions are represented as white and black boxes,
respectively. Positions of P element and GFP-containing piggy-
Bac insertions are indicated in red and green, respectively.
(B) Percentages of GFP− third instar larvae recovered among
the progeny of a cross between Df/CyO-GFP females and
l(2)k13638/CyO-GFP, l(2)SH1217/CyO-GFP, NP6293/
CyO-GFP, or NP6293pr.ex/CyO-GFP males. 33% of GFP−
animals are expected in absence of lethality. NP6293pr.ex is the
NP6293 chromosome obtained after precise excision of the
P element. The third bar corresponds to rescued Df/l(2)k13638;
UAS-GFP-Bsg-fl /tub-Gal4 larvae. Note that ?80% (16/20) of
these larvae developed into pharate adults, of which 38%
(6/16) hatched. At least 177 larvae were scored per cross.
Statistical comparisons to the “expected” control: **, P <
0.001 (χ test). (C) Western blot of wild-type and mutant body
wall extracts, probed with anti-Bsg antibodies. Ponceau stain-
ing is shown as a loading control. (D) Wild-type (top) and
Df/NP6293 (bottom) larvae stained with anti-Bsg and anti-
HRP antibodies. It was necessary to use diluted and purifi ed
anti-Bsg serum to visualize the difference in expression levels.
Both pictures were taken from muscle 4 NMJs using identical
confocal settings. Bar, 20 μm.
BSG IN SYNAPTIC VESICLE DISTRIBUTION AND RELEASE • BESSE ET AL.847
A conserved juxtamembrane basic motif
is crucial for Basigin function in vivo
Depending on the cell type and/or the protein partners, different
domains of mammalian Bsg are required for its activity (Guo
et al., 1997; Kirk et al., 2000; Sun and Hemler, 2001). Thus, to
determine which domains of D. melanogaster Bsg are required
for its function at the larval NMJ, we generated GFP-tagged
truncated variants (Fig. 5 A) and assayed their capacity to res-
cue Df/bsg6293 morphological defects.
Bsg lacking the most C-terminal part of the intracellular
domain (∆intra) rescues defects in bouton size and number
similarly to the full-length tagged form (fl ) when expressed
presynaptically (Fig. 5, D and E). In contrast, forms composed
of the two Ig domains only (Extra) or of the two Ig domains of
Bsg fused to the transmembrane and intracellular domains of
rat CD2 (Bsg-CD2) do not signifi cantly rescue the decrease
in bouton number observed in bsg mutants and only poorly
rescue bouton growth defects (Fig. 5, D and E). Thus, Bsg
transmembrane and/or juxtamembrane cytoplasmic domains
appear crucial for regulation of NMJ bouton growth and bud-
ding by Bsg.
The cytoplasmic juxtamembrane region of Bsg contains
a conserved cluster of positively charged residues (KRR; Fig.
5 B). We found that when KRR is substituted with NGG, the
mutated protein only poorly rescues the reduced bouton num-
ber and only to a low extent the increased bouton size of bsg
larvae (Fig. 5, D and E). Moreover, ubiquitous expression
of the KRR®NGG mutated protein does not significantly
rescue the early lethality of the strong mutant combination
Df/l(2)k13638, whereas full-length Bsg does (Fig. 5 C), further
indicating a crucial and previously unknown role of this motif
for Bsg function.
Figure 4. bsg regulates NMJ growth. (A–F)
NMJs at muscle 4 (A, C, and E) or muscles
6/7 (B, D, and F) of w (A and B), Df/bsg6293
(C and D), and Df/bsg1217 (E and F) larvae
stained with anti-CSP (red) and anti-HRP (green)
antibodies. Asterisks in B, D, and F mark the
branch terminals chosen for magnifi cation (in-
sets). Bars: (A, C, and E) 20 μm; (B, D, and F)
60 μm. (G) Distribution of synaptic boutons
according to their size (in μm2). At least 97
boutons were scored per genotype. (H) Quan-
tifi cation of muscle 6/7 type Ib bouton number.
Bouton numbers were normalized to muscle
surface area, and the reference was set to
100 for w control larvae. Note that removal
of one copy of bsg causes a mild reduction of
bouton number, indicating a dosage sensitiv-
ity of the phenotype. Because of the smaller
muscle size of Df/bsg1217 larvae, their bou-
ton number is increased artifi cially upon nor-
malization (numerical values below the bars
represent the raw data, which are also shown
in Table S1, available at http://www.jcb
Statistical comparisons to the w controls: **,
P < 0.001 (t test). n represents the number
of junctions scored per genotype. Error bars
indicate SEM. (I–K) NMJs at muscle 6/7 of Df/
bsg6293; mhc-Gal4/UAS-bsg (I), elav-Gal4/+;
Df/bsg6293; UAS-bsg/+ (J), and elav-Gal4/+;
Df/bsg6293; mhc-Gal4, UAS-bsg/+ (K) larvae
double stained with anti-CSP (red) and anti
HRP (green) antibodies. Bar, 60 μm. (L) Per-
centage of synaptic boutons >12 μm2 in Df/
bsg6293 larvae and different rescue contexts.
At least 114 boutons were scored per geno-
type. (M) Quantifi cation of muscle 6/7 type Ib
bouton numbers in Df/bsg6293 larvae and dif-
ferent rescue contexts. Bouton numbers were
normalized to muscle surface area, and the
reference set to 100 for w control larvae. Sta-
tistical comparisons to Df/bsg6293 animals: *,
P < 0.05; **, P < 0.001 (t test). n represents
the numbers of junctions scored per genotype.
Error bars indicate SEM. (N) Cell aggregation
assays using S2 cells transfected with either a
GFP control construct (GFP-Golgi) or GFP-Bsg.
