Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion

Abstract

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs.

Keywords: locomotion; mechanosensation; proprioception.

Fig. S1.

Protein sequence alignment between representative members of the TMC family, including human TMC2 (NP_542789), mouse TMC1 (NP_083229), Xenopus TMC1 (predicted, XP_002935639.1), zebrafish TMC1 (AIK19895.1), C. elegans TMC1 (NP_508221), and Drosophila TMC (AFH04369). Blue bars above the sequences indicate predicted transmembrane segments. Green shading indicates the predicted TMC domains. Transmembrane and TMC domains are largely conserved. Drosophila TMC is much longer than that in other TMC family members.

Fig. S1.

Protein sequence alignment between representative members of the TMC family, including human TMC2 (NP_542789), mouse TMC1 (NP_083229), Xenopus TMC1 (predicted, XP_002935639.1), zebrafish TMC1 (AIK19895.1), C. elegans TMC1 (NP_508221), and Drosophila TMC (AFH04369). Blue bars above the sequences indicate predicted transmembrane segments. Green shading indicates the predicted TMC domains. Transmembrane and TMC domains are largely conserved. Drosophila TMC is much longer than that in other TMC family members.

Fig. 1.

Generation of tmc reporter and mutant alleles. (A) Targeting schemes for the generation of the tmc^Gal4 mutant allele by homologous recombination and the generation of the tmc-Gal4 transgene. Black bars under CG46121-PD indicate the predicted transmembrane segments of Drosophila TMC (33). Red Bars above CG46121-PD indicate the predicted TMC domain (32). (B) Confirmation of the tmc^Gal4 mutation as a null allele for transcript expression by RT-PCR.

Fig. 2.

Expression pattern of tmc. (A) Labeling via tmc-Gal4 and endogenous expression of Drosophila TMC in class I and class II da neurons and bd neurons. (B) Labeling via tmc^Gal4 revealing the loss of expression of Drosophila TMC in class I and class II da neurons and bd neurons. (C) Restoration of Drosophila TMC expression in class I and class II da neurons and bd neurons by expressing Drosophila TMC. Arrowheads: bd neuron. Triangle: class I da neuron. Arrows: class II da neuron. Green indicates GFP signals, and red indicates TMC immunofluorescent signals. (A, Left) “D” indicates dorsal, and “P” indicates posterior. (Scale bar, 50 μm.)

Fig. S2.

tmc-Gal4 expression in larvae and adult. tmc-Gal4 labels class I and class II da neurons and bd neurons. Arrowhead: bd neuron. Triangle: class I da neuron. Arrow: class II da neurons. There was no detectable expression of tmc-Gal4 in the larval CNS. In the adult fly, tmc-Gal4 labels sensory neurons in the mouth, wing, haltere, arista, the third segment of the antenna, and the Johnston organ.

Fig. S3.

Validation of the TMC antibody. (A) Immunostaining of Drosophila TMC ectopically expressed in the fly brain. Drosophila TMC was expressed in excitatory cholinergic neurons via Cha-Gal4. Fly genotype: Cha-Gal4, UAS-GFP; UAS-tmc. (Inset) Zoom-in of the cell-body region documenting enrichment of TMC around the cell membrane. (B) Immunostaining of TMC ectopically expressed in Drosophila S2R+ cells. Cells were transfected with TMC (Lower) or without TMC (Upper).

Fig. 3.

tmc is important for Drosophila larval locomotion. (A) Crawling trajectories of wild-type w¹¹¹⁸ larvae, tmc^Gal4 mutants, UAS-tmc controls, and UAS-tmc; tmc^Gal4 larvae. (Scale bar, 1 cm.) Restoring tmc expression rescued the locomotion defects in locomotion speed (B), head curl duration in 90 s (C), and backward wave numbers (D) in 90 s. One-way ANOVA followed by Tukey’s HSD post hoc test was used to test for the statistical significance of the differences between wild-type w¹¹¹⁸, tmc^Gal4, UAS-tmc, and rescue (UAS-tmc; tmc^Gal4) larvae. ***P < 0.001. (E) Head curl duration (in 90 s) and (F) backward wave numbers (in 90 s) are comparable between tmc^Gal4 and Df(3L)BSC576/tmc^Gal4 larvae, with significant increase compared with wild-type control. One-way ANOVA was used to test for the statistical significance of the differences between tmc^Gal4, UAS-tmc, and rescue (UAS-tmc; tmc^Gal4) larvae, followed by Tukey’s HSD post hoc test. ***P < 0.001. NS, not significant. (G) Head curl duration in 90 s and (H) backward wave numbers in 90 s of tmc-Gal4, UAS-tmc-RNAi, and tmc knockdown (UAS-tmc-RNAi; tmc-Gal4) larvae. One-way ANOVA was used to test for the statistical significance of the differences between tmc-Gal4, UAS-tmc-RNAi, and tmc knockdown (UAS-tmc-RNAi; tmc-Gal4) larvae, followed by Tukey’s HSD post hoc test. *P < 0.05. NS, not significant. n > 10.

Fig. 4.

Mechanosensitive calcium responses in the axon terminals of TMC-expressing neurons. (A) Experimental setup for imaging bending-evoked calcium signals in the axon terminals of TMC-expressing neurons. Bending was evoked using a glass probe. (B) Abdominal bending-evoked calcium signals in wild-type larvae and tmc mutant larvae. There is a GCaMP signal background difference between tmc-Gal4 and tmc^Gal4 due to the different expression levels between them. (C) Statistical analysis of the calcium responses in wild-type and tmc mutant larvae. Two-tailed unpaired Student’s t test was used to test the difference between wild-type w¹¹¹⁸ and tmc^Gal4. ***P < 0.001. n ≥ 9.

Fig. S4.

Dendrite morphology of class I da neurons and axon projection of tmc-positive neurons in ventral nerve cord. Neither (A) dendrite morphology of class I da neurons nor (B) axon projection of tmc-positive neurons in ventral nerve cord shows obvious defects in tmc mutant or tmc rescue flies.

Fig. S5.

Example traces of electrophysiological recordings from cultured S2 cells transfected with NOMPC or Drosophila TMC, documenting responses to mechanical stimuli in the former but not the latter.

Fig. 5.

Mammalian TMC1 and TMC2 rescue locomotion defects in tmc mutants. (A) Head curl duration (in 90 s) and (B) backward wave numbers (in 90 s) of wild-type, UAS-mtmc2; tmc^Gal4, UAS-mtmc1/UAS-mtmc2; tmc^Gal4, and UAS-mtmc1; tmc^Gal4 larvae are significantly less than those of tmc^Gal4 larvae. One-way ANOVA was used to test for the statistical significance of the differences, followed by Tukey’s HSD post hoc test. ***P < 0.001. NS, not significant. n ≥ 8.