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First published online 18 March 2009
doi: 10.1242/dev.029983
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1 Key Laboratory of Molecular and Developmental Biology, Institute of Genetics
and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,
China.
2 College of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
* Author for correspondence (e-mail: yqzhang{at}genetics.ac.cn)
Accepted 19 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, HRD, Spastin, TBCE (CG7861), Tubulin chaperone
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
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
- and one β-tubulin polypeptide. The formation of -β
tubulin 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 -tubulin
(Lewis et al., 1997;
Tian et al., 2006).
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 -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 systems.
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 HRD 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 MT formation when the 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 it promotes MT formation in vivo.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
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-spastin line was from K. Broadie
(Trotta et al., 2004). The
chemical mutagen ethyl methanesulfonate (EMS)-induced nonsense mutation
Z0241 in 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
KG09112 from Bloomington has a P element insertion in the
intergenic region between CG14591 and tbce
(Fig. 1B). Before mobilizing
KG09112 as mediated by P transposase 
2-3, 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
Drosophila Genomics Resource Center
(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 the tbce second
exon was fused to the corresponding genomic sequence plus intron 2 as a spacer
(see Fig. 1B) to make a hairpin
RNAi construct targeting nucleotides 337-842 of GenBank sequence NM_136353. An
independent RNAi line targeting a different sequence (nucleotides 919-1280 of
NM_136353) of tbce was obtained from the Vienna Stock Center.
Although multiple independent lines of transgenic flies carrying UAS-TBCE or
RNAi were generated, here we report on UAS-TBCE and UAS-RNAi insertions on the
third chromosome with 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.
Behavioral analyses
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 took to 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 2x loading
buffer. Half a fillet was used for each loading. Primary antibodies used were
anti-TBCE (1:200), anti--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, Amersham).
Whole-mount embryos were fixed and stained with anti-FASII [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--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. Nuclei were visualized 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 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.
Physiological assays
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 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.
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Statistical analyses
All statistical comparisons were performed using GraphPad InStat 5
software. P-values were calculated by two-tailed Student's
t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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As a first step to understanding the in vivo functions 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
assessed by 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, tbce is essential for viability, as the
independent mutations Z0241 and 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 monoclonal antibody, 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 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), confirming
the specificity of the antibody. The expression of TBCE decreased as 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 epidermal cells
(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).
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-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 maintenance.
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-FASII, 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-FASII 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 antibody showed 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 tbce nulls suggest that TBCE is required for axonal
growth in Drosophila.
To understand the physiological functions of TBCE, we performed behavioral assays. As tbce nulls are embryonic lethal, we examined the behavior of larvae in which TBCE expression had been genetically 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, 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. Drosophila
NMJ synapses are a commonly used system to examine protein function at
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 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.
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TBCE regulates neurotransmission at NMJ synapses
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 which TBCE
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 mEJP by 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 (an increase of 69%,
P<0.05) (Fig. 6H).
Presynaptic knockdown of tbce caused a significant increase in mEJP
frequency, 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 tbce on 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.
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). 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 -tubulin, Futsch (the fly
ortholog of mammalian MAP1B; MTAP1B) and 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--tubulin staining (Fig.
7B-B'). Overexpression of tbce, however, led to
smooth and continuous -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
-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.
TBCE antagonizes Spastin to regulate MT formation
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 MTs when 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).
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-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 two.
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. 9Aa with
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 9Aa-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 formation.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Zhang et al.,
2001; Zhang and Broadie,
2005), the most common form of inherited mental retardation.
Fragile X mental retardation protein (FMRP, encoded by 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 (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.
|
). 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 of increased 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 tbce is
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 tbce on
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 Budnik,
2006).
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 tbce had 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.
TBCE antagonizes Spastin in regulating MT dynamics
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.
|
|
;
Trotta et al., 2004) and in
cultured cells (Errico et al.,
2002; Roll-Mecak and Vale,
2005) caused dramatically 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 reported in 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 appearance in 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, tbce nulls
are embryonic lethal, whereas knockdown of tbce leads to a
dramatically reduced MT network in Drosophila neuromusculature (Figs
7,
8,
9). Thus, in contrast to the
nuanced role of endogenous Spastin, TBCE plays a crucial role in MT
formation.
