Orco selectively localises to the olfactory cilia

Drosophila antenna contains three morphologically distinct typed of cuticular projections, called sensilla, each innervated with the cilia of 2–4 OSNs [22,25]. Bipartite cilia extend from the distal ends of dendrites of the OSNs and grow into the hollow cuticular shafts of the sensilla (Fig 1A) [20]. Orco expresses in all the OSNs innervating sensilla basiconica and sensilla trichodea and localises in the cilia (Fig 1A). Association of Or22a and Or43a, respectively, with Orco, is essential for the Orco/ORx entry into the cilia [24]. Accordingly, ectopic overexpression of GFP:Orco, GFP:Or47b and GFP:Or43a (ORxs) in cholinergic neurons enriched the GFP-tagged proteins in both the basiconic and trichoid cilia (Fig 1Ad-f). Contrary to the expectation, the ectopically expressed Or43a and Orco were excluded from the mechanosensory cilia on chordotonal neurons, innervating the Johnston’s organ in the second antennal segment (Fig 1B). Together these suggested that Orco/ORx specifically enters the olfactory sensory cilia, and molecular systems unique to the OSNs facilitate the ciliary access. A similar phenomenon was observed for different G-protein coupled receptors in C. elegans [8].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. The developmental profile of Orco localisation in the cilia.

(A) Organization of olfactory sensory neurons (OSNs) in the adult third antennal segment marked by chaGal4>UAS-tdTomato (a), and enlarged views of the s. trichodea (b) and s. basiconica (c) regions expressing both Endo-Orco:GFP (orco^fTRG) and chaGal4>UAS-tdTomato. A few OSN cell bodies are circled white, and the labelled arrows indicate cilia inside small and large s. basiconica (SB and LB type) and s. trichodea (T3 type) shafts. Ectopic expressions of GFP:Orco (d), GFP:Or47b (e), and GFP:Or43a (f), respectively, by the chaGal4, marked the cilia (arrows) inside all SB and T3-type sensilla. (B) Chordotonal neurons of the Johnston’s organ expressing soluble GFP and GFP:Orco along with GFP:Or43a, respectively, by chaGal4. Arrowheads mark the dendritic knobs, and arrows mark the cilia region. (C) Schematic illustrates cilia organisation on OSNs innervating s. basiconica. (D) TEM images of transverse sections through the base of an LB-type sensillum show the basal bodies (BB) and the connecting cilium (CC) of four innervating cilia (a). Each connecting cilium (CC) carries nine doublet microtubule (9+0) bearing axoneme (arrow, b). Sections of the inner segment (IS, c) and outer segment (OS, d, e) shows the arrangement of singlet microtubules within a membrane sleeve (yellow arrow). (E) tdTomato, GFP:Tubulin84B, mCD8:GFP and Endo-Orco:GFP localisations in the cilia inside ab1-type s. basiconica (arrows- ciliary OS, and arrowheads- ciliary base). (F) Endo-Orco:GFP and the recombinant bacterial channelrhodopsin (ReaChR:YFP) localisations in the ab1-type s. basiconica and that of ectopic GFP:Orco in the or42b cilium. ReaChR:YFP expressed using ocroGal4 was used as a negative control. (G) Total fluorescence intensities (mean ± S.D.) of Endo-Orco:GFP and Orco:GFP enrichments in the cilia inside ab1-type sensilla at different ages after eclosion. The pairwise significance of difference was estimated using one-way ANOVA test, p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots. Images are shown in a false colour intensity heat map (FIRE, ImageJ). Scale bars indicate 50 μm (A-a), 10 μm (A- b-f, B, and E), and 2 μm (F), respectively, and the number of sensilla (n) used for the quantifications is indicated on the plots with appropriate colour coding.

