InteractiveFly: GeneBrief

Doublecortin-domain-containing echinoderm-microtubule-associated protein: Biological Overview | References

Mechanoreceptor cells develop specialized mechanosensory organelles (MOs), where force-sensitive channels and supporting structures are organized in an orderly manner to detect forces. It is intriguing how MOs are formed. This issue was addressed by studying the MOs of fly ciliated mechanoreceptors. The main structure of the MOs is shown to be a compound cytoskeleton formed of short microtubules and electron-dense materials (EDMs). In a knock-out mutant of microtubule associated protein Doublecortin-domain-containing echinoderm-microtubule-associated protein (DCX-EMAP), this cytoskeleton is nearly absent, suggesting that DCX-EMAP is required for the formation of the MOs and in turn fly mechanotransduction. Further analysis reveals that DCX-EMAP expresses in fly ciliated mechanoreceptors and localizes to the MOs. Moreover, it plays dual roles by promoting the assembly/stabilization of the microtubules and the accumulation of the EDMs in the MOs. Therefore, DCX-EMAP serves as a core ultrastructural organizer of the MOs, and this finding provides novel molecular insights as to how fly MOs are formed (Song, 2023).

Mechanoreceptor cells start the neural pathway of mechanosensation by converting physical stimuli (e.g., force or deformation) into cellular signals. To accomplish this task, they develop specialized mechanosensory organelles (MOs), which are structural-mechanical entities that consist of force-sensitive channels and supporting components, such as the cytoskeleton and extracellular matrix. While force-sensitive ion channels are key signal transducers, supporting components were thought to refine the sensory features of mechanoreceptor cells (e.g., sensitivity and dynamic range). For example, vertebrate inner ear hair cells grow stereocilia that contain intracellular actin bundles and extracellular tip-links, which serve to match the mechanical impedance when stereocilia deflection is converted into a conformational change of the mechanotransduction channels. The touch receptors of C. elegans form a specialized sensory complex containing the extracellular matrix (e.g., MEC-5), membrane channels (MEC-4 and MEC-10), and 15-protofilament microtubules (MEC-7 and MEC-12) to sense tactile signals. Recent studies suggest that Piezo, a force-sensitive channel that contributes to the perception of various mechanical stimuli, may be tethered to and regulated by F-actin in cells (Wang, 2022). Therefore, MOs are structurally specialized to match the sensory modality of the mechanoreceptors. This raises the question of how the MOs are formed (Song, 2023).

The MOs of Drosophila ciliated mechanoreceptors (i.e., type I mechanoreceptors) have been extensively studied to understand the structural basis of mechanotransduction. Early ultrastructural analysis showed that the main intracellular structure of the MOs is a compound cytoskeletal structure consisting of arrayed microtubules and electron-dense materials (EDMs). Later, it was shown that NompC force-sensitive channels are structurally linked to the microtubules and also formed into arrays in the MO membrane. These observations suggest that the entire MO acts as an integrated mechanosensor with a nanoscopic size. In recent work, it was revealed that short microtubules are required for the formation of the MOs and that the 'kat-60L1/Patronin' module is required to generate the short microtubules (Sun, 2021). An ensuing question is how these short microtubules are stabilized and organized in the MOs (Song, 2023).

Previous work showed that knocking down the expression level of Patronin, a microtubule minus-end-stabilizing protein, reduces the amount of the MO microtubules (Sun, 2021), suggesting that Patronin stabilizes the MO microtubules. However, this study also found that in addition to Patronin, there is a MO-specific microtubule-stabilizing mechanism (Sun, 2021). A previous study showed that DCX-EMAP, a doublecortin (DCX) domain-containing protein, is highly enriched in fly haltere tissue based on the DNA array analysis and that a piggyBac insertion mutant of DCX-EMAP, which may express a truncated protein (Liang, 2014), contains a disrupted microtubule array in the outer segment of campaniform mechanoreceptors. This implies that DCX-EMAP may be key for the assembly or stability of microtubules inside the mechanosensory cilia. However, due to the lack of further genetic and cell biological analysis, the cellular functions and biochemical mechanism of DCX-EMAP remain elusive, especially in the context of the recently resolved structure of the mechanosensory cilia (Song, 2023).

