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First published online 1 November 2006
doi: 10.1242/dev.02650
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1 Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts
Avenue 68-230B, Cambridge, MA 02139, USA.
2 Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston,
MA 02115, USA.
3 Biology Department, Brandeis University, MS-008, 415 South Street, Waltham, MA
02454, USA.
* Author for correspondence (e-mail: pgarrity{at}brandeis.edu)
Accepted 18 September 2006
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: BEACH domain, Vesicle trafficking, Neuromuscular junction, Bouton, Bristle development, Drosophila
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Su et al., 2004). Furthermore,
mutations in several BEACH genes cause disease in humans. LYST
(lysosomal trafficking regulator), the first BEACH gene to be discovered, is
disrupted in Chédiak-Higashi syndrome - an often-fatal disease
characterized by severe immunodeficiency, albinism, poor blood coagulation and
neurologic involvement (Introne et al.,
1999). Another BEACH family member, neurobeachin (NBEA), has been
implicated as a candidate gene for autism
(Castermans et al., 2003).
Furthermore, upregulation of LRBA (LPS-responsive and beige-like
anchor) is seen in several types of cancer and appears to facilitate cancer
growth (Wang et al.,
2004).
Localization of several BEACH proteins to subcellular membranes, as well as
loss-of-function phenotypes that affect organelle morphology and function,
suggest that these proteins may play roles in membrane trafficking. For
instance, the hallmark of cells mutant in the Lyst gene is the
presence of large intracellular granules of lysosomal origin, which probably
result from increased lysosome fusion
(Harris et al., 2002;
Nagle et al., 1996). However,
the mechanisms by which Lyst and other BEACH proteins regulate vesicle
trafficking are not understood.
In the present study we have found a genetic interaction between
bchs (blue cheese), a BEACH family member recently described in
Drosophila (Finley et al.,
2003), and rab11. The Rab family of small GTPases has a
well-established involvement in membrane traffic; Rabs can regulate vesicle
formation, motility, docking and fusion
(Zerial and McBride, 2001).
Rabs, like other GTPases, act as molecular switches: active in the GTP-bound
form and inactive in the GDP-bound form. Moreover, each Rab must be associated
with its particular subset of cellular membranes in order to carry out its
function (Pfeffer and Aivazian,
2004; Seabra and Wasmeier,
2004). Thus, Rabs impart specificity to membrane trafficking
events.
Rab11 localizes to the pericentriolar recycling endosome, the
trans-Golgi network and post-Golgi vesicles
(Chen et al., 1998;
Ullrich et al., 1996), and
plays a role in both the exocytic biosynthetic pathway and the recycling
pathway (Chen et al., 1998;
Ren et al., 1998;
Satoh et al., 2005;
Ullrich et al., 1996). In
Drosophila, recent work has established a role for rab11 in
multiple developmental events, including polarization of the oocyte,
cellularization of the embryo and morphogenesis of the rhabdomere, the
photosensing organelle of photoreceptor neurons
(Dollar et al., 2002;
Pelissier et al., 2003;
Riggs et al., 2003;
Satoh et al., 2005). In
addition, subcellular localization of Rab11 may contribute to asymmetric cell
division (Jafar-Nejad et al.,
2005).
In this paper we describe the characterization of Drosophila Bchs and its interaction with Rab11. We find that Bchs is highly expressed in the nervous system, where it is associated with vesicles and concentrated in synaptic regions. We show that reductions in bchs function suppress the effects of loss-of-function rab11 mutations in multiple developmental contexts. In particular, bchs mutations suppress a newly described anatomical phenotype of rab11 at the neuromuscular synapse. Our data identify a role for these regulators of vesicle trafficking in developmental events, such as synaptic morphogenesis, and provide insight into how BEACH proteins could be involved in vesicle trafficking.
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MATERIALS AND METHODS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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) were
obtained from Rebay lab (Whitehead Institute, Cambridge, MA).
rab11P2148, rab1193Bi, Df(2L)cl7, pr[1]
cn[1]/CyO, and the Drosophila DK1, DK2 and DK3 deficiency kits
(2001) were obtained from the Bloomington Stock Center,
rab11ex1/TM3, Sb and
rab11ex2/TM3, Sb from R. Cohen
(University of Kansas). w, Canton S flies were use as control, unless
otherwise indicated.