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
vivo evidence demonstrating that Drosophila TBCE is both required and
sufficient for MT formation (Figs
7,
8,
9), supporting early in vitro
biochemical studies that showed that TBCE assists in -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 -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 these different 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
evolution.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/dev.029983/DC1
|
ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Bartolini, F., Tian, G., Piehl, M., Cassimeris, L., Lewis, S. A.
and Cowan, N. J. (2005). Identification of a novel
tubulin-destabilizing protein related to the chaperone cofactor E.
J. Cell Sci. 118,1197
-1207.
Bhamidipati, A., Lewis, S. A. and Cowan, N. J.
(2000). ADP ribosylation factor-like protein 2 (Arl2) regulates
the interaction of tubulin-folding cofactor D with native tubulin.
J. Cell Biol. 149,1087
-1096.
Bodily, K. D., Morrison, C. M., Renden, R. B. and Broadie,
K. (2001). A novel member of the Ig superfamily, turtle, is a
CNS-specific protein required for coordinated motor control. J.
Neurosci. 21,3113
-3125.
Bommel, H., Xie, G., Rossoll, W., Wiese, S., Jablonka, S.,
Boehm, T. and Sendtner, M. (2002). Missense mutation in the
tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor
neuronopathy, a model of human motoneuron disease. J. Cell
Biol. 159,563
-569.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.[Abstract]
Budnik, V., Koh, Y. H., Guan, B., Hartmann, B., Hough, C.,
Woods, D. and Gorczyca, M. (1996). Regulation of synapse
structure and function by the Drosophila tumor suppressor gene dlg.
Neuron 17,627
-640.[CrossRef][Medline]
Budnik, V., Gorczyca, M. and Prokop, A. (2006).
Selected methods for the anatomical study of Drosophila embryonic and larval
neuromuscular junctions. In The Fly Neuromuscular Junction:
Structure and Function, 2nd edn (ed. V. Budnik and C.
Ruiz-Canada), pp. 323-365. New York: Academic
Press.
Errico, A., Ballabio, A. and Rugarli, E. I.
(2002). Spastin, the protein mutated in autosomal dominant
hereditary spastic paraplegia, is involved in microtubule dynamics.
Hum. Mol. Genet. 11,153
-163.
Feng, Y., Ueda, A. and Wu, C. F. (2004). A
modified minimal hemolymph-like solution, HL3.1, for physiological recordings
at the neuromuscular junctions of normal and mutant Drosophila larvae.
J. Neurogenet. 18,377
-402.[CrossRef][Medline]
Grishchuk, E. L. and McIntosh, J. R. (1999).
Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an
essential role in mitotic microtubule assembly. J. Cell
Sci. 112,1979
-1988.[Abstract]
Grynberg, M., Jaroszewski, L. and Godzik, A.
(2003). Domain analysis of the tubulin cofactor system: a model
for tubulin folding and dimerization. BMC
Bioinformatics 4,46
.[CrossRef][Medline]
Hoyt, M. A., Macke, J. P., Roberts, B. T. and Geiser, J. R.
(1997). Saccharomyces cerevisiae PAC2 functions with CIN1, 2 and
4 in a pathway leading to normal microtubule stability.
Genetics 146,849
-857.[Abstract]
Iacoangeli, A., Rozhdestvensky, T. S., Dolzhanskaya, N.,
Tournier, B., Schutt, J., Brosius, J., Denman, R. B., Khandjian, E. W.,
Kindler, S. and Tiedge, H. (2008). On BC1 RNA and the fragile
X mental retardation protein. Proc. Natl. Acad. Sci.
USA 105,734
-739.
Jan, L. Y. and Jan, Y. N. (1976). Properties of
the larval neuromuscular junction in Drosophila melanogaster. J.
Physiol. 262,189
-214.
Kalidas, S. and Smith, D. P. (2002). Novel
genomic cDNA hybrids produce effective RNA interference in adult Drosophila.
