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: firstname.lastname@example.org)
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
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TBCE functions at synapses and promotes microtubule formation