DEVELOPMENT AND DISEASERESEARCH ARTICLE
Microtubules (MTs), one of the major building blocks of cells, play
a crucialrole in a diverse array of biological functions including cell
division, cell growth and motility, intracellular transport and the
maintenance of cell shape. As MTs are important in all eukaryotes,
it is not surprising that defects in MTs are associated with a number
of severe human diseases, including Fragile X mental retardation
and autosomal dominant hereditary spastic paraplegia (AD-HSP)
(Lewis and Cowan, 2002; Penagarikano et al., 2007; Reid, 1997;
Roll-Mecak and Vale, 2005; Sherwood et al., 2004; Trotta et al.,
2004; Zhang and Broadie, 2005). MTs are formed by polymerization
of tubulin heterodimers consisting of one α- and one β-tubulin
polypeptide. The formation of α-βtubulin heterodimers is mediated
by a group of five tubulin chaperones, TBCA-TBCE (Tian et al.,
1996) (for reviews, see Lewis et al., 1997; Nogales, 2000). TBCA
and TBCD assist in the folding of β-tubulin, whereas TBCB and
TBCE facilitate the folding of α-tubulin (Lewis et al., 1997; Tian et
A group of rare, recessive and fatal congenital diseases,
collectively called hypoparathyroidism, mental retardation and
facial dysmorphism (HRD), is caused by mutations in the gene
encoding TBCE (Parvari et al., 2002). TBCE contains three
functional domains: a glycine-rich cytoskeleton-associated protein
domain (CAP-Gly) that binds α-tubulin, a series of leucine-rich
repeats (LRR), and an ubiquitin-like (UBL) domain; the latter two
mediate protein-protein interactions (Bartolini et al., 2005; Grynberg
et al., 2003; Parvari et al., 2002). Identification of the HRD disease
gene revealed a 12 bp deletion in TBCE that leads to the expression
of a mutated TBCE protein lacking four amino acids in the CAP-Gly
domain (Parvari et al., 2002).The mutation causes lower MT density
at the MT organizing center, perturbed MT polarity and decreased
precipitable MT, while total tubulin remains unchanged (Parvari et
al., 2002). Remarkably, overexpression of TBCE in cultured cells
also results in disrupted MTs (Bhamidipati et al., 2000; Sellin et al.,
2008; Tian et al., 2006). Thus, both loss-of-function mutations and
overexpression of TBCE disrupt the MT network in mammalian
Two independent studies have demonstrated that a Trp524Gly
substitution at the last residue of mouse TBCE results in progressive
motor neuronopathy (PMN), which has been widely used as a model
for human motor neuron diseases (Bommel et al., 2002; Martin et
al., 2002). Similar to what has been reported for cells from human
PMN patients, the point mutation in mouse Tbce leads to a reduced
number of MTs in axons (Bommel et al., 2002). Isolated motor
neurons from mutant mice exhibit shorter axons and irregular axonal
swellings (Martin et al., 2002). More specifically, axonal MTs are
lost progressively from distal to proximal, which correlates with
dying-back axonal degeneration in mutant mice (Schaefer et al.,
2007). This demonstrates a mechanistic link between TBCE-
mediated tubulin polymerization and neurodegeneration.
TBCE is well conserved across species, from yeast to human.
Genetic analyses of the TBCE homolog in S. pombe, Sto1P, showed
that it is essential for viability and plays a crucial role in the
formation of cytoplasmic MTs and in the assembly of mitotic
spindles (Grishchuk and McIntosh, 1999; Radcliffe et al., 1999). S.
cerevisiae mutants of the TBCE homolog PAC2 show increased
sensitivity to the MT-depolymerizing agent benomyl (Hoyt et al.,
1997). Similarly, tbce mutants of Arabidopsis have defective MTs,
leading to embryonic lethality (Steinborn et al., 2002).
The Drosophila genome contains a TBCE ortholog, listed as
CG7861 in FlyBase (http://flybase.org), but no studies of it have
been reported. To gain a mechanistic insight into the in vivo
functions of TBCE, we introduced different mutations into
Drosophila tbce. Drosophila tbce nulls are embryonic lethal,
indicating that it is an essential gene. We also examined the
developmental, physiological and pharmacological consequences
with regard to neuromuscular synapses and MTformationwhen the
Drosophila Tubulin-specific chaperone E functions at
neuromuscular synapses and is required for microtubule
Shan Jin1,2, Luyuan Pan1, Zhihua Liu1, Qifu Wang1, Zhiheng Xu1and Yong Q. Zhang1,*
Hypoparathyroidism, mental retardation and facial dysmorphism (HRD) is a fatal developmental disease caused by mutations in
tubulin-specific chaperone E (TBCE). A mouse Tbce mutation causes progressive motor neuronopathy. To dissect the functions of
TBCE and the pathogenesis of HRD, we generated mutations in Drosophila tbce, and manipulated its expression in a tissue-specific
manner. Drosophila tbce nulls are embryonic lethal. Tissue-specific knockdown and overexpression of tbce in neuromusculature
resulted in disrupted and increased microtubules, respectively. Alterations in TBCE expression also affected neuromuscular synapses.
Genetic analyses revealed an antagonistic interaction between TBCE and the microtubule-severing protein Spastin. Moreover,
treatment of muscles with the microtubule-depolymerizing drug nocodazole implicated TBCE as a tubulin polymerizing protein.
Taken together, our results demonstrate that TBCE is required for the normal development and function of neuromuscular synapses
and that it promotes microtubule formation. As defective microtubules are implicated in many neurological and developmental
diseases, our work on TBCE may offer novel insights into their basis.
KEY WORDS: Drosophila, HRD, Spastin, TBCE (CG7861), Tubulin chaperone
Development 136, 0000-0000 (2009) doi:10.1242/dev.029983
1Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
2College of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
*Author for correspondence (e-mail: email@example.com)
Accepted 19 February 2009
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expression of TBCE was altered specifically in neurons or muscles
using the UAS-Gal4 system (Brand and Perrimon, 1993). We found
that TBCE is required for the normal development and function of
neuromuscular synapses and that itpromotesMT formation in vivo.