(left) Light microscopy pictures; (right) number
of clusters containing 10–20 cells, 20–40
cells, or >40 cells (per 5 × 104 cells). Four
samples, from two independent transfections,
were analyzed per construct.
JCB • VOLUME 177 • NUMBER 5 • 2007 848
The specifi cation of active zones and
periactive zones is normal in basigin mutants
Given that bsg mutants exhibit defective NMJ morphology, we
next tested whether the assembly and/or maintenance of pre-
and postsynaptic specializations might also be altered. As shown
in Fig. S2 (A and B; available at http://www.jcb.org/cgi/content/
full/jcb.200701111/DC1), the overall distribution and com-
plementary accumulation of markers specifi c to perisynaptic
zones and PSDs seems to be normal at bsg junctions. Moreover,
no alteration of SSR integrity could be detected at the light
microscopy level (Fig. S2, C and D) or at the ultrastructural
level (Fig. 6 E).
Next, we assayed the distribution of receptor fi elds and
active zones, using antibodies recognizing the glutamate recep-
tor subunit GluRIID (Qin et al., 2005) in combination with anti-
BRP NC82 antibodies (Wagh et al., 2006). As illustrated in
Fig. 6 (A and B), the distribution of these two markers is normal
at bsg junctions: BRP and GluRIID remain concentrated in
individual puncta of normal intensity and distribution (density
of BRP+ puncta: 1.35 ± 0.3/μm2 and 1.19 ± 0.2/μm2 for w and
Df/bsg6293 third instar larvae, respectively; P > 0.05). Moreover,
as described for wild-type animals, BRP+ release sites are re-
producibly found in direct apposition to postsynaptic glutamate
receptor clusters in bsg larvae (Fig. 6, A and B, insets; and not
depicted). Consistent with these observations, transmission EM
showed that active zones are found at normal frequency and that
their characteristic electron-dense specializations (T-bars) are
of normal morphology (Fig. 6, D–I). Quantifi cation, however,
indicated a slight increase in the electron-dense PSD diameter
(Fig. 6 I), which correlates with a slight, but signifi cant, increase
in the mean size of GluRIID clusters observed using light
microscopy (w = 0.76 ± 0.01 μm, n = 525; Df/bsg6293 = 0.84 ±
0.01 μm, n = 501; P < 0.001; Fig. 6 C). Thus, these results
suggest that, although Bsg is involved in defi nition of receptor
fi eld size, its function is not essential for specifying active and
periactive zone domains.
Presynaptic actin cytoskeleton organization
is altered in basigin boutons
Given that D. melanogaster Bsg has been suggested to regulate
cell architecture, possibly by modulating the cell cytoskeleton
(Curtin et al., 2005), we checked the integrity of the actin-based
cytoskeleton at bsg NMJs. α-Spectrin (α-Spec) closely asso-
ciates with the NMJ juxtamembrane actin-rich cytoskeleton
(Ruiz-Canada et al., 2004). Although it is mainly enriched in
the postsynaptic peribouton area, α-Spec is also found at the in-
ner presynaptic bouton cortex (Fig. 7, A and C1; Pielage et al.,
2005). In bsg larvae, even though no major alterations of the post-
synaptic Spectrin cytoskeleton are observed, α-Spec aggregates
are detected within the bouton lumen (Fig. 7, B, D1, and E1) in
?38% of NMJ branches (Fig. 7 L). These aggregates are ?0.5 μm
large and contain other α-Spec–associated proteins, such as
β-and βH-Spectrin (not depicted), as well as the actin-associated
protein Wasp (Fig. 7, G and G′). In contrast, no enrichment of
microtubule-associated proteins was observed in these aggregates
(Fig. 7, I′ and I″). To more directly and specifi cally visualize the
presynaptic F-actin network, we expressed the F-actin–binding
domain of Moesin fused to GFP (GFP-GMA) exclusively in
neurons (Dutta et al., 2002). As shown in Fig. 7 J, GFP-GMA
accumulates at the cortex of wild-type synaptic boutons. In bsg
mutants, although a cortical actin network is still clearly detected
at the periphery of boutons, clusters of F-actin fi laments are also
Figure 5. Differential rescue capacity of vari-
ous mutated Bsg proteins. (A) Scheme of Bsg
variants. ∆intra corresponds to a form lacking
the last 14 amino acids but including the juxta-
membrane KRR stretch, Extra to a form lacking
both the transmembrane and the cytoplasmic
domains, KRR®NGG to a full-length protein
where the KRR residues have been substituted
to NGG, and Bsg-CD2 to a chimeric protein
composed of the two Ig domains of Bsg fused
to the transmembrane and cytoplasmic do-
mains of rat CD2. (B) Alignment of Bsg trans-
membrane and intracellular domains. H.s.,
Homo sapiens; M.m., Mus musculus; D.r.,
Danio rerio; Dr. m., D. melanogaster; A.g.,
Anopheles gambiae. The black box indicates
the amino acids deleted in the ∆intra construct.
(C) Percentages of non–CyO-GFP third instar
larvae recovered among the non-TM6 progeny
of a cross between Df/CyO-GFP; UAS-GFP-
Bsg*/TM6 females and l(2)k13638/CyO-
GFP; tub-Gal4/TM6 males. 33% of non–CyO-GFP
animals are expected in case of complete rescue
(left bar). Numbers correspond to the total
numbers of non-TM6 larvae scored in the entire
progeny of each cross. Statistical comparison
to GFP-Bsg-fl #28 animals: **, P < 0.001
(χ test). (D) Quantifi cation of muscle 6/7 type
Ib bouton numbers in Df/bsg6293 larvae and different rescue contexts (driver used for rescue: elav-Gal4). Statistical comparisons to Df/bsg6293 animals:
**, P < 0.001 (t test). The number of junctions scored for each genotype is represented in white. Error bars indicate SEM. fl #7 and fl #28 represent two
independent insertions of the GFP-tagged full-length transgene. (E) Percentage of synaptic boutons >12 μm2 in Df/bsg6293 larvae and different rescue
contexts. Statistical comparison to w animals: **, P < 0.001 (χ test).