DCX-EMAP belongs to the EMAP (echinoderm-microtubule-associated proteins) family, the members of which are regulators for microtubule dynamics. All EMAP proteins share a conserved TAPE (tandem atypical propeller in EMLs) domain at the carboxyl-terminal, including the HELP (hydrophobic echinoderm-microtubule-associated-like protein) domain and multiple WD40 repeats. The amino-terminal part of EMAP proteins often contains a coiled-coil domain that binds to microtubules through trimerization. Among all EMAP family members, DCX-EMAP is unique because the coiled-coil domain is replaced by a tandem of two DCX domains (Bechstedt, 2010), which has a microtubule-binding/stabilizing activity. It was found that the DCX-domain-containing (DCDC) proteins express in a wide range of cells and show diverse cellular functions. Therefore, it is intriguing to understand how DCX-EMAP couples the functions of the DCX and EMAP families in a specific cellular process, such as the formation of a modified cilium (Song, 2023).

This work studied the formation of fly MOs by analyzing the cellular functions of DCX-EMAP, an essential molecule for fly mechanosensation. The results show that DCX-EMAP specifically expresses in fly mechanosensory cilia and acts as a core organizer for the ultrastructure of the MOs, thereby having a direct contribution to fly mechanotransduction. These findings help take an important step forward in understanding how fly MOs are formed. Additionally, this study provides implications to understand the cellular roles of the DCDC and EMAP family members in ciliary assembly and maintenance (Song, 2023).

This study resolved the 3D ultrastructural organization of the compound cytoskeleton in fly MOs. By studying the function and working mechanism of DCX-EMAP, novel insights were provided into understanding how fly MOs are formed. The key finding is that DCX-EMAP, an essential molecule for fly mechanotransduction, serves as the core ultrastructural organizer of the MOs by locally stabilizing and organizing the microtubule-EDM complex (Song, 2023).

The results demonstrate that the DCX tandem and the TAPE domain (i.e., the HELP + WD40 domains) are all required for the in vivo function of DCX-EMAP. First, in vitro analysis shows that the DCX tandem of DCX-EMAP has a microtubule-binding/stabilizing activity, in which both DCX domains and the structural linkage in between are required. In vivo experiments show that the mutant DCX-EMAP with no or only one DCX domain cannot rescue the cellular and functional phenotypes of DCX-EMAPKO, suggesting that the intact microtubule-binding/stabilizing activity is required for the formation of the MOs. Second, the HELP domain is conserved among all EMAP family members. The HELP domain of DCX-EMAP was shown to be key for the MO-specific localization. However, it is not yet clear how the HELP works at the molecular level. It might interact with other components of the MOs (such as the EDMs) or ciliary motors that are responsible for the directional transport to the MOs. Third, the WD40 domain, a known molecular platform to mediate protein-protein interactions, is also conserved in the EMAP family. It is noted that ΔWD40 could still localize to the MO but the 3D porous structure of the EDMs is absent, suggesting that the WD40 domain of DCX-EMAP is key for the local organization of the EDMs. To refine the in vivo working mechanism of the HELP and WD40 domains in this model, it would be essential to identify more components of the EDMs in future studies (Song, 2023).

Based on these findings, it is proposed that DCX-EMAP serves as a component in the structural link between the microtubules and EDMs in the MOs. More specifically, the DCX tandem promotes the assembly and stabilization of short microtubules in the MOs. The HELP and WD40 domains mediate the localization signal and organize the EDMs. In this model, DCX-EMAP promotes the assembly and stability of the MO microtubules, which would facilitate local accumulation of the EDMs and DCX-EMAP by providing more landing or binding sites. Then, an increase in the amount of DCX-EMAP would in turn promote the assembly or stability of more MO microtubules, thereby mediating a positive feedback loop (Song, 2023).

This study showed that short microtubules, generated by the 'kat-60L1-patronin' module, provide constructional flexibility in the formation of the compound cytoskeleton within the nanoscopic space of the MOs. As a concurrent mechanism, the positive feedback loop mediated by DCX-EMAP would facilitate the full assembly of all other components into a compound cytoskeleton in the MOs. The sensillar structures, in particular the MO membrane and extracellular sheath, could serve as a physical boundary to constrain this positive feedback and control the overall shape/size of the MOs. This point is supported by the observation that in the nompA mutants, where the extracellular contact of the MOs is lost, the morphology of the MOs is largely altered. Based on these considerations, it is concluded that DCX-EMAP acts as a core ultrastructural organizer for the MOs of fly ciliated mechanoreceptors (Song, 2023).