In situ hybridization
Both sense and antisense RNA probes were made using DIG RNA labeling kit
(Roche); the template was made by PCR from genomic fly DNA using the following
primers:
5'-GAATTAATACGACTCACTATAGGGAGAGCACACAAAGTTCGATCTTGAC-3'and
5'-AATTAACCCTCACTAAAGGGAGAGTTCGCCTACAAGCACATCG-3'. Embryo in situ
hybridization was done as in Tautz and Pfeifle
(Tautz and Pfeifle, 1989).
Mouse northern blot
Template for mouse RNA probe corresponding to 4737-5413 bp of
Wdfy3 cDNA was made by PCR from mouse cDNA (Gertler Laboratory, MIT),
primers used 5'-T3CCTAAGCCTGTCGCCACTACTTTAC-3'and
5'-T7CCAAACTTCTTCTTCTGCTCCCG-3'. Probe was synthesized using
Strip-EZ RNA (Ambion) and a-P32UTP 800 ci/mM (Amersham).
Hybridization was done according to the manufacturer's instructions
(Ambion).
EMS mutagenesis and sequencing of the bchs locus
Ethyl methanesulfonate (EMS) mutagenesis was performed as described
(Lewis and Bacher, 1968).
bchs alleles were PCR amplified from genomic DNA and sequenced by the
MGH Core Facility. The following primers were used.
Pair 1: 5'-CAAACCCCACGGACATGC-3'and 5'-GCTGGTGTGGACTGACGCC-3'.
Pair 2: 5'-GCACGCTCCCTCCGTTCG-3'and 5'-CAAACTTGGAGCACTGCCTGAG-3'.
Pair 3: 5'-CAACCAGTTACAGGGTCGGAATC-3'and 5'-GCGCTGACCACTTTTGTAGTCTG-3'.
cDNA cloning and transgene constructs
Full-length bchs cDNA was assembled by combining a partial cDNA
(clone LD02084) with cDNA produced via RT-PCR from S2 cell RNA (provided by
Pardue Laboratory, MIT). RT (RETROscript, Ambion) and PCR (Expand High
Fidelity PCR System, Roche) were done according to the manufacturer's
instructions. The following primers were used:
5'-CGGGATCCATGAATGTAATGCGTAAGCTGCG-3',
5'-CGGAATTCGCCACCAAGGACTTGATGATTTCG-3',
5'-CGGAATTCTGCTTCGCACCACGCAGGTC-3',
5'-CGGGATCCCGAGCGGACAACAAAAGCATTG-3',
5'-ACGCGTCGACCAGATTCCGACCCTGTAACTGG-3',
5'-GCAACCACGAGTTGGAATTCATTGGC-3'and
5'-ATTTGCGGCCGCCCTAATTGTCCAACGAGTTCGTGC-3'.
Fragments were sequenced before assembly into full-length cDNA, which was
modified with 5'HA tag in pcDNA6/V5-His (Invitrogen) and inserted into
pUASt (Brand and Perrimon,
1993).
Antibody production
Bchs aa 2237-2590 were expressed as 6XHis fusion protein in bacteria and
purified according to the manufacturer's instructions (Amersham). Polyclonal
antisera were produced in rats (Covance).
Western blotting
Each lane of a 6% SDS-polyacrylamide gel contained nine adult heads
homogenized in 1xLaemmli buffer in PBS (130 mmol/l NaCl, 175 mmol/l
Na2HPO4, 60 mmol/l NaH2PO4). After
electrophoresis, proteins were transferred to Hybond-P membrane (Amersham
Pharmacia) and membranes were blocked in 5% nonfat milk and probed with
anti-Bchs (1:1000) and rat anti-Elav (1:1000), followed by HRP-conjugated goat
anti-rat antibody (1:5000) (Jackson).