BSG IN SYNAPTIC VESICLE DISTRIBUTION AND RELEASE • BESSE ET AL.849
frequently present within them (Fig. 7, K and L). Altogether, these
observations indicate that the organization of the presynaptic
F-actin network is altered at bsg NMJs.
Diffuse distribution of synaptic vesicles
at basigin NMJs
In the course of our ultrastructural analysis, we observed that
abnormally large vesicles (diameter of up to ?300 nm) are pre-
sent in Df/bsg6293 boutons (Fig. 6, E and H) but are only rarely
observed after presynaptic reexpression of Bsg in this back-
ground (not depicted). The exact nature of these vesicles remains
unclear, as we have not observed any concomitant alteration
in the distribution and/or size of the FYVE-GFP+ endosomal
compartment (Wucherpfennig et al., 2003) at the light micros-
copy level (unpublished data).
To determine whether these defects could be associated
with an alteration of the synaptic vesicle compartment, we ana-
lyzed synaptic vesicle distribution using specifi c vesicle markers.
In wild-type boutons, synaptic vesicles are clearly enriched at
the cortex but are largely excluded from their central core (Fig. 8,
A and A′). In contrast, in bsg larvae, preferential association of
vesicles with the bouton cortex is lost in ?60% of NMJ branches
(Fig. 8 D), and CSP+ (cysteine string protein) vesicles fi ll parts
of (Fig. 8, B–B″) or even the entire lumen (Fig. 8. C–C″) of the
bouton. CSP staining is also abnormally strong in axonal tracts
connecting boutons and appears more granular than in control
animals (Fig. 8, B and C). An essentially identical mislocaliza-
tion was observed using two other independent markers of syn-
aptic vesicles, Synaptotagmin and Synapsin (Fig. 8, E–H). These
defects do not indirectly result from the increase in bouton size
observed in bsg mutants, as synaptic vesicle localization appears
normal in fase76 hemizygous larvae, which also form abnormally
large boutons (Schuster et al., 1996a; Stewart et al., 1996; un-
published data). Together with the fact that such a diffuse distri-
bution can be observed upon tracking of freshly endocytosed
synaptic vesicles (FM 1–43 loading assay; Fig. S3, C and D, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200701111/DC1),
our data suggest that Bsg specifi cally regulates the spatial dis-
tribution of synaptic vesicles and, in particular, their proper
anchoring to the cortex of synaptic boutons.
Presynaptic down-regulation of Basigin
causes excessive, atypically delayed
To address whether the observed changes in the distribution
of synaptic vesicles might be linked to functional changes in
transmitter release, we recorded postsynaptic currents at larval
NMJs. As shown in Fig. 9 A, the amplitude of the postsynaptic
response to the fusion of single vesicles (minis) is increased
above wild-type levels in bsg mutants (Fig. 9 A, bottom right;
Fig. S3 B; and Table S2, available at http://www.jcb.org/cgi/
content/full/jcb.200701111/DC1). This effect is most likely re-
lated to the observed enlargement of the postsynaptic glutamate
receptor clusters (Fig. 6 C), given that no increase in the size of
Figure 6. Pre- and postsynaptic specializa-
tions in bsg mutants. (A and B) w (A) and
Df/bsg6293 (B) NMJs stained with anti-GluRIID
(A and B) and anti-BRP (A′ and B′) antibodies.
Insets show cross-sections of single boutons.
Bar, 10 μm. (C) Frequency distribution of post-
synaptic glutamate receptor cluster sizes in w
(black) and Df/bsg6293 (gray) third instar lar-
vae. (D and E) Electron micrographs of w (D)
and Df/bsg6293 (E) type Ib boutons. Arrows
point to the SSR, whereas black and white
arrowheads indicate active zones, with and
without T-bars, respectively. Asterisks indicate
atypically large vesicles. Although, in E, syn-
aptic vesicles are also localized in the bouton
center, an unambiguous identifi cation of ec-
topically localized synaptic vesicles was more
diffi cult in other samples. Bars, 250 nm. (F–H)
Magnifi cation of w (F) and Df/bsg6293 (G)
active zones. (H) Example of an abnormally
large vesicle in proximity to a Df/bsg6293 ac-
tive zone (defi ned by its electron-dense plasma
membrane and synaptic vesicle cluster).
(I) Transmission EM–based quantifi cation of
active zone and PSD parameters.
JCB • VOLUME 177 • NUMBER 5 • 2007 850
synaptic vesicles was found in bsg mutants compared with w
controls (Df/bsg6293, 34.4 ± 6.6 nm; w, 34.8 ± 7.2 nm; P > 0.5).
Notably, the frequency of spontaneous release events is strongly
elevated in bsg mutants (Fig. 9 A, bottom left), and these events
often occur clustered in “exocytotic bursts” (Fig. 9 A, asterisk).