The functions of other DCDCs have also been implicated in cilia assembly and ciliopathy. For example, a missense mutation in dcdc2, which encodes DCDC2, causes human recessive deafness, likely by interfering with the structures of sensory hair cells and the supporting cells. Similar to DCX-EMAP, DCDC2 has a tandem pair of DCX domains at the amino terminus and an unstructured tail of over 200 residues at the carboxyl terminus, where the pathogenetic mutation is. Moreover, it also tends to localize to the distal end of cilia, similar to DCX-EMAP. Although the cellular functions and working mechanism of DCDC2 still await further studies, the expression of the deaf mutant of DCDC2 leads to disrupted ciliary structure, such as cilium branching and dysregulation of ciliary length, suggesting that DCDC2 has an essential role in organizing ciliary structures. This is to some extent similar to the function of DCX-EMAP in fly mechanosensory cilia. The resemblance in the cell biological features of DCDC2 and DCX-EMAP may suggest a common way of how DCDCs work in regulating ciliary structure, e.g., the ciliary tip compartment (Song, 2023).

A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia

Mechanoreceptors are sensory cells that transduce mechanical stimuli into electrical signals and mediate the perception of sound, touch and acceleration. Ciliated mechanoreceptors possess an elaborate microtubule cytoskeleton that facilitates the coupling of external forces to the transduction apparatus. In a screen for genes preferentially expressed in Drosophila campaniform mechanoreceptors, This study identified DCX-EMAP, a unique member of the EMAP family (echinoderm-microtubule-associated proteins) that contains two doublecortin domains. DCX-EMAP localizes to the tubular body in campaniform receptors and to the ciliary dilation in chordotonal mechanoreceptors in Johnston's organ, the fly's auditory organ. Adult flies carrying a piggyBac insertion in the DCX-EMAP gene are uncoordinated and deaf and display loss of mechanosensory transduction and amplification. Electron microscopy of mutant sensilla reveals loss of electron-dense materials within the microtubule cytoskeleton in the tubular body and ciliary dilation. These results establish a catalogue of candidate genes for Drosophila mechanosensation and show that one candidate, DCX-EMAP, is likely to be required for mechanosensory transduction and amplification (Bechstedt, 2010).

Using DNA microarray analysis, a list of Drosophila genes was compiled in which the expression was higher in mechanoreceptor-rich tissue than in mechanoreceptor-poor tissue. Comparative qRT-PCR of RNA from the two tissues provides a partial validation of this list. Moreover, direct validation is provided by the overrepresentation of genes expressed in mechanoreceptors and genes associated with basal bodies and ciliated receptors. Using this list, one new candidate gene (DCX-EMAP) as the first Drosophila gene that is likely to be required for both auditory transduction and amplification, narrowing down the role of EMAP family members in the process of mechanosensation18 and illustrating that the list contains novel molecules for mechanotransducer function (Bechstedt, 2010).

Among the candidate genes, it was shown that the microtubule-associated protein DCX-EMAP likely has a key role in Drosophila microtubule-based mechanosensation. Mechanosensation is impaired in f02655 homozygous mutants as shown by a loss of coordination and the absence of CAPs in the auditory organ. Mechanical measurements demonstrate that both mechanotransducer gating and amplification are abolished in DCX-EMAP mutants. By establishing a genetic link between mechanosensory transduction and amplification, this study corroborates theoretical studies that suggest that, at the molecular level, both processes are mechanistically linked. This provides evidence that in Drosophila, not just mechanosensory transduction but also amplification is mediated by the transducer-channel complex (Bechstedt, 2010).

According to this analysis, the function of this transduction complex requires DCX-EMAP. One possibility is that DCX-EMAP is involved in intraflagellar transport (IFT), and that the flight and hearing phenotypes are due to the failure of transduction components to be correctly localized within the modified cilia. This is thought unlikely, on the basis of three arguments. First, DCX and EMAP are microtubule-associated proteins that are not known as IFT components. Second, in DCX-EMAP mutant flies, the complete cilium including dilation is present in Johnston's organ and the overall morphology of the tubular body is normal. If DCX-EMAP was an essential component of the IFT machinery, distal ciliary structures would be expected to be severely disrupted. Third, the Inactive ion channel, which is mislocalized in IFT mutants, was found to be correctly localized in the proximal cilium of the DCX-EMAP mutant background. Thus, rather than being involved in the transport of the complex, it is argued that DCX-EMAP could (1) be a part of the complex, (2) be required for proper localization of the complex, (3) participate in the coupling of mechanical forces to this complex, or (4) provide mechanical support for the complex (Bechstedt, 2010).

