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First published online 18 March 2009
doi: 10.1242/dev.029983

Development 136, 1571-1581 (2009)
Published by The Company of Biologists 2009
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Drosophila Tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation

Shan Jin1,2, Luyuan Pan1, Zhihua Liu1, Qifu Wang1, Zhiheng Xu1 and Yong Q. Zhang1,*

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.


Figure 1
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  		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., 2005Go). 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 {alpha}-tubulin expression was detected when tbce expression was altered. Actin was used as a loading control.

 

Figure 2
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  		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: 20 µm.

 

Figure 3
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  		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-{alpha}-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-FASII, 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 bars: 10 µm.

 

Figure 4
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  		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 larvae tested for each genotype is indicated. **P<0.01, ***P<0.001; error bars indicate s.e.m.

 

Figure 5
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  		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: 5 µm. (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.

 

Figure 6
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  		Fig. 6. Altering the presynaptic expression of tbce causes increased neurotransmission. (A-E) Representative 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 indicate s.e.m.

 

Figure 7
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  		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-{alpha}-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 indicate terminal boutons with weak or no Futsch staining. Scale bars: 10 µm. (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 indicate s.e.m.

 

Figure 8
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  		Fig. 8. tbce antagonizes MT-severing spastin. (A-H') Drosophila larval muscles were stained with anti-{alpha}-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: 10 µm.

 

Figure 9
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  		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-{alpha}-tubulin (green); nuclei are stained with PI (red). Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2009