Neuron 33,177
-184.[CrossRef][Medline]
Keller, C. E. and Lauring, B. P. (2005).
Possible regulation of microtubules through destabilization of tubulin.
Trends Cell Biol. 15,571
-573.[CrossRef][Medline]
Lewis, S. A. and Cowan, N. J. (2002). Bad
chaperone. Nat. Med. 8,1202
-1203.[CrossRef][Medline]
Lewis, S. A., Tian, G. and Cowan, N. J. (1997).
The alpha- and beta-tubulin folding pathways. Trends Cell
Biol. 7,479
-484.[CrossRef][Medline]
Lu, R., Wang, H., Liang, Z., Ku, L., O'Donnell, W. T., Li, W.,
Warren, S. T. and Feng, Y. (2004). The fragile X protein
controls microtubule-associated protein 1B translation and microtubule
stability in brain neuron development. Proc. Natl. Acad. Sci.
USA 101,15201
-15206.
Martin, A. R. (1955). A further study of the
statistical composition on the end-plate potential. J.
Physiol. 130,114
-122.
Martin, L., Fanarraga, M. L., Aloria, K. and Zabala, J. C.
(2000). Tubulin folding cofactor D is a microtubule destabilizing
protein. FEBS Lett. 470,93
-95.[CrossRef][Medline]
Martin, N., Jaubert, J., Gounon, P., Salido, E., Haase, G.,
Szatanik, M. and Guenet, J. L. (2002). A missense mutation in
Tbce causes progressive motor neuronopathy in mice. Nat.
Genet. 32,443
-447.[CrossRef][Medline]
Molon, A., Di Giovanni, S., Chen, Y. W., Clarkson, P. M.,
Angelini, C., Pegoraro, E. and Hoffman, E. P. (2004).
Large-scale disruption of microtubule pathways in morphologically normal human
spastin muscle. Neurology
62,1097
-1104.
Nogales, E. (2000). Structural insights into
microtubule function. Annu. Rev. Biochem.
69,277
-302.[CrossRef][Medline]
Packard, M., Koo, E. S., Gorczyca, M., Sharpe, J., Cumberledge,
S. and Budnik, V. (2002). The Drosophila Wnt, wingless,
provides an essential signal for pre- and postsynaptic differentiation.
Cell 111,319
-330.[CrossRef][Medline]
Pan, L., Woodruff, E., 3rd, Liang, P. and Broadie, K.
(2008). Mechanistic relationships between Drosophila fragile X
mental retardation protein and metabotropic glutamate receptor A signaling.
Mol. Cell. Neurosci. 37,747
-760.[CrossRef][Medline]
Parvari, R., Hershkovitz, E., Grossman, N., Gorodischer, R.,
Loeys, B., Zecic, A., Mortier, G., Gregory, S., Sharony, R., Kambouris, M. et
al. (2002). Mutation of TBCE causes
hypoparathyroidism-retardation-dysmorphism and autosomal recessive
Kenny-Caffey syndrome. Nat. Genet.
32,448
-452.[CrossRef][Medline]
Penagarikano, O., Mulle, J. G. and Warren, S. T.
(2007). The pathophysiology of fragile x syndrome.
Annu. Rev. Genomics Hum. Genet.
8, 109-129.[CrossRef][Medline]
Radcliffe, P. A., Hirata, D., Vardy, L. and Toda, T.
(1999). Functional dissection and hierarchy of tubulin-folding
cofactor homologues in fission yeast. Mol. Biol. Cell
10,2987
-3001.
Reeve, S. P., Lin, X., Sahin, B. H., Jiang, F., Yao, A., Liu,
Z., Zhi, H., Broadie, K., Li, W., Giangrande, A. et al.
(2008). Mutational analysis establishes a critical role for the N
terminus of fragile X mental retardation protein FMRP. J.
Neurosci. 28,3221
-3226.
Reid, E. (1997). Pure hereditary spastic
paraplegia. J. Med. Genet.
34,499
-503.