The mean amplitude of nerve-evoked excitatory junc-
tional currents (eEJCs) is also increased at bsg NMJs (Fig. 9 B),
which largely correlates with the observed enlargement of minis
(Table S2). However, the temporal profi le of bsg mutant eEJCs
is strikingly lengthened, refl ecting a dramatic and atypically
delayed release of vesicles. Indeed, although the charge carried
by bsg mutant minis is only moderately increased (1.5-fold
increase; Table S2), a near eightfold elevation of the charge trans-
ferred to the postsynapse after exocytosis occurs upon initial
nerve stimulation (Fig. 9 C and Table S2). Notably, this value
decreases progressively after further low-frequency stimulation,
which may result from the exhaustion of the abnormally re-
cruited pool of vesicles responsible for the atypically delayed
release component. Averaging the charge transferred over 15
consecutive sweeps nonetheless reveals a near fi vefold increase
in bsg mutants (Table S2); therefore, a more than threefold ele-
vation of the number of vesicles released per action potential
(quantal content) is estimated to occur.
These defects refl ect a requirement for Bsg within the pre-
synaptic terminal, as sole presynaptic expression of wild-type
Bsg in the mutant background rescues both the asynchronous
evoked release (Fig. 9 B) and the high frequency of spontaneous
release (Fig. 9 A, bottom left), whereas its sole postsynaptic re-
expression does not (Fig. 9, A–C). Interestingly, the presynaptic
reexpression of Bsg even decreases the amplitude of eEJCs and
the frequency of minis below control levels, indicating a dose-
dependent role of presynaptic Bsg in restricting vesicle release.
In this study, we have identifi ed the small transmembrane Ig
CAM Bsg as a new component of perisynapti c zones of D.
melanogaster NMJs. We have shown that Bsg function is required
in pre- and postsynaptic compartments for the formation and
growth of synaptic boutons and that Bsg controls different as-
pects of synapse structure, including distribution of synaptic
vesicles and organization of the presynaptic terminal cortical
actin network. Bsg behaves as a canonical Ig CAM, as it pro-
motes cell–cell adhesion and has a conserved motif in its cyto-
plasmic tail essential for its function in vivo. We propose that
Bsg is part of a transsynaptic complex regulating synaptic
growth and structural organization. Moreover, and very origi-
nally for an Ig CAM, we found that Bsg is essential for inhibit-
ing transmitter release and that this function is restricted to the
Basigin controls synaptic terminal growth
and synaptic organization
In D. melanogaster, the fi nal pattern of larval motoneuron con-
nections and the establishment of synapses are complete by the
end of embryogenesis, yet NMJs are highly dynamic during lar-
val development, expanding through sprouting of new branches
and addition of new synaptic boutons (Schuster et al., 1996a).
Here, we have shown that down-regulation of Bsg levels at the
D. melanogaster NMJ strongly affects bouton growth and bud-
ding, resulting in a decrease in bouton number. This effect is
probably independent of the increased transmitter release ob-
served in bsg larvae because (1) it is already observed in early
second instar larvae and (2), in contrast to the increased neuro-
transmission phenotype, it corresponds to a requirement for bsg
Figure 7. Distribution of actin cytoskeleton markers is altered in bsg larvae.
(A and B) Wild-type (A) and Df/bsg6293 (B) muscle 4 NMJs stained with
anti–α-Spec antibodies. Bar, 10 μm. (C–E) Heterozygous control (C)
and Df/bsg6293 (D and E) boutons stained with anti–α-Spec (C1, D1, and
E1), anti-HRP (C2, D2, and E2), and anti-Dlg (C4, D4, and E4) antibodies.
Bar, 5 μm. (F and G) w (F) and Df/bsg1217 (G) bouton stained with anti-
Wasp (F and G) and anti–α-Spec (F′ and G′, red) antibodies. (H and I) w
(H) and Df/bsg6293 (I) boutons stained with anti–α-Spec (H and I) and anti-
Fusch (H′ and I′) antibodies. Bar, 5 μm. (J and K) Synaptic boutons of wild-
type (J) and Df/bsg6293 (K) larvae expressing a fusion of GFP with the
F-actin binding domain of Moesin (GMA), under the control of elav-Gal4.
GFP-GMA expression is shown in J and K, and is in green in J′ and K′. HRP
staining is shown in red in J′ and K′. Bar, 5 μm. Images A and B corre-
spond to z projections of serial confocal sections throughout entire boutons
(step size: 0.3 μm), and images C–K correspond to single optical slices
taken through bouton centers. (L) Graph showing the percentage of NMJ
6/7 branches containing presynaptic Spec+ or GMA+ aggregates. **,
P < 0.001 (χ test).
BSG IN SYNAPTIC VESICLE DISTRIBUTION AND RELEASE • BESSE ET AL.851
function in both pre- and postsynaptic compartments. This may
refl ect a role of Bsg in regulating adhesion between pre- and
postsynaptic membranes, as described for the Ig CAM Fas II
(Schuster et al., 1996a). Bsg function is, however, not restricted
to modulation of synaptic membrane adhesiveness, as mutant
forms lacking the transmembrane and/or juxtamembrane cyto-
plasmic domains can promote cell–cell aggregation (Fig. S1 B)
but function poorly in vivo. Bsg may thus also signal toward the
cell cytoplasm and/or regulate the actin cytoskeleton (see the
In addition, Bsg controls synaptic architecture: it modu-
lates the size of postsynaptic glutamate receptor fi elds and,
more strikingly, is required for the anchoring of synaptic vesi-
cles to the presynaptic terminal cortex. This suggests that Bsg
could be a key component coupling organization of the plasma
membrane and cytoplasmic vesicular compartments. Consistent
with such a role, we have observed defects in “plasma mem-
brane versus internal membrane” sorting of presynaptic trans-
membrane components (for internal accumulation of HRP
epitopes, see Fig. 7, D and E; not depicted). We thus propose that
Bsg may be part of a transsynaptic complex surrounding active
zones and involved in the coordinated development of pre- and
postsynaptic membranes, as well as in the functional coupling of
plasma membrane and cytoplasmic vesicles. Notably, Bsg re-
cently has been identifi ed within synaptic vesicle preparations
(Takamori et al., 2006).