Immunohistochemistry
Immunohistochemistry of larval and adult brains was performed as previously
described (Garrity et al.,
1999). Anti-Bchs was preabsorbed using bchs17
animals and used at 1:500. Primary antibodies: mouse MAb 24B10 anti-Chaoptin
(Fujita et al., 1982) (1:200),
rabbit anti-Syt1 (T. Littleton, MIT) (1:500) and mouse anti-HA (Covance)
(1:1000) were used. Secondary antibodies were goat anti-mouse HRP (1:200),
goat anti-rat Cy3 (1:500), goat anti-rabbit FITC (1:200) and goat anti-mouse
Cy3 (1:1000) (Jackson). Fluorescent samples were visualized using a Nikon
PCM2000 confocal microscope.
Larval body wall dissections were done in PBS and fixed in 4% PFA in PBS. Anti-Bchs was preabsorbed using bchs null animals and used at 1:500. Mouse anti-Rab11 antibody (BD Biosciences) was used at 1:200. Subtracted goat anti-rat Cy3 at 1:100 (Jackson), goat anti-mouse Alexa-488 at 1:100. Cy5- and FITC-conjugated anti-HRP antibodies at 1:100 (Jackson) were used. Confocal data was acquired as single images or image stacks of multi-tracked, separate channels with a Zeiss LSM 510 microscope.
Screen for modifiers of Bchs overexpression
Each stock from the Drosophila Deficiency Kits (195 stocks that
together delete over 85% of the genome) was crossed to GMR-GAL4;
EP-bchs flies to determine whether heterozygosity for particular genomic
region modified the adult eye phenotype caused by Bchs overexpression. Smaller
deficiency chromosomes and mutations within genomic regions of interest were
obtained (Bloomington Stock Center) and examined for interactions with
GMR-GAL4;EP-bchs.
Quantification of survival and bristle phenotypes
To measure viability, 60 larvae (second instar) of each genotype were
placed in identical vials at 25°C and monitored daily for survival to
adulthood. Bristle loss was quantified by counting the numbers of
bristle-filled and empty sockets in the last row of abdominal tergites
(segments) 2, 3 and 4 in adults (
1-day old). Fraction of bristle-filled
sockets was calculated. At least ten animals per genotype were counted. All
P-values were determined using two-tailed unpaired Student's
t-tests.
Subcellular fractionation and western blotting
Sucrose gradient was prepared by Bill Adolfsen (Littleton Laboratory, MIT)
as described in Adolfsen et al.,
2004 (Adolfsen et al.,
2004). Western blots were as described above using: rat anti-Bchs
1:1000, mouse anti-Rab11 (BD Biosciences) 1:1000, mouse anti-Rop (4F8)
(Harrison et al., 1994) (H.
Bellen, Baylor) 1:1000, and rabbit anti-Synaptotagmin (rabbit) (T. Littleton,
MIT) 1:500. HRP-conjugated goat anti-rat, mouse, rabbit and guinea pig
secondary antibodies (Jackson) were used at 1:5000.
Quantification of the bouton density and branching phenotypes
Confocal stacks through third instar NMJ 6/7 were flattened into
projections using the LSM 510 software. Each bouton was marked on the image by
a colored dot using Adobe Photoshop and the resulting dots were counted using
ImageJ software. The number of branching boutons was counted in the same
manner. Muscle surface area was estimated as described
(Schuster et al., 1996).
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DISCUSSION
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). One
of these lines, EP(2)2299 (henceforth termed EP-bchs), contained an
EP transposon insertion adjacent to bchs. When bchs was
highly expressed in the developing retina by the GMR-GAL4 driver,
photoreceptor growth cones had an unusually bulbous central region and were
less expanded than controls, although they appeared to grow normally toward
their targets (Fig.
1A'A'',B',B''). While patterning of the
third instar eye disc appeared normal in these GMR-GAL4;EP-bchs
animals, suggesting that early eye development was not significantly affected
(R.K., unpublished), overexpression of bchs in the retina resulted in
a smaller, glazed adult eye (Fig.
1A,B).