MATERIALS AND METHODS
Drosophila husbandry and stocks
Flies were cultured in standard cornmeal media at 25°C, unless specified.
w1118was used as the wild-type control. Other stocks used include muscle-
specific C57-Gal4 (Budnik et al., 1996), pan-neuronal elav-Gal4, and
deficiencies Df(2R)ED1484 and Df(2R)ED1482, which remove tbce
completely (Bloomington Stock Center). The spastin-null mutant spastin5.75
was from N. Sherwood (Sherwood et al., 2004), and a UAS-spastinline was
from K. Broadie (Trotta et al., 2004). The chemical mutagen ethyl
methanesulfonate (EMS)-induced nonsense mutation Z0241in tbce(see Fig.
1B) was obtained from a TILLING (targeting induced local lesions in
genomes) service at Seattle (http://tilling.fhcrc.org).
P element-mediated excision was used to generate small deletions in tbce
following a standard protocol. The original stock KG09112from Bloomington
has a Pelement insertion in the intergenic region between CG14591and tbce
(Fig. 1B). Before mobilizing KG09112asmediated by Ptransposase Δ2-3, we
isogenized the original stock. w+deletion lines with the P insertion excised
either precisely (LH198) or imprecisely (LH260 and LH15) were initially
screened by PCR followed by DNA sequencing, in conjunction with
immunochemical analyses to confirm the mutations at the protein level.
Production of UAS and RNAi transgenic flies
For overexpression studies, a UAS-TBCE construct was made by
amplifying the full-length tbce cDNA from EST clone GM13256, obtained
from the DrosophilaGenomics
(https://dgrc.cgb.indiana.edu/vectors), and cloned into the transformation
vector pUAST. For tissue-specific knockdown assays, an RNAi construct
was made according to a previously described procedure (Kalidas and
Smith, 2002). Specifically, a cDNA fragment from thetbcesecond exon was
fused to the corresponding genomic sequence plus intron 2 as a spacer (see
Fig. 1B) to make a hairpin RNAi construct targeting nucleotides337-842 of
GenBank sequence NM_136353. An independent RNAi line targeting a
different sequence (nucleotides 919-1280 of NM_136353) of tbce was
obtained fromthe Vienna Stock Center. Although multiple independent lines
of transgenic flies carrying UAS-TBCE or RNAi were generated, here we
report on a UAS-TBCE insertion on the third chromosome and on UAS-
RNAi of apparent effect. To ensure high efficiency of overexpression or
knockdown of TBCE by the UAS-Gal4 system, flies, including wild-type
controls, were raised at 28°C instead of 25°C for all the assays involving the
UAS and RNAi transgenic lines.
Production of monoclonal antibody against TBCE
His-tagged peptide corresponding to the N-terminal 512 amino acids of
TBCE (full-length TBCE is 542 amino acids) produced in E. coli was used
as antigen. Immunization and screening of antibody producing cells were
performed according to standard procedures. Several positive clones were
identified, but the antibody produced by clone 8E11 is specific for both
western and immunostaining.
The larval roll-over assay was performed largely according to published
protocols (Bodily et al., 2001; Pan et al., 2008). Before the assay, larval
culture and agar plates were placed at room temperature for 2 hours to
acclimatize. For each assay, an individual animal was placed on a 1% agar
plate and allowed to move freely for 2 minutes. The test animal was rolled
over using a soft brush to a completely inverted position, indicated by the
ventral midline facing up. The time that the animal tookto totally right itself
was recorded. Three assays were performed continuously without any
resting time for each animal, and then averaged to produce one data point.
Immunochemical analyses and confocal microscopy
For western analyses, third instar larvae were dissected in PBS with all
internal organs removed, followed by homogenization in 2?loading buffer
(xxxxx constituents or reference? xxxxx). 1/2 fillet was used for each
loading. Primary antibodies used were anti-TBCE (1:200), anti-α-tubulin
(1:50,000; mAb B-5-1-2, Sigma) and anti-actin (1:50,000; mAb1501,
Chemicon). The blots were detected with horseradish peroxidase (HRP)-
coupled secondary antibodies using a chemiluminescent method (ECL Kit,
Whole-mount embryos were fixed and stained with anti-FAS2 [1:100;
Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa]
and BP102 antibody (1:200; DSHB) using standard procedures. For
immunostaining of first instar larvae, animals were dissected and processed
following an established protocol (Budnik et al., 2006). The medical
adhesive Compont (Shunkang, Beijing, China) was used to glue the
epidermis of larvae to Sylgard-coated coverslips. Dissection and antibody
staining of third instar larvae are described elsewhere (Zhang et al., 2001).
Primary antibodies used include: anti-α-tubulin (1:1000; Sigma), anti-TBCE
(1:1), Texas Red-conjugated goat anti-HRP(1:50; Jackson Laboratory), anti-
Futsch (1:1000; DSHB) and anti-Discs large (DLG) 4F3 (1:1000; DSHB).
All primary antibodies were visualized using Alexa 488- or Cy3-conjugated
goat anti-mouse IgG (1:200; Invitrogen). To examine the MT network in
muscles, muscle 2 in abdominal segment A4 was analyzed as it has fewer
tracheal branches to obscure the observation of MTs. Nucleiwerevisualized
by staining with propidium iodide (PI; 1.25 μg/ml) for 30 minutes at room
temperature. Images were collected with a Leica SP5 confocal microscope
and processed using Adobe Photoshop.
NMJ quantifications largely followed published procedures (Zhang et al.,
2001). All images analyzed were three-dimensional projections from
complete z-stacks through the entire NMJ4 of abdominal segment A3.
Synaptic boutons were defined according to anti-HRP (presynaptic) and
anti-DLG (postsynaptic) staining. Branches originating directly from the
nerve entry point were defined as primary branches, and each higher-order
branch was counted only when two or more boutons in a string could be
observed. For bouton area analyses, ImageJ 3.0 (NIH) was used to define
anti-HRP-stained individual boutons. The software output reports the area
for each bouton automatically. At least 22 NMJ4 terminals of different
genotypes were analyzed.