Bsg might act directly, or through interaction with trans-
membrane and cytoplasmic partners. Consistent with this latter
hypothesis, we have shown that conserved amino acids found in
the cytoplasmic tail of Bsg are crucial for the function of the
protein in vivo and that they may thus mediate transduction of a
signal toward the cell cytoplasm and/or interaction with the cor-
tical cytoskeleton (see the following section).
Basigin and the actin cytoskeleton
The F-actin/Spectrin cytoskeleton underlying pre- and postsyn-
aptic membranes seems essential for different aspects of synap-
tic terminal growth and plasticity, including terminal expansion,
organization of presynaptic vesicle pools, and postsynaptic re-
ceptor clustering (Dillon and Goda, 2005; Pielage et al., 2006;
Ruiz-Canada and Budnik, 2006). Here, we have shown that Bsg
modulates the organization of the presynaptic actin cytoskele-
ton, as revealed by the presence of ectopic aggregates of F-actin
and actin-associated proteins within the lumen of bsg synaptic
boutons. Although we could not detect any obvious alterations
of the postsynaptic actin cytoskeleton, which is intermingled
with the dense membraneous network of the SSR, it is nonethe-
less possible that Bsg also regulates this cytoskeleton. Our ob-
servations further support previous reports showing that Bsg
colocalizes with the actin cytoskeleton specifi cally at cell–cell
contacts and that expression of Bsg in cultured cells results in
reorganization of the F-actin network and consequent formation
of lamellipodia (Schlosshauer et al., 1995; Curtin et al., 2005).
At the NMJ, Bsg may modulate actin cytoskeleton orga-
nization indirectly, by interacting with integrin subunits at the
plasma membrane. Indeed, mammalian Bsg has been found in
Figure 8. Synaptic vesicle distribution is altered
in bsg larvae. (A–C) w (A) and Df/bsg6293 (B
and C) larvae stained with anti-CSP (A–C and
A′–C′, red) and anti-HRP antibodies (A′–C′,
green). (A″–C″) Fluorescence intensity profi les
of CSP staining along a section of the terminal
bouton indicated in A–C (white marks). (D)
Graph showing the proportion of NMJ 6/7
branches showing either a cortical or luminal
accumulation of CSP. **, P < 0.001 (χ test).
(E and F) Wild-type (E) and Df/bsg6293 (F) lar-
vae expressing GFP-tagged synaptotagmin
and stained with anti-Synapsin (E and F) and
anti-GFP (E′ and F′) antibodies. Images in A–F
correspond to z projections of serial confocal
sections throughout entire boutons (see Fig.
S3 A, available at http://www.jcb.org/cgi/
content/full/jcb.200701111/DC1, for serial
individual pictures corresponding to pictures
E and F). Bar, 10 μm. (G and H) Single confocal
sections of bsg6293/+ and Df/bsg6293 larvae
double stained for Synaptotagmin-GFP (Syt; G)
or Synapsin (Syn; H) and HRP. Optical slices all
traverse bouton centers (defi ned by the section
with the largest bouton diameter and the ring-
like appearance of HRP staining). Bars, 4 μm.
JCB • VOLUME 177 • NUMBER 5 • 2007 852
a complex with β1-integrin (Berditchevski et al., 1997; Xu and
Hemler, 2005), and both α- and β-integrin subunits colocalize
with D. melanogaster Bsg at larval NMJs (Beumer et al., 1999;
unpublished data). Furthermore, although we have not detected
a genetic interaction between bsg and myspheroid (mys, which
encodes the main D. melanogaster β-integrin subunit) during
larval NMJ development, we identifi ed several mys missense
mutations displaying a junction undergrowth phenotype very
similar to that of bsg mutants (Jannuzi et al., 2004; unpublished
data). Another attractive hypothesis is that Bsg, through its
juxtamembrane cytoplasmic motif, recruits Spectrin or other
actin-associated proteins and thereby directly participates in or-
ganizing a cortical actin network. Interestingly, different mem-
bers of the FERM domain protein family have been shown to
link cell surface glycoproteins and the actin cytoskeleton by
directly binding to both the intracellular region of transmem-
brane proteins and to actin or Spectrin (Tsukita and Yonemura,
1999; Bretscher et al., 2002). In particular, Moesin directly in-
teracts with the intracytoplasmic domains of mammalian CD43,
CD44, and intercellular adhesion molecule 2, through a posi-
tively charged amino acid cluster found in the juxtamembrane
region of these proteins (Legg and Isacke, 1998; Yonemura
et al., 1998). The striking conservation and functional importance
of the KRR juxtamembrane motif of Bsg suggests that such cy-
toplasmic proteins may physically link cell-surface Bsg to the
underlying F-actin network and mediate organization of cortical
domains at the NMJ.
Basigin controls synaptic vesicle release
Down-regulation of bsg at D. melanogaster NMJ terminals
causes a dramatic increase in transmitter release, which, to our
knowledge, is unique among Ig CAM mutants. This phenotype
corresponds to a specifi c presynaptic function of Bsg and may
be explained by (1) an alteration of the excitability of the synap-
tic terminal or (2) an altered defi nition of the different func-
tional synaptic vesicle pools.
Mammalian Bsg has been shown to promote translocation
of transporter proteins to the plasma membrane, as well as
regulate the activity of multiprotein transmembrane complexes
(Kirk et al., 2000; Zhou et al., 2005). At the D. melanogaster
NMJ, Bsg may thus be required for the proper distribution and/
or clustering of ion channels regulating Ca2+ dynamics. In this
context, it was recently demonstrated that the presynaptic scaf-
folding protein BRP is required for the clustering of Ca2+ chan-
nels and for their spatial coupling with synaptic vesicles at
the D. melanogaster active zone. This process appears to be
required for the rapid evoked component of synaptic vesicle
release but not for spontaneous release (Kittel et al., 2006).