https://doi.org/10.1371/journal.pgen.1009752.g001

The olfactory cilium extends from a basal body (BB) placed at the distal end of the OSN dendrite (Fig 1C). Each cilium inside the s. basiconica shaft, henceforth called basiconic cilia, has a bipartite organisation well-resolved by confocal microscopy. The base consists of a 9+0 arrangement of microtubule doublets (Fig 1D), which supports a short (~0.5 μm) connecting cilium (CC) between the ciliary inner-segment (IS) and the basal body (BB). The outer-segment (OS) is extended from the IS and housed within the porous cuticle shaft. It consists of membranous branches supported by singlet microtubules (Fig 1Dd-e). The OS contains two distinct structural domains. The proximal domain with fewer branches containing multiple microtubules each, and the distal domain where each branch has a singlet microtubule tightly invested with the ciliary membrane (Fig 1Dd-e). The ab1-type large s. basiconica are innervated with four OSNs, and one of these expresses OR42b [26]. We used these sensilla because of their characteristic features and high levels of Orco enrichment (Fig 1E and 1F) for all quantitative analyses in this study.

The bulk of the Orco enters cilia in two brief episodes, shortly after eclosion

Olfactory cilia develop in stages, and the process completes by 90-hours after pupa formation (APF) [20]. Consistent with anti-Orco immunostaining analysis (S1A Fig), the GFP-tagged Orco, expressed from its endogenous promoter in the orco^fTRG stock [27], hereafter, referred to as Endo-Orco:GFP, marked a few basiconic cilia after 90 hours APF. As the development progressed, Endo-Orco:GFP appeared in an increasing number of basiconic cilia (S1B and S1C Fig). The onset of Orco expression, which begins from 80 hours APF [28], could determine the timing of its entry into the cilium. To test the conjecture, we ectopically expressed GFP:Orco, GFP:Or47b, and coexpressed GFP:Orco with GFP:Or43a using chaGal4, which marked the OSNs from an earlier stage of development. In all these cases, the GFP-tagged transgenic proteins were restricted to the cell body until 96-hours APF (S1D Fig). In comparison, the expression of soluble GFP and mCD8:GFP using the same driver marked the entire ciliary volume and membrane, respectively, throughout the pupal stages (S1D Fig). These results suggested that the Orco/ORx entry into the olfactory cilium is regulated independently of their gene expression profiles. A similar phenomenon was observed using orcoGal4>GFP:Orco background (S1E Fig).

The ciliary levels of Endo-Orco:GFP reached the steady-state within 6 hours after eclosion (AE; S1F–S1G Fig), indicating that the bulk of Orco/ORx enters the cilia within a short period immediately after the adult emerged from the pupal case. We further resolved the entry period to two distinct phases in the ab1-type sensilla (Figs 1G and S2A). The same pattern was observed in the cilium expressed on Or42b OSN (Fig 1G). The episodic Orco entry pattern was exclusive. The bulk membrane content, monitored using the heterologous bacterial transmembrane protein, ReaChR:YFP, a chimeric channelrhodopsin (Fig 1G), and the ciliary volume remained unaltered during the Orco entry period (S2B and S2C Fig). Altogether, these observations suggested that Orco/ORx independently enters the cilium after it is fully developed and the process is developmentally gated. Hence, it raised a new question–how is the Orco entry regulated?

Kinesin-2 is involved in maintaining odour reception independent of the cilia assembly

The cilia structure and function are intimately linked [7]. Genetic studies suggested that the heterotrimeric kinesin-2 is essential for cilia assembly and maintenance [29] and the ciliary enrichment of several transmembrane receptors [3,14,18,30]. For example, total loss-of-function (null) mutation in the Klp64D gene, which codes for the kinesin-2α/Kif3A motor subunit in Drosophila, disrupts the development of olfactory cilia as well as reduces the odour-evoked potentials from the antenna [20]. Therefore, to assess whether the motor is separately engaged in the Orco/ORx localisation and transport, we first utilised a forward genetic strategy to disrupt the olfaction-specific function of kinesin-2. We isolated viable Klp64D alleles with selective olfaction defects in the adult stage (Fig 2A). An initial screen for jump response defects in adult flies isolated five homozygous, viable Klp64D mutant alleles (kj353, kj429, kj925, kj1070, and kj1072) with substantial olfactory defects (Fig 2B and 2C). The chemotactic behaviour of the mutant larvae towards ethyl-acetate and Butanol (S1 Table), the locomotion of the homozygous mutant adults, and the ultrastructure of the cilia on chordotonal neurons in Johnston’s organ were unaffected in the homozygous Klp64D^kj backgrounds (S3A and S3B Fig). These observations confirmed that the Klp64D^kj mutations selectively disrupt adult olfaction, fulfilling the central objective of the screen.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Isolation of homozygous viable Klp64D mutant alleles with specific odour response defects in adults.

