The microtubule-based cytoskeleton is a component of a mechanical signaling pathway in fly campaniform receptors

Abstract

In mechanoreceptors, mechanical stimulation by external forces leads to the rapid opening of transduction channels followed by an electrical response. Despite intensive studies in various model systems, the molecular pathway by which forces are transmitted to the transduction channels remains elusive. In fly campaniform mechanoreceptors, the mechanotransduction channels are gated by compressive forces conveyed via two rows of microtubules that are hypothesized to be mechanically reinforced by an intervening electron-dense material (EDM). In this study, we tested this hypothesis by studying a mutant fly in which the EDM was nearly absent, whereas the other ultrastructural elements in the mechanosensitive organelle were still present at 50% (or greater) of normal levels. We found that the mechanosensory response in this mutant was reduced by 90% and the sensitivity by at least 80%. To test whether loss of the EDM could lead to such a reduction in response, we performed a mechanical analysis and estimated that the loss of the EDM is expected to greatly decrease the overall rigidity, leading to a marked reduction in the gating force conveyed to the channel. We argue that this reduction in force, rather than the reduction in the number of transduction channels, is primarily responsible for the nearly complete loss of mechanosensory response observed in the mutant fly. Based on these experiments and analysis, we conclude that the microtubule-based cytoskeleton (i.e., microtubules and EDM) is an essential component of the mechanical signaling pathway in fly campaniform mechanoreceptor.

Figure 1

Model of the mechanical signaling pathway in the fly campaniform mechanoreceptor. (Left) A schematic of seven linked structures in the mechanoreceptive organelle of fly campaniform receptors (6). The MT and EDMs together are termed the MT-based cytoskeleton. (Right) The transduction channels are activated by the compressive force conveyed by the MTs via the MMCs. External forces are indicated by the gray arrows.

Figure 2

Electrophysiological responses of campaniform mechanoreceptors in wild-type and mutant flies. (A) An image of a fly haltere. The mechanical probe (red) was positioned in the middle of the receptor field in the pedicel (green bracket). The cuticular structures that overlay the campaniform receptors are visible in the pedicel. This image of the haltere is used to indicate the position of the stimulation probe; the actual recording was performed on intact living flies immobilized on the stage. (B) Representative recording traces from a wild-type fly in response to different mechanical stimuli (uppermost curves). Typical response traces to five cycles of stimulation are shown at left. At right, the responses to 50 cycles are aligned with the stimulation cycles and overlaid (gray). The average trace is also shown (red). Note that the individual responses to 15-μm stimuli lose synchronicity with the stimulation (the gray lines do not overlap with each other as they do for 3- to 9-μm stimuli). (C) The distribution of the peak response amplitudes to 150 6-μm stimuli. An example of a response to 50 stimuli is shown above. The broad distribution of response amplitudes can be fit with a multipeaked distribution. (D) Representative recording traces from a mutant fly in response to different mechanical stimuli (note that the scale of the response traces differs from that in B). Typical recording traces of five cycles are shown at left. At right, the responses to 50 cycles are overlaid (gray) and the average trace is shown (red). (E) The distribution of the amplitudes of responses to 15-μm stimuli in a mutant receptor was similar to that to 3-μm stimuli in a wild-type receptor. The histograms have single peaks at P_f02655 = 0.060 mV and P_WT = 0.057 mV. The representative recording traces for 100 ms (10 cycles) are shown above the histograms. Note that wild-type receptors showed multiple spikes, whereas mutant receptors showed only a single spike. (F) The average peak response amplitudes from campaniform receptors in f02655 mutant flies (triangles) were significantly smaller than those in wild-type flies (circles). Data are represented as the mean ± SE (n = 4–6 flies/data point).

Figure 3

Characterization of the f02655 strain. (A) The wild-type (left lane) and f02655 mutant (right lane) transcripts of dcx-emap gene were amplified using RT-PCR. A longer transcript (3604 bp) was detected in the f02655 strain. Note that in the f02655 sample, no wild-type transcript was detected. (B) Protein domain organization in the wild-type (upper) and f02655 mutant DCX-EMAP (lower). The additional sequence from the piggyBAC insertion was predicted to introduce seven amino acids (black arrowhead) after cysteine 532 in the DCX-EMAP protein sequence. An in-frame stop codon after the sequence coding the seven amino acids was predicted to lead to the prematuration of the DCX-EMAP protein, which would only contain the doublecortin and HELP domains, but not the WD40 domain (lower). The blue line (upper) indicates the fragment of DCX-EMAP (amino acids 258–461, 204 amino acids) used as the antigen to generate the DCX-EMAP antibody. (C) In the campaniform mechanoreceptors of the wild-type flies, the DCX-EMAP antibody specifically stained the distal part of the dendrites (left), including the distal tip (upper right) and the tubular body (lower right). The campaniform mechanoreceptors shown at lower right are located in the pedicel of a wild-type haltere (Fig. 2A). (D) In the f02655 strain, the immunofluorescence signal of DCX-EMAP was absent in the distal tips and tubular bodies of the campaniform mechanoreceptors. Some residual signals were observed in some cells (white arrows). Note that the tubulin signal was overexposed to visualize the tubular bodies of the campaniform receptors. The campaniform mechanoreceptors shown are located in the pedicel of an f02655 mutant haltere. (E) The ultrastructure of the distal-tip region in cross section in wild-type (upper panel) and f02655 mutant cells (lower). (F) Magnified images of the cross sections outlined in red in E and their corresponding schematics indicate that there are ultrastructural differences between wild-type cells (left) and mutant cells (right). The EDM was nearly absent in the mutant cells (lower left), whereas the other structural elements were present. Abbreviations: s, sheath; SMC, sheath membrane connector; m, membrane; MMC, membrane MT connector; MT, microtubule; EDM, electron dense material. (G) NompC (red) localization in the campaniform receptors (green) of the f02655 strain. (Inset) NompC localization in the wild-type campaniform receptors. Note that only the distal parts of the campaniform receptors are shown. Scale bar, 2 μm.

Figure 4

Mechanical analysis. (A) A simple mechanical gating model showed that the transduction channel and the mechanical links (C_t) are connected in series (left). In response to the compression stimulus, D, a gating force (F_g) is generated to deflect the channel gate (d), which leads to the opening of the channel. (B) In wild-type cells, the EDM prevents the MTs from approaching each other (left), so most of the deformation D is taken up by the MMCs and the membrane (middle). In f02655 mutant cells, the absence of the EDM allows the two rows of MTs to move toward each other, thereby taking up some of the deformation, D. The additional displacement of the MTs increases the compliance of the links, which would lead to a smaller gating force and a weaker electrical response.