Therefore, both the additional spontaneous and delayed evoked
component of transmitter release in bsg mutants might corre-
spond to the fusion of vesicles lacking a tight association with
Ca2+ channels. An elevated contribution of asynchronous re-
lease has also been reported to occur naturally at particular syn-
apses of the mammalian central nervous system and is thought
to refl ect long-lasting Ca2+ transients and a loose coupling be-
tween Ca2+ sources and vesicles (Hefft and Jonas, 2005). It is
thus conceivable that down-regulation of Bsg alters Ca2+ dy-
namics, leading to an abnormal recruitment of vesicles distant
from Ca2+ sources.
Alternatively, the observed transmitter release phenotype
may not be associated with an alteration of Ca2+ signals, but
rather refl ects a role of Bsg in organizing synaptic vesicle pop-
ulations. It has been suggested that synaptic vesicles are orga-
nized into functionally distinct pools (readily releasable pool,
recycling pool, and reserve pool) with specifi c recycling and
mobilization properties (Rizzoli and Betz, 2004, 2005). Here,
we have shown that down-regulation of bsg leads to an abnor-
mal distribution of vesicles in resting terminals (Fig. 8), as well
as an aberrant traffi cking of vesicles to the center of boutons
(where reserve pool vesicles are thought to reside) under con-
ditions where synaptic vesicle recycling is normally restricted
Figure 9. Presynaptic loss of Bsg provokes asynchronous vesicle release.
(A) Representative traces and quantifi cation of mini frequency and am-
plitude in bsg mutants and rescued animals. *, P ≤ 0.05; **, P ≤ 0.01.
(B) Example traces of eEJCs after 15 stimuli at 0.2 Hz and mean amplitudes.
*, P ≤ 0.05; **, P ≤ 0.01;***, P ≤ 0.001. (C) Presynaptic loss of Bsg
leads to an increased and atypically delayed release of vesicles, refl ected
by the larger charge carried by eEJCs of both bsg mutants and postsynap-
tic rescues. Precise genotypes are as follows: Basigin, Df/bsg6293; control,
elav-Gal4/Y; prerescue, elav-Gal4/Y; Df/bsg6293; UAS-bsg/+; postrescue,
BSG IN SYNAPTIC VESICLE DISTRIBUTION AND RELEASE • BESSE ET AL.853
to the periphery (and to the recycling pool; Fig. S3, C and D).
These data suggest that the defi nition of different synaptic ves-
icle populations may be altered in bsg mutants, which in turn
may explain the observed defects in precise recruitment and
release of vesicles. This is of particular interest given that mam-
malian Bsg has been suggested to physically associate with syn-
aptic vesicles (Takamori et al., 2006). Additionally, presynaptic
actin fi laments have been proposed to provide a physical barrier
impeding vesicle dispersion and, in particular, to regulate the
availability of the reserve pool (Dillon and Goda, 2005). They
have also been suggested to participate in a mechanism restrain-
ing fusion of synaptic vesicles in cultured hippocampal neurons
(Morales et al., 2000). An attractive possibility is therefore that
Bsg controls synaptic vesicle organization and retention through
its effect on the cortical actin cytoskeleton.
Materials and methods
In brief, +/Y; pBac[3xP3-DsRed; GFPexon]/+; pHer[3xP3-ECFP; α-tub-
piggyBacK10]/+ jumpstarter males, carrying the protein-trap transposon
and a source of piggyBac transposase (Horn et al., 2003), but not express-
ing any detectable GFP, were used to mobilize the GFP cassette and create
new insertions. Jumpstarter males were crossed en masse with w virgins,
and embryos were collected at 25°C on apple juice plates for either 5 h
(for late embryo sorts) or overnight (for L1 sorts). They were then aged to
stage 16–17 embryos and L1 larvae, respectively. Dechorionated embryos
or L1 larvae were sorted using an embryo sorter (COPAS Select 500;
Union Biometrica), following a procedure adapted from Furlong et al.
(2001). Sorted larvae were then manually rescreened at later stages using
a stereomicroscope (MZFLIII; Leica) equipped with standard GFP fi lters.
About 350 GFP+ individuals from a total of ?1.5 million animals sorted
(1 million sorted as young larvae and 0.5 million sorted as late embryos)
were selected, which roughly corresponds to a frequency of GFP+ event re-
covery of 1/2,000–2,500 (estimated after taking into account the percent-
age of lethality).
Emerging GFP+ individuals were then crossed individually to w fl ies
to establish independent lines. Third instar larvae from each line were dis-
sected, briefl y fi xed (5 min in 4% PFA), and washed several times in PBT.
Fillet preparations were examined for GFP distribution using an epifl uor-
escence microscope. For lines of interest, the position of the inserted protein-
trap transposon was determined by sequencing of the fl anking genomic
regions using the inverse PCR protocol described by Horn et al. (2003).
The exact positions (in the genomic scaffold AE003619) of the three piggyBac
protein-trap insertions described are the following: 271285 (line 05.02),
277305 (line 91.03), and 278932 (line 79.13). Although two of these in-
sertions (05.02 and 79.13) are homozygous viable, with homozygous larvae
showing a rather normal NMJ morphology, one insertion is homozygous
lethal (91.03). Given that the three insertions are located in the same in-
tron, and thus generate the same fusion protein, this lethality is not associ-
ated with production of the chimeric protein.