(A) Flow diagram summarises the ethyl-methane-sulfonate (EMS) mutagenesis screen used for isolating the viable alleles of Klp64D with jump response defect. Klp64D^k5 is a homozygous lethal allele. The freshly mutagenised genome was screened for jump response defect mutants in trans-heterozygote combinations over the Klp64D^k5 for several generations. Fx (x = 1 to 6) indicates the number of generations of outcross with Klp64D^k5. (B) Jump response indices of wild type (Canton S) and homozygous kj isolates. (C) Electroantennogram (EAG) responses to different odours measured from the homozygous kj mutants (see S2 Table and S3 Fig for detail genetic characterisation of the kj alleles by EAG). The pairwise significance of difference was estimated using two-tailed Student’s T-test, p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots. (D) EAG response analyses at different dilutions of four different odorants across the spectrum show that mutations in the homozygous kj flies lower the odour sensitivity (N ≥10 or as depicted on each set of bars). (E) KLP68D:YFP localisation in the cilia (expressed by chaGal4) in control, and homozygous kj353 and kj925 backgrounds, at 96 hours APF and 0–3 hours AE, respectively. Connecting cilia (CC), and the outer segments (OS, arrows) are shown in each panel (Scale, 10 μm). Images are shown using a false-colour intensity heat map (FIRE, ImageJ). (F) Relative intensity values of the KLP68D:YFP fluorescence in the cilia estimated against the control group at each stage. Number of sensillae (n) and the number of antennae (N) are indicated on the plots.

https://doi.org/10.1371/journal.pgen.1009752.g002

The olfactory sensory responses and the odour-induced neuronal activity are measured using the Jump assay and the Electroantennogram (EAG). The aberrant odour-evoked jump responses (Fig 2B) and EAG defects (Fig 2C) displayed by the homozygous mutant flies were recessive in nature. We mapped them to the Klp64D locus using genetic non-complementation and transgenic rescue analyses (S2 Table). The olfactory defect of the mutants could be rescued by the ectopic expression of Klp64D transgene and not by the Klp68D (kinesin-2β/Kif3B orthologue) expression (S3C–S3E Fig). Together, these established that mutations in all the kj alleles belong to the Klp64D locus. Since the jump response and EAG characteristics of these alleles appeared very similar, we decided to continue further phenotypic characterisations using the kj353 and kj925 alleles. Both these alleles have good homozygous viabilities.

The mutation reduced the EAG responses to wide-ranging odours in the homozygous Kp64D^kj mutants (Fig 2C and 2D). Of these, ethyl acetate (EA), nButanol (nBut), and benzaldehyde (Benz) are detected by OSNs innervating s. basiconica, and propionic acid (PA) by OSNs innervating s. coeloconica [26,31]. These results suggested that the KLP64D function is necessary to develop odour reception in different types of olfactory cilia. A constant downshift of the response voltage at different concentrations of a variety of odorants further suggested that the loss of kinesin-2 function could suppress odour reception at different types of OSNs (Fig 2D).

KLP64D is essential for the heterotrimeric kinesin-2 assembly [32], and the level of KLP68D:YFP is significantly reduced in the cilia in the Klp64D total-loss-of-function allele [20]. Therefore, to understand the nature of the kinesin-2 disruption in Klp64D^kj alleles, we estimated the ciliary levels of KLP68D:YFP during development in wild-type and homozygous Klp64D^kj353 and Klp64D^kj925 backgrounds, respectively. The KLP68D:YFP level in the mutants was comparable to that of the wild-type at 96 hours APF, but it was significantly reduced in the freshly eclosed (0–3 hour AE) homozygous mutant adults (Fig 2E and 2F). The cuticle autofluorescence mostly contributed to the values estimated in the mutants in the adult stage (Fig 2F). This observation suggested that the mutations in homozygous Klp64D^kj alleles selectively reduce kinesin-2 entry into the olfactory cilia only in the adult stage.