Futsch staining intensity relative to HRP staining at NMJ synapses was
quantified largely according to Trotta et al. (Trotta et al., 2004). Staining
intensities of Futsch and HRP from an entire NMJ4 terminal were digitalized
automatically using ImageJ 3.0. Synaptic boutons with different Futsch
staining patterns were quantified following published procedures (Packard
et al., 2002; Sherwood et al., 2004). Synaptic boutons were divided into three
types based on the Futsch staining pattern: (1) continuous (bundle or splayed
bundle), (2) looped, and (3) diffuse (punctate) or no staining. Terminal
boutons were defined as those at the ends of synaptic branches. Fourteen
NMJ4 terminals from seven animals for each genotype were statistically
analyzed for Futsch expression features (see Fig. 7G-I).
For quantification of tubulin staining in muscles, all images analyzed
were three-dimensional projections of serial stacks through the muscle cell.
The perinuclear areas were defined as the coverage that spans 10 μm around
nuclei, which were stained with PI. Tubulin staining signals within the
perinuclear area from muscle 2 of abdominal segment A4 were calculated
using ImageJ 3.0. The software reports the ratio of the tubulin-positive area
divided by the total perinuclear area. At least four readings, one from one
animal, were analyzed for each genotype.
Intracellular recordings were carried out at 18°C following a conventional
procedure (Jan and Jan, 1976). Specifically, wandering third instar larvae
were dissected in Ca2+-free HL3.1 saline (Feng et al., 2004) and recorded
in HL3.1 saline containing 0.25 mM Ca2+. Intracellular microelectrodes
with a resistance of 10-20 M? filled with 3M KCl were used for the
assay. Recordings were performed using an Axoclamp 2B amplifier
(Axon Instruments) in Bridge mode. Data were filtered at 1 kHz, digitized
using a Digitizer 1322A (Axon Instruments) and collected with Clampex
9.1 software (Axon Instruments). EJPs were evoked at 0.3 Hz by a suction
electrode with a depolarizing pulse delivered by a Grass S48 stimulator
(Astro-Grass). EJPs were recorded from muscle 6 of abdominal segment
A2 or A3, followed by mEJP recording for 120 seconds. EJP and mEJP
recordings were processed with Clampfit 9.1 software (Axon
Development 136 ()
Instruments). Quantal content was calculated by dividing the corrected
EJP amplitude by the mEJP amplitude according to a classical protocol
(Martin, 1955). The EJP correction for nonlinear summation was
performed using a reversal potential of 10 mV. At least nine animals were
recorded for each genotype.
Pharmacological treatment of dissected larvae with nocodazole
MT dynamics were examined using the MT-depolymerizing drug
nocodazole. Nocodazole (Sigma) was prepared as a 16.6 mM stock in
dimethyl sulfoxide (DMSO). Third instar larvae were dissected open in
Ca2+-free HL3.1 buffer and incubated with 30 μM nocodazole in
Schneider’s insect medium (Sigma) for 4 hours at 25°C. The drug was then
washed out with the medium and animals further incubated for 1, 2, 5 and
20 minutes for recovery of MTs. The control group was mock treated with
Schneider’s insect medium containing DMSO. The treated samples were
then fixed stained, and imaged as described.
All statistical comparisons were performed using GraphPad InStat 5
software. P-values were calculated by two-tailed Student’s t-test.
Drosophila CG7861 encodes an ortholog of human
Sequence comparison showed that the Drosophilagenome contains
an uncharacterized gene, CG7861, that encodes an ortholog of
human TBCE. Drosophila TBCE is 30% identical and 49% similar
to human TBCE (Fig. 1A). The high degree of conservation between
Drosophila and human TBCE suggests that its function is well
maintained through evolution.
As a first step to understanding thein vivofunctions of TBCE, we
generated null mutations of tbce (Fig. 1B). We obtained an EMS-
induced nonsense mutation, Z0241 (Fig. 1B), by a TILLING
approach. This mutation is likely to be a null, as it is located at the 5?
terminus of the coding region and produced no detectable TBCE as
assessedby immunostaining (Fig. 2B). We also generated a deletion,
LH15, which removes a large part of tbce (Fig. 1B) via P element-
mediated imprecise excision. LH15 is presumably another null allele
of tbce, based on its molecular lesion. Hemizygous or heteroallelic
Z0241 and LH15 mutants are embryonic lethal, whereas LH198, a
precise excision control, is fully viable. Similarly, LH260, which
removes the majority of the flanking gene CG14591, is also viable
with no detectable phenotype(data not shown) (Fig. 1B). In summary,
tbceis essential for viability, as the independent mutations Z0241and
LH15 resulted in embryonic lethality. The embryonic lethality of tbce
nulls prevented straightforward developmental and functional
analyses.To overcome the limitation of early lethality, we constructed
multiple independent UAS and RNAi transgenic lines for tissue-
specific overexpression and knockdown of tbce, respectively, using
the UAS-Gal4 system (Brand and Perrimon, 1993). The efficacy of
the transgenic lines was confirmed by western analysis and
immunostaining with an anti-TBCE monoclonalantibody, 8E11,that
we generated (Fig. 1C; Fig. 2E-G).
TBCE is cytoplasmic and ubiquitously expressed
Previous TBCE overexpression studies in HeLa cells detected
TBCE in the cytoplasm (Bhamidipati et al., 2000; Tian et al., 2006).
Mouse TBCE is enriched in motor neurons and localizes in both
TBCE functions at synapses and promotes microtubule formation
Fig. 1. Drosophila TBCE. (A)Sequence alignment of Drosophila (d) and human TBCE. CAP-Gly, glycine-rich cytoskeleton-associated protein; LRR,
leucine-rich repeat; UBL, ubiquitin-like. The CAP-Gly, LRR and UBL domains are delineated as previously published (Bartolini et al., 2005). Dark and
light gray shading indicate identical and similar amino acids, respectively. Nonsense mutation Z0241 is indicated. (B)Genomic structure of
Drosophila tbce and mapping of mutants. The intron-exon organization of tbce and its flanking gene, CG14591, is shown at the top. Gray boxes,
coding regions; white boxes, untranslated regulatory regions; ‘gaps‘, introns; horizontal line, intergenic region (2220 bp). The two deletion lines
LH260 and LH15, generated by imprecise excision of KG09112, are depicted, as is the precise excision line LH198. The genomic region used to
generate RNAi knockdowns is indicated. (C)Western analysis of tbce overexpression and RNAi knockdown transgenic flies. The anti-TBCE
monoclonal antibody (8E11) that we generated recognizes a single band of the expected size (60 kDa). No change in α-tubulin expression was
detected when tbce expression was altered. Actin was used as a loading control.
crude membrane and cytosolic fractions prepared from spinal cord
(Schaefer et al., 2007). We investigated the expression pattern and
subcellular localization of Drosophila TBCE using our 8E11
monoclonal antibody. The antibody detected a single band of 60
kDa, as expected from the deduced amino acid sequence (Fig. 1C).