Fly stocks and transgenes
The NP6293, l(2)SH1217, and l(2)k13638 insertions, and the defi ciency
covering bsg locus (Df[2L]Exel7034), were obtained from the Kyoto,
Szeged, and Bloomington stock centers, respectively. NP6293 and
l(2)1217 lie at position 267161 and 267380 of 2L, respectively. The
NP6293 chromosome contains a single P element insertion (as verifi ed by
inverse PCR), but contains an additional mutation outside of the region
covered by Df(2L)Exel7034, as indicated by the fact that both NP6293
and NP6293pr.ex. homozygous animals never reach larval stages, whereas
a good number of NP6293/Df(2L)Exel7034 larvae survive until third lar-
val instar. For precise excision of NP6293, NP6293/CyO; ∆2-3, Sb/+
males were individually crossed to If/CyO females. A single w− CyO
male per vial was then selected among the progeny and crossed to If/CyO
virgin females to establish a stock. Excisions were then evaluated at the
molecular level, by PCR amplifi cation and sequencing of genomic DNA of
hemizygous larvae. mhc-Gal4, elavC155-Gal4, tub-Gal4, UAS-syt-EGFP,
UAS-GMA stocks were obtained from the Bloomington stock center. All
crosses were reared at 25°C.
The UAS-bsg transgene was generated by insertion of a 2.7-kb
EcoRI–XhoI fragment obtained by double digestion of the DGC clone
LD19437 into an EcoRI–XhoI digested pCasper3 vector. The following pro-
cedure was used to construct GFP-tagged full-length and mutated Bsg vari-
ants in which GFP insertion mimics that generated upon insertion of the
protein-trap transposon. Total RNA was isolated from bsg protein-trap
larval body wall preparations by standard Trizol extraction and used for
reverse transcription with Superscript II RT (Invitrogen) and oligo dT primers.
Reverse transcription products were then subjected to further PCR and
cloned into a pENTR/DTOPO vector (Gateway technology; Invitrogen).
One of the full-length GFP-tagged Bsg clones obtained was used as a tem-
plate to construct the other Bsg variants. Insertion of the different Bsg-
tagged proteins into pUASt vector was achieved through a LR recombination
reaction (Gateway technology; Invitrogen) using pTW as a destination vec-
tor (Drosophila Gateway Vector Collection; http://www.ciwemb.edu/
labs/murphy/Gateway%20vectors.html). Lines expressing similar levels of
GFP-tagged mutant proteins were used for the rescue experiments de-
scribed in Fig. 5 (not depicted).
Dissections and immunostainings were performed as described by Qin
et al. (2005), using the following antibodies: rabbit anti-GFP (1:1,000; Invit-
rogen), mouse anti-Dlg (1:1,000; Developmental Study Hybridoma Bank
[DSHB]), mouse anti-CSP (1:40; a gift from E. Buchner, Theodor-Boveri-
Institute, Würzburg, Germany), mouse NC82 anti-BRP (1:50; [Wagh et al.,
2006]), mouse anti-Synapsin (1:20; a gift from E. Buchner), rabbit anti–
α-Spectrin (1:100; a gift from R. Dubreuil, University of Illinois at Chicago,
Chicago, IL), mouse anti–α-Spectrin (1:50; clone 3A9; DSHB), mouse anti-
Futsch (1:50; clone 22C10; DSHB), guinea pig anti-Wasp (1:400; Bogdan
et al., 2005), FITC- and Cy5-conjugated anti-HRP (Cappel and Jackson
ImmunoResearch Laboratories, respectively). Alexa Fluor 488 (Invitrogen),
Cy3 or Cy5 (Jackson ImmunoResearch Laboratories) conjugated second-
ary antibodies were used at a 1:500 dilution. Unless specifi ed, confocal
pictures are those of muscles 6/7 NMJs (segments A2–A4) and were taken
with a microscope (DMR-E; Leica) equipped with a scan head (TCS SP2
AOBS; Leica) and an oil-immersion 63× 1.4 NA objective.
For bouton number quantifi cations, type Ib boutons of muscles 6/7
on segment A2 were counted using anti-CSP and anti-HRP double stain-
ings. Muscles were photographed at 20× magnifi cation and then traced
and measured using ImageJ. The normalized bouton number was calcu-
lated by the dividing bouton number by the muscle surface area (data
are expressed as a percentage of the w controls in each experiment).
Although Df/bsg6293 larvae exhibited muscles of a rather normal size, Df/
bsg1217 larvae have smaller muscles. Larvae with extremely thin muscles
were excluded from the quantifi cation of normalized bouton numbers. For
bouton surface area quantifi cation, confocal pictures of muscle 4 NMJs
(segment A2) stained with anti-HRP antibodies were taken with a 63×
magnifi cation. Sections along the z axis were projected, and individual
type Ib boutons were then manually traced and measured using ImageJ.
For measurement of BRP+ puncta density, sections of NMJ 6/7 branches
were projected along the z axis, and the density was calculated as the
ratio between the total number of puncta and the projected surface of the
branch (using Image J).
Generation of antibody and Western blotting
Rat polyclonal anti-Bsg antibodies were raised against an N-terminal
synthetic peptide (Q S L D K L V P N Y D ) obtained from Thermo Scientifi c. Crude
serum was used at a 1:200 dilution for immunostainings and at a 1:1,500
dilution for Western blot analysis. Insertion of GFP within the epitope used
to generate anti-Bsg antibodies probably explains the lower signal
observed for tagged versus untagged Bsg proteins (Fig. 1 C). The highest
molecular weight isoform detected in total larval extracts is not detected in
body wall extracts (Fig. 3 C) and might correspond to a glycosylated form,
as described for mammalian Bsg. The following antibodies were used for
Western blot analysis: rabbit anti-GFP (1:500 [Santa Cruz Biotechnology,
Inc.] or 1:2,500 [Torrey Pines]) and HRP-conjugated anti–mouse, –rat, or
–rabbit (1:2,000; GE Healthcare) antibodies.