Bchs is evolutionarily conserved and neuronally expressed
Bchs has a predicted mass of 390 kDa and has been evolutionarily conserved
from the slime mold Dictyostelium to humans
(De Lozanne, 2003). In all
BEACH proteins, including Bchs, the BEACH domain is preceded by a
pleckstrin-homology (PH) domain and followed by four to six WD40 repeats (five
in Bchs) (Fig. 2A). The large
size of most BEACH proteins combined with the presence of multiple WD40
domains, which serve as protein-protein interaction motifs, suggest that BEACH
proteins could be involved in the assembly of large protein complexes. BEACH
proteins have been subdivided into five classes
(De Lozanne, 2003;
Wang, N. et al., 2002).
Whereas the PH-BEACH-WD40 module is conserved across the family, each BEACH
protein has additional regions of homology particular to members of its class.
There are five predicted BEACH proteins in Drosophila, and Bchs falls
in a class that includes LvsA in Dictyostelium, several
uncharacterized proteins in Arabadopsis and Caenorhabditis
elegans, and two human members: Alfy (autophagy-linked FYVE protein;
WDFY3 - Human Genome Nomenclature Database) and the uncharacterized molecule
KIAA1607. Bchs has extensive homology to Alfy throughout the entire protein
length: 51-84% amino acid identity in a carboxyl portion that includes the
PH-BEACH-WD40 regions and 41% identity in the remainder of the protein,
hereafter called CRAB (conserved region in Alfy and Bchs)
(Fig. 2A). In addition to the
PH-BEACH-WD40 module, Bchs and Alfy also have a C-terminal FYVE (Fab1p, YOTB,
Vac1p and EEA1) domain (Fig.
2A), a motif that can mediate interactions with
phosphatidylinositol 3-phosphate (Misra et
al., 2001).
Both bchs and genes expressing the Alfy protein are expressed in
the nervous system. RNA in situ hybridization indicated that bchs
mRNA was enriched in, but not restricted to, the embryonic brain and ventral
nerve cord (Fig. 2B) (see also
Kraut et al., 2001).
Similarly, transcripts for murine Alfy (Wdfy3 - Mouse Genome
Informatics) were expressed highly, but not exclusively, in the brain
(Fig. 2C). Thus, Bchs and its
homologs are neuronal, but are likely to also function in other cell
types.
bchsmutants do not exhibit observable loss-of-function phenotypes
We reasoned that mutations disrupting bchs function would decrease
or eliminate the ability of Bchs to alter the structure of the adult eye when
overexpressed. Therefore, we mutagenized EP-bchs animals using EMS
and isolated 17 independent EP-bchs chromosomes that no longer caused
strong eye defects when combined with GMR-GAL4
(Fig. 1 and
Table 1). DNA sequencing
identified mutations in the bchs gene in eight of these strains
(Table 1). Examination of the
larval photoreceptor axons in these animals showed that those mutations that
strongly suppressed the adult eye phenotype also strongly suppressed the
growth cone phenotype (Fig.
1C). The identification of these mutations within bchs
not only provided loss-of-function bchs alleles for further analysis,
but also confirmed that the growth cone and eye phenotypes resulted from
bchs overexpression.
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We examined multiple bchs alleles for mutant phenotypes, including the putative protein null alleles bchs12, bchs17 and bchs58 in combination with a deficiency chromosome, Df(2L)cl7, missing the bchs locus. Mutants in bchs were viable, fertile and had no overt defects. Furthermore, no defects in axon guidance or growth cone morphology could be detected in the larval visual system or the embryonic central nervous system (CNS) and motor axons (R.K., unpublished).
rab11 is a modifier of Bchs overexpression
To gain insight into the function of Bchs, we searched for genes that could
alter the adult eye phenotype caused by Bchs overexpression. We examined a
collection of 195 chromosomal deficiencies that deleted defined portions of
the Drosophila genome (in total, covering 
85% of the genome).
Five chromosomal deficiencies dominantly suppressed the
GMR-GAL4;EP-bchs eye phenotype, while eight dominantly enhanced it.
In particular, deficiency Df(3R)e-R1 acted as a strong enhancer.
Genes within the interval deleted by Df(3R)e-R1 were examined, and
mutations in rab11 were found to strongly enhance the Bchs
overexpression phenotype (Fig.