Immunostaining of embryos with the anti-TBCE antibody showed
that TBCE is ubiquitously expressed, with particular enrichment in
the central nervous system (CNS) and muscles (Fig. 2A). No
expression was detected in Z0241 homozygous mutants (Fig. 2B),
confirmingthe specificity of the antibody. The expression of TBCE
decreasedas the animal developed from embryo to larva. In the third
instar larva, weak expression with a perinuclear enrichment was
observed in muscles (Fig. 2C), whereas substantial expression was
observed in epidermalcells (Fig. 2D). In both cell types, TBCE was
clearly cytoplasmic and excluded from the nucleus (Fig. 2C,D).
Low-level expression of TBCE was also seen in the central neurons
and peripheral axons of the wild-type (WT) larva (Fig. 2E).
Corresponding changes in TBCE levels were observed in the central
neurons of a ventral ganglion when tbce was overexpressed or
knocked down by elav-Gal4(Fig. 2F,G), demonstrating the efficacy
of the UAS and RNAi transgenes. Like endogenous TBCE (Fig.
2C,D), overexpressed TBCE was also cytoplasmic (Fig. 2F).
TBCE is required for microtubule formation,
axonal growth and coordinated larval locomotion
Drosophila tbce nulls are embryonic lethal, with a few escapers
developing to first instar larvae. To reveal the effects of TBCE on
MTs, we stained mutant first instar larvae with anti-α-tubulin. The
MT network was greatly decreased, with fewer and shorter MT
fibers in mutant muscle cells as compared with the dense and
evenly distributed MT network in the WT (compare Fig. 3D with
3B), indicating that TBCE is required for MT formation or
A mutated Tbce in mouse leads to retarded axonal growth and
axonal degeneration (Bommel et al., 2002; Martin et al., 2002;
Schaefer et al., 2007). To assess the role of TBCE in neuronal
development, we stained stage 16 embryos with anti-FAS2, which
detects a set of three longitudinal axon bundles, and with BP102
antibody, which recognizes the anterior and posterior commissures
and longitudinal connectives of the ventral nerve cord (VNC). As
shown in Fig. 3E, anti-FAS2 staining of WT embryos showed three
parallel longitudinal axon bundles at either side of the body.
Compared with the WT, Z0241/Df [Df(2R)1482 or Df(2R)1484
removes tbce completely] and LH15/Df mutants showed
longitudinal axon bundles that crossed at the midline (Fig. 3F,G).
Interrupted axon bundles were also observed (Fig. 3G). Heteroallelic
Z2041/LH15 had comparable axonal defects (data not shown). WT
embryos stained with BP102 antibodyshowed a regular ladder-like
pattern of axon bundles in the VNC (Fig. 3H). However, the regular
pattern of axons was grossly disrupted in tbce mutants with
interrupted or missing longitudinal bundles (Fig. 3I,J). The dramatic
axonal defects in tbcenulls suggest that TBCE is required for axonal
growth in Drosophila.
To understand the physiological functions of TBCE, we
performed behavioral assays. As tbcenulls are embryonic lethal, we
examined the behavior of larvae in which TBCE expression had
beengenetically altered in a tissue-specific fashion by the UAS-Gal4
system. Tissue-specific knockdown of TBCE in neurons by elav-
Gal4 or in muscles by C57-Gal4 produced fully developed larvae
(they were late pupal lethal and fully viable, respectively) with
normal rhythmic peristalsis and crawling activity (data not shown).
However, a larval roll-over assay revealed obvious and profound
locomotion defects when TBCE expression was altered. As a
genetic control, transgenic flies of elav-Gal4, C57-Gal4, UAS-tbce
and UAS RNAi without alterations in TBCE expression showed
indistinguishable roll-over time from the WT (Fig. 4). However,
TBCE knockdown in neurons and muscles caused significantly
slower locomotion compared with the WT (taken as 100%), with the
average roll-over time increased to 205% and 246%, respectively
(Fig. 4). Similarly, neuronal and muscular overexpression of TBCE
also caused a significantly compromised roll-over performance
compared with the WT, with average roll-over time increased to
168% and 190%, respectively (Fig. 4). The abnormal behavior of
animals with altered TBCE expression indicates that TBCE is
required for the physiological function of the neuromusculature.
TBCE regulates the development of
neuromuscular junction synapses
Abnormal synapses are associated with misregulated MTs (Roos et
al., 2000; Sherwood et al., 2004; Trotta et al., 2004). To understand
the molecular pathogenesis of HRD, we examined the development
of neuromuscular junction (NMJ) synapses in flies in which tbce
expression had been manipulated by the UAS-Gal4 system.
DrosophilaNMJ synapses are a commonly used system to examine
protein functionat synapses, as they are large, simple and amenable
to various morphological and functional assays.
Three NMJ synapse features – synaptic branching, bouton
number and average bouton area – were statistically analyzed (Fig.
5). For synaptic branching, both elav-Gal4-driven presynaptic and
C57-Gal4-driven postsynaptic knockdowns of tbce displayed a
significant over-branching compared with the WT control (Fig. 5A-
C,F). However, overexpression of tbce pre- or postsynaptically did
Development 136 ()
Fig. 2. TBCE is cytoplasmic and ubiquitously expressed in
neuromusculature. (A,B)Wild-type (WT) and Z0241 mutant
Drosophila embryos stained with the 8E11 anti-TBCE monoclonal
antibody. WT embryos showed specific and substantial expression of
TBCE in the ventral nerve cord (CNS) and muscles (A), whereas mutant
embryos showed no specific expression (B). (C,D)Immunostaining of
larval muscles (C) and epidermal cells (D) showed that TBCE protein is
localized in the cytoplasm and excluded from the nucleus. (E)Weak
expression of TBCE was observed in the central neurons (asterisk) and
peripheral axons (arrow). (F,G)Cytoplasmic TBCE in the ventral
ganglion neurons was clearly seen when TBCE was pan-neuronally
overexpressed using elav-Gal4 (F), whereas no appreciable expression of
TBCE was observed when RNAi was driven by elav-Gal4 (G). Scale bars:
not show the opposite phenotype to RNAi knockdown. Instead,
presynaptic overexpression of TBCE resulted in normal NMJ
branching, whereas postsynaptic overexpression showed mild but
significant over-branching (P=0.04) (Fig. 5D-F). Thus, except for
presynaptic overexpression of TBCE, which caused normal
branching, all other manipulations of TBCE expression resulted in
increased branching of NMJ synapses.
