Kinesin-2 transports Orco into the olfactory cilium of Drosophila melanogaster at specific developmental stages

Author summary Cilia are antenna-like organelles of eukaryotic cells, which mediate cellular signalling. Dysregulation of cilia assembly and the functionalisation process leads to numerous human diseases, including ciliopathies and cancer. In sensory cilia, specific sub-compartments are established to support multiple signal transduction processes, such as photoreception, olfaction, and mechanosensation. In the last two decades, several reports described how a cilium is built and functions. However, a detailed understanding of the timing and modulation of receptor cargo transport to the cilium remains unclear. Here, we show that Orco, a signalling coreceptor for a family of insect olfactory receptors, specifically enters the cilia expressed on olfactory sensory neurons, and the process is restricted to a narrow developmental window in the fruit fly, Drosophila. The coreceptor is kept out of the cilia until it reaches its structural maturity shortly before Drosophila completes its metamorphosis and emerges from the pupal casing. This time-gated Orco entry would enable the adult fly to smell the environment as it emerged from the pupal case. We also find that the heterotrimeric kinesin-2, a core evolutionarily conserved anterograde motor essential for assembling the ciliary structure, is critical for functionalising the olfactory cilia. It interacts with Orco and transports the receptor into the cilium in an IFT-independent fashion. In addition, the motor appeared to deliver and maintain Orco at its designated slot in the ciliary outer-segment. Altogether, our results describe how activation of the olfactory cilia in a multicellular organism is tightly regulated. This system also provides an excellent tool and platform to understand the complex regulation of signalling components in cilia functionalisation, maintenance, and plasticity. The cilium, the sensing centre for the cell, displays an extensive repertoire of receptors for various cell signalling processes. The dynamic nature of ciliary signalling indicates that the ciliary entry of receptors and associated proteins must be regulated and conditional. To understand this process, we studied the ciliary localisation of the odour-receptor coreceptor (Orco), a seven-pass transmembrane protein essential for insect olfaction. Little is known about when and how Orco gets into the cilia. Here, using Drosophila melanogaster, we show that the bulk of Orco selectively enters the cilia on adult olfactory sensory neurons in two discrete, one-hour intervals after eclosion. A conditional loss of heterotrimeric kinesin-2 during this period reduces the electrophysiological response to odours and affects olfactory behaviour. We further show that Orco binds to the C-terminal tail fragments of the heterotrimeric kinesin-2 motor, which is required to transfer Orco from the ciliary base to the outer segment and maintain within an approximately four-micron stretch at the distal portion of the ciliary outer-segment. The Orco transport was not affected by the loss of critical intraflagellar transport components, IFT172/Oseg2 and IFT88/NompB, respectively, during the adult stage. These results highlight a novel developmental regulation of seven-pass transmembrane receptor transport into the cilia and indicate that ciliary signalling is both developmentally and temporally regulated.

Automated summary

Cilia, antenna-like organelles of eukaryotic cells, play important roles in cellular signaling. Research shows that the entry of Orco into the cilia on olfactory sensory neurons in fruit flies is restricted to a narrow developmental window. Heterotrimeric kinesin-2 is critical for functionalizing olfactory cilia and interacts with Orco to transport it into the cilium.