KIF17, the homodimeric kinesin-2, has been reported to facilitate the localisation of the cyclic nucleotide gated channel comprising CNGA2 and CNGB1b subunits in the mammalian olfactory cilia [3]. Therefore, we reasoned that the loss of kinesin-2 entry into the cilia could affect the Orco/ORx transport resulting in the loss of odour reception. However, Kif17 is redundant for the primary cilia assembly, whereas the heterotrimeric kinesin-2 also transports tubulin into the olfactory cilia [33] and powers the IFT [29]. Both are essential for maintaining the cytoskeletal integrity in the cilia. Hence, alternatively, disruption of the ciliary cytoskeleton due to the loss of kinesin-2 function could cause the EAG defect as reported earlier [20].

Loss of kinesin-2 in the adult stage reduced the cilia Orco enrichment

To distinguish between these two possibilities, we compared the ciliary enrichment of GFP:Orco, GFP:Tubulin84B, and a non-specific membrane marker (mCD8:RFP), respectively, in wild type and homozygous Klp64D^kj mutants. The GFP:Orco levels, independently expressed in the OSNs by chaGal4 and orcoGal4, were significantly reduced in homozygous Klp64D^kj353 adults (Fig 3A and 3B), and the expression of KLP64D transgene in the Klp64D^kj353 mutant background rescued the defect (Fig 3C). The levels were unaffected at 96 hours APF. These observations suggested that the mutations in the Klp64D gene restricted the GFP:Orco enrichment in adult cilia. Also, we found that the total GFP:Tubulin84B level and the ciliary volume were unaltered, and there was a marginal increase in the cytoplasmic GFP and mCD8:RFP levels in the cilia in homozygous Klp64D^kj353 background (S4A Fig).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Mutations in the Klp64D gene reduced Orco localisation in the adult olfactory cilia.

(A) GFP:Orco localisation in cilia of s. basiconica (arrow), in 96-h APF and two-day-old control and homozygous Klp64D^kj353 mutant antennae. The GFP:Orco transgene was separately expressed using chaGal4 (a) and orcoGal4 (b) to test the effects of different levels of recombinant protein expression on its cilia localisation. (B) Total fluorescence intensity of GFP:Orco, driven by chaGal4 (a) and orcoGal4 (b) in the cilia of s. basiconica of two-days-old control and homozygous Klp64D^kj353 mutant adults. (C) GFP:Orco localisation in the ciliary OS of control (chaGal4>UAS-GFP:Orco/+; Klp64D^kj353/+), hemizygous kj353 (chaGal4>UAS-GFP:Orco/+; kj353/Klp64D^k1) and rescued (chaGal4>UAS-GFP:Orco/UAS-KLP64D-TevHis; Klp64D^kj353/Klp64D^k1) adults (a). Total fluorescence intensity (mean ± S.D.) measured in the different genetic backgrounds (b). The pairwise significance of difference was estimated using two-tailed Student’s T-test, p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots. All Images are shown in a false colour intensity heat map (FIRE, ImageJ), and scale bars indicate 10 μm. (D-E) Ultrastructure of the basiconic cilia in the wild-type and homozygous Klp64D^kj antennae. Schematic illustrates the microtubule organisation in cilia inside s. basiconica. TEM images of sections through the basal bodies, connecting cilia (CC) and outer segments (OS) of cilia inside s. basiconica from one-day-old wild-type (D) and homozygous kj353 (E) antennae. F, G) Histograms describe the total number of singlet MTs (F) and the distribution of different types of branches (G) found in the OS in wild type and the mutants. The pairwise significance of difference was estimated using one-way ANOVA test, p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots. Error bars represent as ± S. D. The n values indicate number of sensilla in panels B and C, and the N values in panel F indicate number of TEM sections obtained from at least 3 different heads.