Synaptic bouton number was affected similarly to synaptic
branching for the four genotypes assayed. Both the elav-Gal4-driven
and C57-Gla4-driven TBCE knockdown caused a significant
increase in bouton number compared with the control (Fig. 5A-
C,G). However, overexpression of TBCE by elav-Gal4 resulted in
normal bouton numbers, whereas C57-Gal4-driven overexpression
caused a significant increase in bouton number (Fig. 5D,E,G). For
synaptic bouton area, C57-Gal4-driven knockdown or
overexpression of tbce showed significantly decreased bouton size
(Fig. 5A,C,E,H). elav-Gal4-driven knockdown also showed
decreased bouton size (P<0.001), but presynaptic overexpression
exhibited normal bouton size (Fig. 5D,H). These NMJ phenotypes
are not due to the Gal4 driver, UAS or RNAi insertions, as these
showed wild-type NMJ synapses (data not shown). In summary,
quantification analyses showed that except for presynaptic
overexpression of tbce, the remaining three manipulations of tbce
caused increased branching number, increased bouton number and
decreased bouton size at NMJ synaptic terminals. These results
demonstrate that TBCE plays a crucial role in NMJ synapse
development, which might underlie the mental retardation seen in
TBCE regulates neurotransmission at NMJ
As shown above, TBCE regulates the development of NMJ
synapses (Fig. 5). We then investigated whether TBCE plays a role
in synaptic function. We found no change in the amplitude of
excitatory junction potentials (EJPs) in animals in whichTBCE had
been knocked down or overexpressed postsynaptically (Fig. 6A,D-
F). However, compared with the WT, both knockdown and
overexpression of TBCE presynaptically elevated the EJP
amplitudes significantly, by 29% and 40%, respectively (Fig. 6A-
C,F). We also examined the miniature excitatory junction potentials
(mEJPs), i.e. the amplitude of the response to a single vesicle
release, also known as quantal size. The mEJP for the WT was
0.95±0.05 mV. Knockdown and overexpression of TBCE
presynaptically increased the mEJPby 35% and 27%, respectively
(Fig. 6A-C,G). The increase in EJP and mEJP was not due to elav-
Gal4, as it displayed normal neurotransmissions (data not shown).
Alterations in TBCE on the postsynaptic side caused no significant
change in mEJP as compared with the WT (Fig. 6D,E,G). The
quantal content –the number of vesicles released per evoked event,
calculated by dividing the corrected EJP amplitude by the mEJP
amplitude – was affected only when tbce was overexpressed
presynaptically (anincrease of 69%, P<0.05) (Fig. 6H). Presynaptic
TBCE functions at synapses and promotes microtubule formation
Fig. 3. TBCE is required for microtubule formation and axonal
growth. (A,C)Phase-contrast images of muscles 6 and 7 of WT (A) and
Z0241/Df(2R)ED1484 mutant (C) first instar Drosophila larvae.
(B,D)Dissected larvae were double-stained with anti-α-tubulin (green)
and with propidium iodide (PI, red) to label nuclei. tbce mutants have a
greatly reduced microtubule (MT) network and shorter MT fibers (D)
compared with the WT (B). (E-J)Embryos were stained with anti-FAS2,
which labels three parallel longitudinal axon bundles (E-G), and with
BP102 antibody, which labels the anterior and posterior commissures
and longitudinal connectives of the ventral nerve cord (H-J). (E,H)WT;
(F,I) Z0241/Df; (G,J) LH15/Df. Asterisks indicate midline crossing;
arrowheads indicate broken longitudinal connectives. ac, anterior
commissure; pc, posterior commissure; lc, longitudinal connective. Scale
Fig. 4. Alterations in TBCE expression in neuromusculature lead
to defective locomotion. The roll-over assay was performed to
examine coordinated locomotion in larvae of different genotypes.
Knockdown or overexpression of tbce specifically in muscles by C57-
Gal4, or in neurons by elav-Gal4, resulted in a significant increase in
roll-over time. As a control, elav-Gal4, C57-Gal4, UAS or RNAi
transgenic flies without alteration of tbce expression showed normal
roll-over, as in the WT (P>0.05). The number of flies tested for each
genotype is indicated. **P<0.01, ***P<0.001; error bars indicate
knockdown of tbcecauseda significant increase in mEJPfrequency,
but other manipulations of tbce expression showed no significant
changes (Fig. 6I).
In summary, altering the dosage of tbce on the postsynaptic side
had no effect on the neurotransmission parameters we examined.
But, a precisely controlled expression of tbceon the presynaptic side
was necessary for the normal function of NMJ synapses. Taken
together, these analyses demonstrate that TBCE functions
presynaptically to control neurotransmission at NMJ synapses.
TBCE is required for MT formation in presynaptic
A previous study revealed decreased MT density in mutant cells
from HRD patients (Parvari et al., 2002). In Tbce mutant mice,
axonal MT loss proceeds retrogradely in parallel with the axonal
dying-back process (Martin et al., 2002; Schaefer et al., 2007). To
investigate the effect of TBCE on MTs in the nervous system, we
stained third instar larvae with antibodies against α-tubulin,
Futsch (the fly ortholog of mammalian MAP1B; MTAP1B) and
the neuronal marker HRP (Fig. 7). As shown in Fig. 7B, tbce
knockdown in presynaptic neurons resulted in obviously
decreased, interrupted or even missing MTs at the distal part of
the synaptic terminal detected by anti-α-tubulin staining (Fig. 7B-
B?). Overexpression of tbce, however, led to smooth and
continuous α-tubulin staining, compared with the WT (compare
Fig. 7C? with 7A?). Anti-Futsch is a useful marker to reveal
stabilized MTs specifically in neurons (Fig. 7D-F). Similar to the
α-tubulin staining, a much weaker and thinner staining with anti-
Futsch, with weak or no staining in the terminal boutons, was
observed when tbce was knocked down (Fig. 7E?). Statistical
analyses showed that the Futsch staining intensity relative to that
of HRP was significantly decreased in the knockdown and
increased upon overexpression of TBCE in presynaptic neurons,
as compared with the WT (Fig. 7D-F,G).