DCX-EMAP localizes exclusively to subcompartments of the sensory cilia, namely, the tubular body in campaniform receptors and the ciliary dilation in Johnston's organ. In DCX-EMAP mutants, these ciliary structures display specific ultrastructural defects: interfering with the DCX-EMAP function abolishes the electron-dense material between the microtubules in the tubular body and ciliary dilation while the shape of these subciliary structures is preserved. DCX-EMAP is a microtubule-binding protein and contains 10 WD domains that are known to serve as interaction platforms for other proteins. It was thus hypothesized that DCX-EMAP binds to microtubules within the modified sensory cilia and organizes a protein network that is visible as the electron-dense material and that may consist of diverse proteins. The defects caused by mutations in DCX-EMAP document a molecular equivalence of these materials for different types of ciliated mechanoreceptor cells (Bechstedt, 2010).

For campaniform receptors, the distal fan-shaped region of the tubular body has been hypothesized to be the site of transduction. According to this hypothesis, the cuticle functions as a pair of pincers that exerts compressive force on the sensory dendrite, leading to compression of the filaments linking the dendritic sheath and plasma membrane to microtubules. The current results support this hypothesis by showing that a gene, the mutation of which leads to a mechanotransduction phenotype, encodes a protein that localizes to the tubular body, and leads to ultrastructural alterations when mutated (Bechstedt, 2010).

For chordotonal receptors, the ciliary dilation has been proposed as one of the candidate sites for transduction. The localization of DCX-EMAP, together with the presence of a similar electron-dense material seen in campaniform receptors, and its requirement for transduction support this idea. What role might the dilation, together with its electron-dense material, have in mechanotransduction? Because of its shape, the ciliary dilation is likely to be the weakest mechanical element along the cilium. The ciliary dilation is expected to deform when the cilium is stretched and compressed. Stretch leads to excitation of chordotonal neurons in Johnston's organ, whereas compression leads to hyperpolarization. The electron-dense material may then have the role of a rigid substrate against which the channels are gated when the dilation constricts. Gating will be abolished if this mechanical support is lost, consistent with the effects seen in DCX-EMAP mutants. These observations thus lead to a hypothesis about the mechanism of transduction channel gating in chordotonal mechanoreceptors (Bechstedt, 2010).

Search PubMed for articles about Drosophila DCX-EMAP

Bechstedt, S., Albert, J. T., Kreil, D. P., Muller-Reichert, T., Gopfert, M. C., Howard, J. (2010). A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia. Nat Commun, 1(1):11 PubMed ID: 20975667

Jabali, A., Hoffrichter, A., Uzquiano, A., Marsoner, F., Wilkens, R., Siekmann, M., Bohl, B., Rossetti, A. C., Horschitz, S., Koch, P., Francis, F., Ladewig, J. (2022). Human cerebral organoids reveal progenitor pathology in EML1-linked cortical malformation. EMBO reports, 23(5):e54027 PubMed ID: 35289477

Kefalov, V. J. (2022). EML1 is essential for retinal photoreceptor migration and survival. Sci Rep, 12(1):2897 PubMed ID: 35190581

Song, X., Cui, L., Wu, M., Wang, S., Song, Y., Liu, Z., Xue, Z., Chen, W., Zhang, Y., Li, H., Sun, L., Liang, X. (2023). DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles. J Cell Biol, 222(10) PubMed ID: 37651176

Sun, L., Cui, L., Liu, Z., Wang, Q., Xue, Z., Wu, M., Sun, T., Mao, D., Ni, J., Pastor-Pareja, J. C., Liang, X. (2021). Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia. J Cell Biol, 220(1) PubMed ID: 33263729

Uzquiano, A., Cifuentes-Diaz, C., Jabali, A., Romero, D. M., Houllier, A., Dingli, F., Maillard, C., Boland, A., Deleuze, J. F., Loew, D., Mancini, G. M. S., Bahi-Buisson, N., Ladewig, J., Francis, F. (2019). Mutations in the Heterotopia Gene Eml1/EML1 Severely Disrupt the Formation of Primary Cilia. Cell Rep, 28(6):1596-1611 e1510 PubMed ID: 31390572

Wang, J., Jiang, J., Yang, X., Zhou, G., Wang, L., Xiao, B. (2022). Tethering Piezo channels to the actin cytoskeleton for mechanogating via the cadherin-beta-catenin mechanotransduction complex. Cell Rep, 38(6):110342 PubMed ID: 35139384

date revised: 16 March 2024

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