Roll-Mecak, A. and Vale, R. D. (2005). The
Drosophila homologue of the hereditary spastic paraplegia protein, spastin,
severs and disassembles microtubules. Curr. Biol.
15,650
-655.[CrossRef][Medline]
Roos, J., Hummel, T., Ng, N., Klambt, C. and Davis, G. W.
(2000). Drosophila Futsch regulates synaptic microtubule
organization and is necessary for synaptic growth.
Neuron 26,371
-382.[CrossRef][Medline]
Ruiz-Canada, C. and Budnik, V. (2006). Synaptic
cytoskeleton at the neuromuscular junction. Int. Rev.
Neurobiol. 75,217
-236.[Medline]
Schaefer, M. K., Schmalbruch, H., Buhler, E., Lopez, C., Martin,
N., Guenet, J. L. and Haase, G. (2007). Progressive motor
neuronopathy: a critical role of the tubulin chaperone TBCE in axonal tubulin
routing from the Golgi apparatus. J. Neurosci.
27,8779
-8789.
Sellin, M. E., Holmfeldt, P., Stenmark, S. and Gullberg, M.
(2008). Op18/Stathmin counteracts the activity of overexpressed
tubulin-disrupting proteins in a human leukemia cell line. Exp.
Cell Res. 314,1367
-1377.[CrossRef][Medline]
Sherwood, N. T., Sun, Q., Xue, M., Zhang, B. and Zinn, K.
(2004). Drosophila spastin regulates synaptic microtubule
networks and is required for normal motor function. PLoS
Biol. 2,e429
.[CrossRef][Medline]
Steinborn, K., Maulbetsch, C., Priester, B., Trautmann, S.,
Pacher, T., Geiges, B., Kuttner, F., Lepiniec, L., Stierhof, Y. D., Schwarz,
H. et al. (2002). The Arabidopsis PILZ group genes encode
tubulin-folding cofactor orthologs required for cell division but not cell
growth. Genes Dev. 16,959
-971.
Tarrade, A., Fassier, C., Courageot, S., Charvin, D., Vitte, J.,
Peris, L., Thorel, A., Mouisel, E., Fonknechten, N., Roblot, N. et al.
(2006). A mutation of spastin is responsible for swellings and
impairment of transport in a region of axon characterized by changes in
microtubule composition. Hum. Mol. Genet.
15,3544
-3558.
Tian, G., Huang, Y., Rommelaere, H., Vandekerckhove, J., Ampe,
C. and Cowan, N. J. (1996). Pathway leading to correctly
folded beta-tubulin. Cell
86,287
-296.[CrossRef][Medline]
Tian, G., Lewis, S. A., Feierbach, B., Stearns, T., Rommelaere,
H., Ampe, C. and Cowan, N. J. (1997). Tubulin subunits exist
in an activated conformational state generated and maintained by protein
cofactors. J. Cell Biol.
138,821
-832.
Tian, G., Huang, M. C., Parvari, R., Diaz, G. A. and Cowan, N.
J. (2006). Cryptic out-of-frame translational initiation of
TBCE rescues tubulin formation in compound heterozygous HRD. Proc.
Natl. Acad. Sci. USA 103,13491
-13496.
Trotta, N., Orso, G., Rossetto, M. G., Daga, A. and Broadie,
K. (2004). The hereditary spastic paraplegia gene, spastin,
regulates microtubule stability to modulate synaptic structure and function.
Curr. Biol. 14,1135
-1147.[CrossRef][Medline]
Zhang, Y. Q. and Broadie, K. (2005). Fathoming
fragile X in fruit flies. Trends Genet.
21, 37-45.[CrossRef][Medline]
Zhang, Y. Q., Bailey, A. M., Matthies, H. J., Renden, R. B.,
Smith, M. A., Speese, S. D., Rubin, G. M. and Broadie, K.
(2001). Drosophila fragile X-related gene regulates the MAP1B
homolog Futsch to control synaptic structure and function.
Cell 107,591
-603.[CrossRef][Medline]
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22).
*P<0.05, **P<0.01,
***P<0.001; error bars indicate s.e.m.