To further define the effect of TBCE on MTs, we quantified
synaptic boutons based on the Futsch staining pattern. TBCE
knockdown in presynaptic neurons caused significantly decreased
numbers of synaptic boutons that had organized (continuous and
looped) Futsch, and increased numbers of boutons with diffuse or
no Futsch signals (82.28%), as compared with the WT (23.57%)
(Fig. 7H). By contrast, boutons with Futsch loops were significantly
increased, and boutons with diffuse or no Futsch staining were
decreased, when TBCE was overexpressed (Fig. 7H). These
differences were also reflected in terminal boutons. Only 69% of
terminal boutons in TBCE knockdown animals had Futsch-positive
boutons, compared with 98% in the WT, whereas TBCE
overexpression showed a similar number of Futsch-positive boutons
as the WT (Fig. 7I). In summary, knockdown of tbce in presynaptic
neurons resulted in decreased MTs, whereas overexpression of tbce
led to increased MTs in synaptic terminals.
Development 136 ()
Fig. 5. TBCE regulates NMJ synapse development.
NMJs from wandering third instar Drosophila larvae were
stained using anti-HRP (red) and anti-DLG (green)
antibodies, to reveal the pre- and postsynaptic domains,
respectively. Representative images of the NMJ on muscle
4 of abdominal segment A3 are shown. (A)WT control.
(B)elav-Gal4-driven presynaptic RNAi knockdown.
(C)C57-Gal4-driven postsynaptic RNAi knockdown.
(D)elav-Gal4-driven overexpression of TBCE. (E)C57-
Gal4-driven overexpression of TBCE. Scale bar: 5μm. (F-
H)Quantification of NMJ branch number (F), bouton
number (G), and bouton area (H), for the different
genotypes (n?22). *P<0.05, **P<0.01, ***P<0.001;
error bars indicate s.e.m.
TBCE antagonizes Spastin to regulate MT
To better understand how TBCE affects MTs, we studied its genetic
interaction with Spastin. Spastin severs the MT network in cultured
cells and Drosophila neuromusculature (Errico et al., 2002; Roll-
Mecak and Vale, 2005; Sherwood et al., 2004; Trotta et al., 2004).
We first confirmed that Spastin severs MTswhen it is overexpressed
in muscles (see Fig. 8D) (see also Sherwood et al., 2004; Trotta et
al., 2004), although no obvious abnormality in the MT network was
observed in spastin nulls (Fig. 8E).
Compared with the thin presynaptic neuronal terminals (Fig.
7), muscle cells enabled a much higher resolution visualization of
MTs (Fig. 8). In the large multi-nucleated muscle cells (80?400
μm) from a third instar larva, a remarkable MT meshwork was
revealed by anti-α-tubulin staining, with the highest intensity of
staining around the nucleus (Fig. 8A). The intensity of perinuclear
MT staining was quantified for various genotypes (see Fig. S1 in
the supplementary material). Compared with the WT,
overexpression of tbce in muscles increased the MT network
dramatically, with a prominent perinuclear enrichment (Fig. 8B).
Indeed, the perinuclear MTs had to be overexposed in order to see
individual MT fibers in the area distal to the nucleus (Fig. 8B).
Conversely, RNAi knockdown of tbce decreased the network,
with sparser and shorter MT fibers (Fig. 8C). The decreased MT
network was confirmed with an independent RNAi line from the
Vienna Stock Center. We then examined the interaction between
tbce and spastin in various combinations. When tbce and spastin
were co-overexpressed, the resulting phenotype was similar to
that of spastin overexpression alone (compare Fig. 8F with 8B,D).
When tbce was knocked down while spastin was overexpressed,
the phenotype was again more like that of spastin overexpression
alone, with small MT fragments (compare Fig. 8G with 8C,D).
The apparent spastin overexpression phenotype of shorter MT
fragments in animals with altered tbce expression suggests that
the function of spastin is dominant over that of tbce. When tbce
was knocked down in a spastin-null background, the RNAi
phenotype of sparser and shorter MT fibers was clearly
ameliorated, although not completely rescued (compare Fig. 8H
with 8C,E), indicating an antagonistic interaction between the
TBCE is acutely required for MT polymerization
To further elucidate the requirement for TBCE in MT formation, we
treated dissected animals with the MT-depolymerizing drug
nocodazole to disassemble the MTs completely, and then followed
MT reformation after drug washout. We noticed that after mock
treatment for 4 hours with a buffer containing the DMSO solvent,
the MT network in muscles, especially in WT and tbce-
overexpressing muscles, was not as dense as in the untreated cells
(compare Fig. 9A,B with the corresponding Fig. 8A,B). As
expected, 30 μM nocodazole treatment of WT animals for 4 hours
eliminated the MT network, and only residual MT buds could be
seen in muscles (compare Fig. 9Aawith 9A). Appreciable recovery
of MTs, represented by denser perinuclear tubulin staining and
longer MT fibers, could be detected 2 minutes after drug washout in
the WT (Fig. 9Ab). By 5 minutes, near complete recovery of MTs
was observed (compare Fig. 9Ac with 9A). tbce-overexpressing
animals showed a similar pattern of MT recovery as the WT
(compare Fig. 9Bb-Bd with the corresponding 9Ab-Ad). The
recovery of MTs was much slower, however, when TBCE was
knocked down by RNAi (compare Fig. 9Ca-Cd with the
corresponding Aa-Ad). By 20 minutes, recovery of MTs was
appreciable, but there was still a large number of MT buds or fibers
that had not yet formed a MT network (Fig. 9Cd). By 1 hour after
washout, the MTs had still not completely recovered (data not
shown). For statistical analyses of MT recovery after the drug
treatment, see Fig. S2 in the supplementary material. This
experiment showed that tbce is acutely required for MT network
Misregulated MTs are associated with human diseases such as
Fragile X syndrome, hereditary spastic paraplegia and HRD. We
have been using Drosophila as a model system to unravel the
molecular pathogenesis of Fragile X syndrome (Reeve et al., 2008;
Zhang et al., 2001; Zhang and Broadie, 2005), the most common
form of inherited mental retardation. Fragile X mental retardation
protein (FMRP; FMR1) plays an important role at synapses
(Penagarikano et al., 2007; Zhang and Broadie, 2005). At the
molecular level, the MT-associated protein MAP1B is a target of
FMRP and is upregulated in Fmr1 knockout mice and mutant flies
TBCE functions at synapses and promotes microtubule formation
Fig. 6. Altering the presynaptic expression
of tbce causes increased
traces of excitatory junction potentials (EJPs)
(upper row) and miniature excitatory junction
potentials (mEJPs) (lower row) of NMJ synapses
from WT (A), presynaptic RNAi (B), presynaptic
overexpression (C), postsynaptic RNAi (D) and
postsynaptic overexpression (E) of tbce. (F-
I)Quantification of EJP amplitudes (F), mEJP
amplitudes (G), quantal content (H) and mEJP
frequencies (I) for the different genotypes
(n?9). *P<0.05, ***P<0.001; error bars
(Iacoangeli et al., 2008; Lu et al., 2004; Zhang et al., 2001). Since
defective MTs are implicated in both Fragile X syndrome and HRD,
and both diseases show mental retardation, we sought to unravel the
in vivo functions of TBCE with regard to synapse development and
MT formation. We provide in vivo evidence demonstrating that
TBCE and the MTs that it regulates function at NMJ synapses, and
that at the molecular level, TBCE is both necessary and sufficient to
promote MT formation.
