First published online 1 November 2006
doi: 10.1242/dev.02650
Development 133, 4655-4665 (2006)
Published by The Company of Biologists 2006
Bchs, a BEACH domain protein, antagonizes Rab11 in synapse morphogenesis and other developmental events
Rita Khodosh1,2,
Adela Augsburger1,
Thomas L. Schwarz2 and
Paul A. Garrity1,3,*
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.
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Fig. 1. Bchs overexpression disrupts eye development and photoreceptor growth
cone morphology. (A-C) Adult eyes. (A) Control (GMR-GAL4)
eye has normal rows of ommatidia. (B) Overexpression of wild-type Bchs in the
eye (GMR-GAL4;EP-bchs) produces a small eye lacking distinct
ommatidia. (C) An early stop codon in the bchs58 allele
prevents the overexpression phenotype, despite the presence of the
EP-bchs insertion in this allele
(GMR-GAL4;bchs58). (A'-C')
Photoreceptor (R-cell) axons in larval brain, labeled with anti-Chaoptin.
Subsets of R-cell axons enter via the optic stalk to terminate in the lamina
and medulla, from which the indicated region is enlarged in
A''-C''. Compared with control brains
(A',A''), overexpression of wild-type Bchs (B',B'') does
not disrupt axon pathfinding, but causes photoreceptor growth cones
(arrowheads) to have larger central areas and appear less expanded than
controls. This phenotype is also prevented by the stop codon in
bchs58 (C',C''). Scale bars: 100 µm in A-C;
10 µm elsewhere. la, lamina; me, medulla; os, optic stalk.
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Fig. 2. Structure and expression of Bchs protein. (A) The domains of
Drosophila Bchs and human Alfy are similar in organization and
sequence. The percentage amino acid identity between domains is indicated,
including homology for the extended portion of the protein called CRAB
(Conserved Region in Alfy and Bchs; shown in gray and not shown to scale to
conserve space). Mutations in bchs alleles are marked (nonsense,
stars; missense, triangles). (B) In a stage 13 embryo, bchs
mRNA is highly expressed in embryonic CNS, including brain and ventral nerve
cord, and in the salivary glands (arrowhead). A sense control probe showed no
signal above background (data not shown). (C) Analysis of murine mRNA
indicates mouse Alfy is widely expressed, but enriched in adult brain.
(D) Protein blot probed with antisera raised to amino acids 2237-2590
of Bchs (arrow indicates Bchs; just below Bchs, the anti-sera also detects a
crossreacting protein, whose size and intensity are unaffected in multiple
bchs truncation alleles). GMR-GAL4;EP-bchs animals express
more Bchs protein than controls, including EP-bchs. Overexpression is
abolished by the nonsense mutation in bchs17
(GMR-GAL4;bchs17/+). bchs12,
bchs17 and bchs58 express no detectable
Bchs, while bchs8 still produces substantial amounts of
protein. Antibody against Elav demonstrates equal loading. VNC, ventral nerve
cord.
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Fig. 3. Reductions in rab11 enhance the bchs overexpression
phenotype. (Left) Bchs overexpression produces a small, glazed eye.
(Middle, right) Heterozygosity for rab11 alone has no eye phenotype
(not shown) but further reduces the eye in animals that overexpress Bchs.
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Fig. 6. Bchs localization in presynaptic endings. (A) Bchs
immunoreactivity (red) within the third-instar larval brain and nerve cord is
enriched in the synaptic neuropil, marked with Synaptotagmin1 immunoreactivity
(green). Bchs immunoreactivity is undetectable in bchs12.
(B) When expressed in the adult ellipsoid body, HA-tagged Bchs (red)
accumulates in the axon terminals of EB neurons, while transmembrane mCD8::GFP
(green) distributes evenly throughout these neurons. Stars denote EB neuron
presynaptic terminals, arrowheads point to EB neuron dendrites, arrows point
to EB cell bodies. (C) In a confocal stack of images from a
third-instar NMJ (muscle 6/7), Bchs immunoreactivity (red) is enriched in
synaptic boutons. The neuronal membrane is outlined by anti-HRP (green).
(D) In a single confocal section, Bchs puncta (red) are visible within
the HRP-labeled (green) neuronal ending. The specificity of this
immunoreactivity is confirmed by its absence in bchs58.
Scale bars: 20 µm in C; 5 µm in D. EB, ellipsoid body.
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Fig. 7. Bchs localizes to a distinct compartment within presynaptic terminals at
the NMJ. All GFP-tagged markers (green) were expressed using a
pan-neuronal Elav-GAL4 driver. anti-HRP immunoreactivity (blue) marks the
neuronal membrane. Bchs-immunoreactive puncta (red) do not show significant
overlap with (A) 2XFYVE-GFP or (B) Rab5-GFP, which mark the
early endosomal compartment (Wucherpfennig
et al., 2003 ), (C) Anf-GFP, a dense-core vesicle marker, or
(D) resemble the periactive zone staining of Clathrin-GFP, which marks
areas actively undergoing endocytosis. A single confocal section at NMJ 6/7 is
show in each panel. Scale bars: 5 µm.
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Fig. 8. Bchs and Rab11 partially overlap within the NMJ. (A) Rab11
positive puncta are present in the motor axon (arrowhead), synaptic boutons
(arrow) and muscle (star). The number and brightness of Rab11 puncta are
reduced in rab11ex1/rab1193Bi hypomorph (right
panels) compared with control (left panels). Confocal stacks through NMJs at
muscle 4 are shown. (B)
rab11ex1/rab1193Bi mutants have reduced levels
of Rab11 protein compared with controls. Anti-Elav serves as control for
protein loading. (C) Single confocal slice through muscle 4 NMJ of
third instar larva, double labeled for Rab11 (green) and Bchs (red), reveals
significant overlap. Anti-HRP (blue) outlines neuronal membrane. Scale bars: 5
µm.
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Fig. 10. rab11 mutants have a morphological phenotype at the NMJ that is
suppressed by bchs. (A) Confocal images of muscle 6/7 of
abdominal segment 3 labeled with anti-HRP to outline neuronal membrane.
Compared with controls and bchs mutants,
rab11ex1/rab1193Bi animals have an increased
density of synaptic boutons and an increased percentage of boutons that are
branched (connected to three or more adjacent boutons; arrowheads). Both
defects are suppressed in
bchs17/bchs12;rab11ex1/rab1193Bi
double mutants. (B,C) Quantification of bchs-dependent
rab11 NMJ defects in bouton density (B) and branching (C). Bouton
density (bouton number/µm2 muscle area) in
rab11ex1/rab1193Bi mutants is 225% of control
(P<0.0001). This defect is suppressed in
bchs17/bchs12;rab11ex1/rab1193Bi
double mutants (P<0.01 compared to
rab11ex1/rab1193Bi). In
rab11ex1/rab1193Bi mutants, the fraction of
branching boutons is 266% of control (P<0.0001). This defect is
also suppressed in
bchs17/bchs12;rab11ex1/rab1193Bi
double mutants (P<0.0001 compared to
rab11ex1/rab1193Bi). n=14 for each
genotype. Scale bars: 10 µm.
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© The Company of Biologists Ltd 2006
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