3). Multiple loss-of-function rab11 alleles were tested,
including rab11ex1, rab11ex2,
rab11E(To)3, rab11E(To)11 and
rab11J2D1(also known as rab11P2148)
(Dollar et al., 2002;
Jankovics et al., 2001).
Heterozygocity for rab11 did not cause a disruption in eye
development in otherwise wild-type animals, but all the examined
rab11 mutants showed a strong dominant enhancement of the
bchs overexpression phenotype
(Fig. 3). Thus, Bchs
overexpression makes the eye sensitive to partial reductions in rab11
gene function, raising the possibility that Bchs and Rab11 functionally
antagonize one another.
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;
Jankovics et al., 2001). The
combination of a hypomorphic allele rab1193Bi and a
putative null allele rab11ex1
(Dollar et al., 2002) is
mostly lethal (Jankovics et al.,
2001), but a small number of
rab11ex1/rab1193Bi flies survive to
adulthood. To test whether the loss of bchs had an effect on the
viability of rab11ex1/rab1193Bi
mutants, we introduced into this genotype the putative protein null alleles
bchs12, bchs17 and Df(2L)cl7. Loss of
one or both copies of bchs strongly enhanced the viability of
rab11ex1/rab1193Bi animals
(Fig. 4A,B). For example, in
one experiment, survival to adulthood was improved from 17±4% in
rab11ex1/rab1193Bi mutants to
77±3% in
bchs12/bchs17;rab11ex1/rab1193Bi
double mutants (all values are mean±s.e.m., n=3 independent
vials, P<0.001) (Fig.
4A). In another experiment, survival was improved from 5±1%
for rab11ex1/rab1193Bi mutants to
48±6% for
bchs17/+;rab11ex1/rab1193Bi
animals (n=8 independent vials, P<0.001)
(Fig. 4B). Thus, reduction in,
or loss of, bchs function increases the survival of rab11
mutants. This suggests that Bchs antagonizes essential functions of Rab11.
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). In
addition, the posterior scutellar macrochaetae, large mechanosensory bristles,
are shortened in rab11ex1/rab1193Bi
mutants (Fig. 5B). Mutations in
bchs suppressed both the bristle shortening and the bristle loss
defects of rab11 mutants (Fig.
5A,B). To quantify this suppression we calculated the fraction of
empty sockets in the last row of abdominal tergites 2, 3 and 4 for two
different allelic combinations of rab11 (the viable
rab1193Bi homozygote and the semi-lethal
rab11ex1/rab1193Bi heterozygote) alone
and in combination with bchs alleles. Reductions in bchs
suppressed bristle loss in both rab11 allelic combinations in a
dosage-dependent manner. The removal of one copy of bchs strongly
suppressed bristle loss, and the removal of both copies returned the number of
bristles to control levels (Fig.
5C). As expected from the western blot above,
bchs17 and bchs12 were
indistinguishable in this suppression assay from Df(2L)cl7,
suggesting that they are null alleles. These data further support the
conclusion that Bchs and Rab11 function antagonistically during
Drosophila development.
Interestingly, bchs8, which produces full-length Bchs
protein, suppressed bristle loss to the same extent as the protein null
bchs58 allele (Fig.
5C, bottom panel). This suggests that the missense mutation in
bchs8, which alters a conserved threonine in the interface
between the PH and the BEACH domains of the protein
(Fig. 2A and
Table 1)
(Jogl et al., 2002), strongly
disrupts protein function.
Bchs and Rab11 proteins localize to overlapping membrane regions
We examined the localization of Bchs protein in the larval brain and found
that it was enriched in synaptic regions within the CNS
(Fig. 6A). The specificity of
the Bchs immunoreactivity was demonstrated by its absence from the neuropil of
bchs12.