TBCE regulates NMJ synapse development and
The MT cytoskeleton regulates synapse development and function,
but how MT functions at synapses is poorly understood(Ruiz-Canada
and Budnik, 2006). Futsch, a MT-associated protein, stabilizes MTs
in presynaptic neurons, and futsch mutants show reduced bouton
number and increased bouton size (Roos et al., 2000), whereas spastin
mutants have the opposite phenotype ofincreased bouton number but
decreased bouton size (Sherwood et al., 2004). Our analyses show that
TBCE plays a role at synapses. Except for the presynaptic
overexpression of tbce, all other manipulations of tbce at either side
of the NMJ synapse caused increased branching number, increased
bouton number and decreased bouton size, demonstrating that tbceis
required for normal NMJ synapse development (Fig. 5). Given the
dramatic MT alterations on both pre- and postsynaptic sides (Figs 7
and 8), the NMJ phenotypes appear subtle (Fig. 5). The seemingly
conflicting result that both overexpression and knockdown of tbceon
the postsynaptic side led to similar phenotypes in synapse
development supports an existing hypothesis that abnormal synaptic
growth results from the disruption of MT dynamics, rather than from
an alteration in the absolute quantity of MTs (Ruiz-Canada and
Increased neurotransmission, reflected in both EJP and mEJP
amplitude, was observed upon presynaptic alteration of tbce
expression, whereas postsynaptic manipulations of tbce showed
normal neurotransmission (Fig. 6). This suggests that synaptic
neurotransmission is sensitive to pre- but not postsynaptic MT
alteration, although postsynaptic alterations of tbcehad a significant
effect on synapse development (Fig. 5). Interestingly, both
overexpression and knockdown of tbce on the presynaptic side led
to a similar increase in both EJP and mEJP amplitude (Fig. 6). The
increased EJP amplitude observed upon presynaptic alterations of
TBCE might be accounted for by increased mEJP amplitude (Fig.
6). The increase in mEJP amplitude could be caused by an increase
in presynaptic vesicle size, an increase in the concentration of
vesicular glutamate, or an increase in postsynaptic glutamate
receptor sensitivity. It is interesting to note that the mEJP is also
increased in both Fmr1-null and Fmr1-overexpression NMJ
synapses (Zhang et al., 2001). However, the exact mechanism by
which TBCE, and other MT regulators, affect neurotransmission
remains to be elucidated.
Development 136 ()
Fig. 7. Presynaptic knockdown of tbce results in
decreased MTs in synaptic terminals. (A-C?) TBCE is
required for the formation of the MT cytoskeleton. The NMJ
synapses were double-stained with anti-α-tubulin (red) and
anti-HRP (green). In wild-type NMJ synapses, MTs are present
continuously in synaptic terminals (A-A?). When tbce was
knocked down, the MT bundles were interrupted and not
visible in the distal part of the synaptic terminal (B-B?).
However, when tbce was overexpressed, a continuous and
smooth MT cytoskeleton extending to the very tip of the
terminal was observed (C-C?). (D-F?) Synaptic expression of
Futsch is also regulated by TBCE. NMJ synapses were double-
labeled with anti-HRP (green) and anti-Futsch (red). In
presynaptic tbce knockdown flies, Futsch staining was
dramatically decreased (E-E?), whereas overexpression of tbce
led to an increase in Futsch staining (F-F?), as compared with
the WT (D-D?). Arrowheads in E, xxxxx xxxxx xxxxx xxxxx? Scale
bars: 10μm. (G-I)Futsch staining intensity relative to that of
HRP (G), the percentage of boutons exhibiting continuous,
looped, or diffuse/no Futsch staining (H), and the percentage
of Futsch-positive terminal boutons (I) in the different
genotypes. *P<0.05, **P<0.01, ***P<0.001; error bars
TBCE antagonizes Spastin in regulating MT
Our genetic analyses revealed an antagonistic interaction between
TBCE and Spastin. TBCE promotes MT formation, whereas Spastin
severs MTs. Autosomal dominant hereditary spastic paraplegia
(AD-HSP) is a heterogeneous group of neurodegenerative disorders
characterized by progressive and bilateral spasticity of the lower
limbs, with specific degeneration of the longest axons in the CNS
(Reid, 1997). Forty to fifty percent of all AD-HSP cases are caused
by mutations in spastin. However, the MT-related pathology of
human patients with spastin mutation has not been documented.