Cell aggregation assay
3 ml of a 106 cells/ml culture of met-Gal4–expressing S2 cells were plated
and transfected the next day with 3.5 μg of either UAS-Golgi-GFP or UAS-
GFP-Bsg. After overnight recovery, cells were induced for 30 h with 0.7
mM CuSO4, shortly centrifuged, and resuspended at 2 × 106 cells/ml in
D. melanogaster SFM medium (Invitrogen) containing 18 mM L-Glutamine,
50 U/ml penicillin, and 50 g/ml streptomycin and puromycin. 1 ml of
each suspension was placed in a 2-ml tube and shaken for 1 h at room
JCB • VOLUME 177 • NUMBER 5 • 2007 854
temperature. Aliquots were spotted on slides and examined using Nomarski
optics and epifl uorescence microscopy.
Transmission micrographs were obtained from dissected preparations of
third instar larvae (NMJ 6/7 and 12/13, segment A2/A3), as described
by Wagh et al. (2006). All measurements were done using ImageJ. The
data are reported as mean ± SEM, and where included, p-values denote
the signifi cance according to the Mann-Whitney Rank Sum test. Measure-
ment of synaptic vesicle diameter was performed on populations of vesi-
cles found within a radius of 300 nm around active zone T-bars.
Two-electrode voltage clamp recordings were obtained at 22°C from VLM 6
in segments A2 and A3, of late third instar larvae, essentially as previ-
ously described (Kittel et al., 2006). The composition of the extracellular
haemolymph-like saline (HL-3) was as follows: 70 mM NaCl, 5 mM KCl,
20 mM MgCl2, 10 mM NaHCO3, 5 mM trehalose, 115 mM sucrose,
5 mM Hepes, and 1 mM CaCl2, pH adjusted to 7.2. Minis (voltage clamp at
−80 mV) and eEJCs (voltage clamp at −60 mV) were recorded with intra-
cellular microelectrodes fi lled with 3 M KCl to give fi nal resistances of
8–21 MΩ. The data are reported as mean ± SEM. n indicates the number of
cells examined, and where included, p-values denote the signifi cance ac-
cording to the Mann-Whitney Rank Sum test. In the fi gures, the level of sig-
nifi cance is marked with asterisks: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤
0.001. The quantal content was roughly estimated as the ratio between the
mean charge transferred per action potential and the mean charge carried
by single minis (see Tables S2 for raw values).
FM1-43 labeling assays
The procedure used for FM1-43 labeling was adapted from Kuromi and
Kidokoro (1998). For quantifi cation of FM1-43 distribution upon loading
(Fig. S3 C), fi llets of third instar larvae were dissected in Ca2+-free saline
(130 mM NaCl, 36 mM sucrose, 5 mM KCl, 4 mM MgCl2, and 5 mM
Hepes) and incubated for 6 min with high-K+ saline (45 mM NaCl, 36 mM
sucrose, 90 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM Hepes, and
0.5 mM EGTA) containing 7 μl/ml (?10−5 mol.l−1) of a 1 μg/μl FM1-43X
solution (Invitrogen). Fillets were then briefl y washed two times in Ca2+-free
saline, fi xed for 5 min in 4% formaldehyde, and washed quickly before
being mounting on a slide. FM1-43 fl uorescence was imaged right after
using a confocal microscope (SP2; Leica).
For analysis of FM1-43 loading and unloading, fi llets of third instar
larvae were dissected in Ca2+-free saline, transferred into a microscope
chamber, and incubated for 5 min with high-K+ saline containing 6 μl/ml
(?10−5 mol.l−1) of FM1-43 dye (Invitrogen). Preparations were then briefl y
rinsed three times with Ca2+-free saline, further washed (once for 5 min
and once for 10 min) with Ca2+-free saline, and imaged using confocal mi-
croscopy and a 40× immersion objective (“after loading” pictures). We
could not quantify the amount of FM1-43 dye loaded after the fi rst part of
the procedure, as bsg muscles contract longer than wild-type muscles upon
transfer to Ca2+-free saline, and thus start unloading part of the dye. We
also noticed that contractions provoked by the high-K+ solution are weaker
in muscle 4 than in muscles 6/7, and therefore imaged exclusively muscle
4 NMJs. For unloading, two consecutive rounds of high-K+ saline stimula-
tion (1 min each), separated by washes with Ca2+-free saline (three times
for 5 min), were then applied in the absence of any dye. Junctions were
imaged after further washing (“after unloading” pictures), using the same
confocal settings as for the “after loading” pictures.
Online supplemental material
Fig. S1 shows second instar larvae NMJ morphology and cell–cell aggre-
gation assays performed with Bsg variants. Fig. S2 shows the distribution
of pre- and postsynaptic markers in bsg larvae. Fig. S3 shows the local-
ization of synaptic vesicle–associated proteins (serial confocal sections),
the distribution of mini amplitudes, and FM1-43 labeling assays. Table
S1 shows raw bouton number and size in mutant and rescued contexts.
Table S2 shows recorded electrophysiological properties of mutant and
rescued larvae. Online supplemental material is available at http://www
We thank the laboratories that made their fl y strains and reagents available,
as well as the Developmental Studies Hybridoma Bank at the University of
Iowa for antibodies. We are very grateful to R. Ventzky, A. Cyrklaff, and S.
Lopez de Quinto for their help during the screen; A.M. Voie for fl y injections;
T. Vaccari for the GFP-Golgi control construct; and laboratory members for help
F. Besse was supported by fellowships from the Federation of
Euro pean Biochemical Societies and the Human Frontier Science
Submitted: 19 January 2007
Accepted: 5 May 2007
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