To examine the distribution of Bchs protein within a neuron at greater
resolution, we expressed an HA-tagged version of full-length Bchs in ellipsoid
body neurons using EB1-GAL4
(Wang, J. et al., 2002). EB
neurons extend a single neurite that splits into distinct dendritic and axonal
processes, with the dendrite arborizing in the lateral triangle, and the axon
in the ellipsoid body ring (Hanesch et
al., 1989). Whereas a membrane-associated CD8-GFP fusion
(Chang et al., 2002) was evenly
distributed throughout these neurons, HA-Bchs preferentially accumulated near
the axon terminals (Fig. 6B),
suggesting that Bchs can preferentially localize to the presynaptic regions of
a neuron.
|
),
dense-core vesicles marked with ANF-GFP
(Rao et al., 2001) and
recycling vesicles marked with Clathrin-GFP
(Chang et al., 2002)
(Fig. 7A,B,C,D,
respectively). Rab11 immunoreactivity had not been previously characterized at the NMJ; therefore, we examined its distribution to determine whether it, like Bchs, was enriched at the synapse. Rab11-positive puncta were present in nerves, synaptic boutons and muscles (Fig. 8A). The observed staining was specific for Rab11, as it was greatly reduced in rab11ex1/rab1193Bi mutants, which also showed reduced protein levels (compared with controls) by western blot analysis (Fig. 8A,B). While both Rab11 and Bchs labeled puncta at the NMJ, Bchs was more enriched in the boutons than Rab11, while Rab11 was more abundant in the muscles than Bchs. Double labeling these preparations for Rab11 and Bchs demonstrated that their subcellular localization substantially overlapped both in neurons and muscle cells (Fig. 8C), although some puncta were immunoreactive for only one protein or the other. Within puncta that contained both proteins, Bchs and Rab11 staining was not always perfectly congruent: Bchs immunoreactivity at times predominated to one side of the stained structure and Rab11 to the other (Fig. 8C, arrows). These localization data, showing that Bchs and Rab11 are present in overlapping locations at the NMJ, together with the genetic interaction data, suggest that Bchs and Rab11 function in the same or closely related processes.
The results of biochemical fractionation experiments were also consistent
with the partially overlapping distribution of Bchs and Rab11 proteins
detected by immunohistochemistry at the NMJ. We probed fractions [previously
characterized in Adolfsen et al. (Adolfsen
et al., 2004)] from a 10-30% sucrose velocity gradient of head
extract to determine how Bchs migrated with respect to known membrane
fractions (Fig. 9). Bchs
migrated with an intermediate density characteristic of a membrane-associated
protein, but did not co-migrate with either the plasma membrane marker
Syntaxin 1A (Schulze et al.,
1995) or the synaptic vesicle markers neuronal-Synaptobrevin and
Synaptotagmin1 (DiAntonio et al.,
1993; Littleton et al.,
1993). Rab11 showed a different distribution across the gradient
than Bchs (Fig. 9).
Nonetheless, a significant amount of Rab11 was recovered in fraction 17, the
fraction in which Bchs was most abundant. Such partial co-migration of Bchs
and Rab11 is consistent with localization of these proteins to partially
overlapping membrane compartments.
|
),
bchs loss-of-function mutants had no detectable defects at the NMJ.
rab11 hypomorphs, however, showed significant alterations in synaptic
bouton patterning at all NMJs. The defect was quantified at the muscle 6/7
synapse in abdominal segment A3, where the total number of synaptic boutons
increased by 60% in rab11 mutants compared with controls
(166±11 versus 98±5, n=14 for each genotype;
mean±s.e.m.; P<0.0001). Because synapses at the NMJ of
wild-type larvae grow in proportion to muscle size
(Schuster et al., 1996), we
also calculated the number of boutons per unit of estimated muscle area. When
the decreased size of rab11 muscles was taken into account
(50±2x103µm2 in rab11 versus
67±1x103 µm2 in control), the density of
synaptic boutons/µm2 muscle area in the mutants was more than
double that of controls (3.3±0.2x10-3 versus
1.5±0.1x10-3 boutons/µm2; n=14;
P<0.0001) (Fig.
10A,B).
In addition to the increase in number and density, the boutons in
rab11 mutants were often arrayed in tight clusters resembling bunches
of grapes, rather than the normal beads-on-a-string morphology. Among the
previously described phenotypes at the NMJ, rab11 most closely
resembled that of nervous wreck, a mutation influencing the WASP
signaling pathway (Coyle et al.,
2004). The unusual clustering of boutons in rab11 mutants
was related to an increase in the number of branching boutons - those
connected to more than two neighboring boutons. Normally such branch points
are uncommon, but in rab11 mutants the fraction of boutons that
branch increased 2.6-fold (22.2±0.8% of all boutons versus
8.4±0.4% in control; n=14; P<0.0001)
(Fig. 10A,B). Thus,
rab11 mutants are abnormal in synaptic growth and morphogenesis.