Overexpression of spastin in Drosophila neuromusculature
(Sherwood et al., 2004; Trotta et al., 2004) and in cultured cells
(Errico et al., 2002; Roll-Mecak and Vale, 2005) causeddramatically
fragmented and reduced MTs. Surprisingly, morphologically normal
muscles are present in patients with spastin mutations, although
large-scale disruption of MT pathways was detected at the molecular
level (Molon et al., 2004). No MT defects were reportedin a mouse
model in which the endogenous spastin is truncated (Tarrade et al.,
2006). Similarly, spastin-null mutants of Drosophila show no
dramatic change in MT appearancein muscles (Fig. 8E), suggesting
that Spastin plays a fine-tuning role in MT dynamics. Indeed, spastin
nulls are late pupal lethal with a few adult escapers, further
confirming a subtle role for Spastin in MT regulation. By
comparison, tbcenulls are embryonic lethal, whereas knockdown of
tbce leads to a dramatically reduced MT network in Drosophila
neuromusculature (Figs 7-9). Thus, in contrast to the nuanced role
of endogenous Spastin, TBCE plays a crucial role in MT formation.
TBCE functions at synapses and promotes microtubule formation
Fig. 8. tbce antagonizes MT-severing spastin. (A-H?) Drosophila
larval muscles were stained with anti-α-tubulin to show the MT
network (green) and with PI to show the nucleus (red). A?-H? are higher
magnification views from A-H. The MT network in the muscles is
shown for the WT (A) and for tbce overexpression (B), tbce knockdown
(C), spastin overexpression (D) and spastin-null (E) mutants. Co-
overexpression of tbce and spastin produced a phenotype more like
that of overexpression of spastin alone (compare F with B and D).
Knockdown of tbce while concomitantly overexpressing spastin led to
an enhanced form of the phenotype observed upon spastin
overexpression alone (compare G with D). Knockdown of tbce in the
spastin mutant background ameliorated the tbce RNAi phenotype
(compare H with C and E). Scale bars: 10μm.
Fig. 9. TBCE is required for MT network formation. (A-Cd) Muscle
MTs were examined after treatment with the MT-depolymerizing drug
nocodazole. (A-Ad) WT; (B-Bd) tbce overexpressed; (C-Cd) tbce
knocked down by RNAi. (A-C)Muscle MTs upon mock treatment with
DMSO solvent for 4 hours. Note that the MTs in mock-treated cells of
WT (A) and tbce overexpression (B) flies were consistently less dense
than in their untreated counterparts (compare with Fig. 8A,B). (Aa-Ca)
MTs in muscle cells treated with nocodazole with no washout. Ab-Cb,
Ac-Cc and Ad-Cd show MTs after nocodazole washout for 2, 5 and 20
minutes, respectively. Near complete recovery of MTs by 5 minutes after
washout was observed in the WT (Ac), but only weak recovery of MTs
was observed in tbce knockdown flies (Cc). Even after 20 minutes of
washout, the recovery was still not complete in tbce knockdown flies
(Cd). MTs are labeled with anti-α-tubulin (green); nuclei are stained
with PI (red). Scale bar: 10μm.
TBCE promotes MT formation
Although Drosophila possesses a TBCE ortholog, no previous
studies of it have been reported. Our work shows for the first time
that tbce is essential for early neuromuscular development in
Drosophila(Fig. 3). We also provide in vivoevidence demonstrating
that Drosophila TBCE is both required and sufficient for MT
formation (Figs 7-9), supporting early in vitro biochemical studies
thatshowed that TBCE assists in α-β-tubulin heterodimer formation
(Tian et al., 1996; Tian et al., 1997).
We found that overexpression of tbce produced increased MTs
(Figs 7 and 8). To our knowledge, this is the first report of increased
MT formation when a tubulin chaperone is overexpressed, and is
contrary to reports in other systems. Overexpression of human
TBCE in cultured cells leads to complete disruption of MTs
(Bhamidipati et al., 2000; Sellin et al., 2008; Tian et al., 2006), as
does overexpression of a TBCE-like protein (Bartolini et al., 2005;
Keller and Lauring, 2005; Sellin et al., 2008). It was further
hypothesized that the UBL domains present in TBCE and the
TBCE-like protein might contribute to the degradation of tubulin via
the proteasomal pathway (Bartonili et al., 2005). In addition, the
overexpression of other tubulin chaperones, such as TBCD, results
in a similar disruption of MTs (Bhamidipati et al., 2000; Martin et
al., 2000). These in vivo data are consistent with the early in vitro
observation that TBCD or TBCE in excess destroys tubulin
heterodimers by sequestering the bound tubulin subunit, leading to
the destabilization of the freed partner subunit (Tian et al., 1997). It
is thus believed that in addition to assisting in the folding pathway,
TBCE also interacts with native tubulins to disrupt α-β-tubulin
heterodimers (Bhamidipati et al., 2000). The discrepancy between
our overexpression result and the findings of others could have
several explanations. First, the use of different experimental
systems: transgenic animals in this work and cultured cells in other
studies (Bhamidipati et al., 2000; Sellin et al., 2008; Tian et al.,
2006). Second, different systems might have different expression
levels of tbce, leading to varying effects on MTs. Third, Drosophila
and human TBCE might have diverged functions. Further analyses
are needed to reconcile the conflicts in the effects of TBCE
overexpression in thesedifferent systems. In general, however, tbce
mutant phenotypes are consistent in all species examined so far,
from yeast to human, indicating that the function of TBCE in
promoting MT formation has been well-conserved throughout
We thank K. Broadie for transgenic UAS-spastin flies; N. Sherwood for
spastin5.75; V. Budnik for C57-Gal4; the Bloomington Stock Center for fly
stocks; the Developmental Studies Hybridoma Bank, University of Iowa, for
antibodies; the TILLING service at Seattle for providing the nonsense mutation
Z0241; Dr S. Y. Wang for assistance in characterizing transgenic flies; Dr F.
Huang for advice on physiological assays; and Drs Z. H. Wang, C. L. Yang, X.
Huang and N. Sherwood for critical reading of the manuscript. Fly transgenic
work was carried out in Dr Z. H. Wang’s laboratory. This work is supported by
a grant from the National Science Foundation of China (NSFC) to S.J.
(30871368), and grants from NSFC (30430250, 30525015), the Ministry of
Science and Technology of China (2006AA02Z166, 2007CB947200) and the
Chinese Academy of Sciences (KSCX1-YW-R-69) to Y.Q.Z.
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TBCE functions at synapses and promotes microtubule formation