The NMJ phenotypes observed in rab11 mutants were partially suppressed by loss-of-function mutations in bchs. Muscle size was restored to near control in bchs,rab11 double mutant animals (64±3x103µm2 in bchs;rab11 vs. 67±1x103 µm2 in control, n=14). The bchs,rab11 double mutant and rab11 single mutant had similar numbers of synaptic boutons. However, the density of boutons per muscle area in bchs,rab11 animals was significantly less than in rab11 mutants alone (2.5±0.2x10-3 boutons/µm2; 169% of control in bchs,rab11 versus 3.3±0.2x10-3 boutons/µm2; 225% of control in rab11; n=14, P<0.01). This represents a 44% suppression of the rab11 phenotype (Fig. 10A,B). The percentage of branching boutons in the bchs,rab11 double mutant was also significantly less than in rab11 alone (15.6±1.1% versus 22.2±0.8%; n=14; P<0.0001). This represents a 48% suppression of the rab11 phenotype (Fig. 10A,B). Thus, loss of bchs partially suppressed the increases both in bouton density and in bouton branching exhibited by rab11 mutants. Consequently, Bchs probably has a modulatory role in synaptic development, acting as an antagonist of Rab11.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The subcellular localization of Bchs also supports the hypothesis that this protein functions in membrane traffic. Bchs was present exclusively in membrane fractions and exhibited punctate staining in the presynaptic motoneuron terminals and in the muscles at the NMJ. This pattern is consistent with the localization of Bchs to a membrane-bound organelle. Furthermore, in line with a functional relationship between Bchs and Rab11, we observed significant subcellular co-localization of Bchs and Rab11 at the NMJ, as well as partial overlap in the distribution of Bchs and Rab11 within membrane fractions. These data further support our hypothesis that Bchs regulates vesicle trafficking and that it may do so via an interaction with the Rab11 GTPase.
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).
The ability of mutations in bchs to strongly suppress all
rab11 bristle defects implicates Bchs in bristle morphogenesis and is
consistent with it playing a role in Rab11-mediated vesicle trafficking.
We have also uncovered a crucial role for rab11 in the formation
of the Drosophila NMJ: rab11 mutants exhibit an increase in
the density and branching of synaptic boutons and a decrease in the size of
the muscles. Vesicle trafficking is important in determining the number and
morphology of boutons at the NMJ (Dickman
et al., 2006). In sculpting the synapse, membrane traffic is
needed not only for the addition of new membrane and active zone proteins, but
also for the insertion, removal and signaling of regulatory molecules at the
cell surface (Marie et al.,
2004; Sweeney and Davis,
2002). Furthermore, exocyst-dependent membrane addition is
required for the expansion of NMJs (Murthy
et al., 2003), and Rab11 is a known regulator of exocyst function
(Beronja et al., 2005;
Zhang et al., 2004). Thus,
Rab11 is involved in synaptic morphogenesis at the NMJ, probably via
regulation of vesicle trafficking.
By virtue of suppressing rab11 NMJ phenotypes, Bchs is also implicated in a membrane-trafficking aspect of synaptic morphogenesis. Consistent with such a model, both Bchs and Rab11 showed punctate localization and partial overlap at the NMJ. A functional role of Bchs in presynaptic development may explain its concentration in the axonal rather than dendritic compartment of ellipsoid body neurons (Fig. 7B).
Membrane pathways modulated by Bchs
The link between Bchs and Rab11 function provides initial mechanistic
insights into the trafficking pathways that may involve Bchs. Rab11 is
involved in both biosynthetic exocytic traffic and membrane traffic through
the recycling endosome (Pelissier et al.,
2003; Satoh et al.,
2005). As the loss of bchs suppresses lethality of
rab11 alleles, it is likely to be involved in all the essential
functions of Rab11.
The partial colocalization of Bchs and Rab11 suggests candidate sites for
the function of Bchs. In particular, Rab11 has been observed on the
trans-Golgi network, post-Golgi vesicles, recycling endosomes and
vesicles that travel from the recycling endosome to the plasma membrane
(Chen et al., 1998;
Ullrich et al., 1996;
Ward et al., 2005). In
regulating traffic to the plasma membrane, Rab11 has been shown to physically
interact with members of the exocyst complex
(Beronja et al., 2005;
Zhang et al., 2004). We found
the distribution of Bchs to be highly polarized in neurons and enriched at
synaptic endings, not cell bodies or dendrites. This suggests that
interactions between Bchs and Rab11 may occur in a compartment adjacent to the
presynaptic plasma membrane, rather than near the trans-Golgi
network, the perinuclear recycling endosome or dendritic endosomes.
|
|
). Bchs did not colocalize with early endosomes, marked with
either 2XFYVE-GFP or Rab5-GFP, through which at least some synaptic vesicles
are thought to cycle, nor did Bchs immunoreactivity resemble the distribution
of endocytic vesicles identified by clathrin-GFP. The former was perhaps
surprising, given that Bchs possesses a FYVE domain. These data suggest that
Bchs is unlikely to be involved in early endocytic events. The BEACH domain
protein Lyst has been implicated in lysosomal trafficking; however, we
observed no overlap of Bchs immunoreactivity with lysotracker (R.K.,
unpublished). Bchs is, therefore, not a lysosomal protein. Rather, Bchs
appears to reside on a novel synaptic compartment adjacent to or overlapping a
Rab11-containing organelle. We speculate that this may be a previously
unappreciated presynaptic sorting endosome through which either recycled or
Golgi-derived proteins are trafficked.
The nature of the Bchs-Rab11 interaction
What cellular and molecular mechanisms underlie the extensive genetic
interactions between bchs and rab11? In one scenario, Bchs
could negatively regulate Rab11 activity, perhaps by promoting a Rab11-GAP
that restricts Rab11 function. Bchs could modulate the efficacy of Rab11
function, but might be only one of several negative regulators of Rab11. Such
a model would be consistent with our genetic studies, as loss of bchs
might not cause defects on its own, but bchs overexpression would
shut down the Rab11 pathway. Alternatively, rab11 and bchs
could be involved in competing intracellular pathways. For example, Rab11
might direct endosomal cargos toward the plasma membrane, while Bchs diverts
these cargos elsewhere. This hypothesis receives support from the observation
that Rab11 and Bchs appear to concentrate in partially distinct
subcompartments of those organelles on which they both reside
(Fig. 8C). This pattern of
partially overlapping localizations is reminiscent of other pairs of
regulators of membrane traffic, including Rab4 and Rab5, and Rab4 and Rab11 on
two sequential, yet distinct, populations of endosomes. The distribution of
these Rabs reflects their participation in linked steps of cargo transport
along the recycling pathway (de Renzis et
al., 2002; Sonnichsen et al.,
2000), which may also explain the localization pattern of Bchs and
Rab11.
|
). Mouse
Neurobeachin mutants lack evoked synaptic transmission at the NMJ, implicating
neurobeachin in neurotransmitter release
(Su et al., 2004). Our finding
that Bchs antagonizes Rab11 raises the intriguing possibility that other BEACH
proteins might also interact with Rab family members. It will be interesting
to determine whether the defects observed in Lyst and neurobeachin
mutants reflect the altered activity of particular Rabs and whether
alterations in Rab function could ameliorate defects caused by the absence of
Lyst or Neurobeachin. |
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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3
vials/genotype, unpaired t-test). (B) Two-fold reduction in
bchs gene dosage reduces lethality of rab11 hypomorphs.
Compared to rab11ex1/rab1193Bi:
P<0.005 for
Df(2L)cl7/+;rab11ex1/rab1193Bi, P<0.0005 for
bchs17/+;rab11ex1/rab1193Bi and
P<0.01 for
bchs17/bchs12;rab11ex1/rab1193Bi
(n