https://doi.org/10.1371/journal.pgen.1009752.g003

To understand the underlying cause of this Orco reduction, we studied the ciliary ultrastructure using TEM. The analysis of 1-day-old homozygous Klp64D^kj353 and Klp64D^k925 mutant adults revealed marginal defects in the ciliary ultrastructure (Fig 3D) with a small but significant reduction in the number of singlet microtubules only in the Klp64D^kj353 background (Fig 3F). Also, the number of membrane-bound branches bearing singlet microtubules were considerably reduced in the homozygous Klp64D^kj backgrounds (Fig 3G). The defect was much less severe than those observed in the homozygous Klp64D^k1 background [20], which is recessive lethal and considered to be a null allele. The apparent difference in the microtubule count in the TEM sections and the total GFP:Tubulin84B levels assessed from the confocal images could be attributed to the difference in resolutions of these two techniques. Together, these results suggested that loss of kinesin-2 function in the adult olfactory cilia has a relatively stronger impact on Orco localisation than on the axoneme.

The ciliary tubulin has low turnover during the adult stage [33]. Consistent with this report, very little GFP:Tubulin84B localised in the cilia when expressed using orcoGal4 as compared to the chaGal4 and or42bGal4, respectively (S4B Fig). OrcoGal4 expresses after 90 hours APF, much later than the onset of chaGal4 (30–34 hours APF) and or42bGal4 (60–70 hours APF). Hence, the results suggested that very little tubulin is transported into the cilia in the adult stage. Together, these observations explained the relatively milder impact on microtubule count in the homozygous Klp64D^kj backgrounds. Therefore, we concluded that the loss of kinesin-2 function in the adult OSN would not significantly affect the ciliary cytoskeleton in the near term. The loss of membrane branches was, however, intriguing.

Orco entry and transport into the ciliary outer-segment requires kinesin-2

To assess the function of kinesin-2 in the bi-phasic increment of Orco during 0–6 hours AE, we reduced the kinesin-2 levels just before eclosion. The KLP64D and KLP68D dsRNA were expressed using orcoGal4 in the orco^fTRG background, to suppress the target genes in the late pupa (after 90 hours APF) and adult stages. We found a significant reduction of Endo-Orco:GFP level in the cilia at 0 hour AE (~105 hours APF) in both the Klp64D and Klp68D RNAi backgrounds. Although the level appreciated to some extent in the subsequent stages, it remained significantly lower as compared to the control until 12 hours AE (Fig 4B, 4C and 4E). A similar profile was obtained in the Klp64D^kj353 homozygous background (Figs 4D, 4E and S5A). The reduction of Endo-Orco:GFP in the cilia in the kinesin-2 RNAi and mutant backgrounds was associated with its episodic accumulation at the dendritic-knob region at 2, 4, and 6 hours AE (Fig 4B–4D), suggesting that the reduction of kinesin-2 levels affect Orco movement from the dendritic base into the cilium. In comparison, the Jupiter:GFP levels, an indicator of the ciliary microtubule content, remained unaffected in these backgrounds (S5C Fig). Together, these observations implicated kinesin-2 in the Orco movement through the transition zone.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Kinesin-2 is necessary for Orco entry into the olfactory cilia.

(A-D) Total fluorescence intensity (mean ± S.E.M) of Endo-Orco:GFP in the ciliary OS and the dendritic knob region of OSNs in Control (A), Klp64D RNAi (B), Klp68D RNAi (C) and homozygous Klp64D^kj353 mutant (D) backgrounds, respectively. The pairwise significance of difference as compared to the wild type control and between different stages was estimated using one-way ANOVA test, and the p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots with appropriate colour codes. Inset shows representative images of the cilia at corresponding stages. (E) Images depict Endo-Orco:GFP localisation in the ab1-type s. basiconica from Control (orcobGal4, Endo-Orco:GFP/+; UAS-Dicer/+), Klp64D RNAi (orcoGal4, Endo-Orco:GFP/UAS-Klp64D RNAi; UAS-Dicer/+), Klp68D RNAi (orcoGal4, Endo-Orco:GFP/UAS-Klp68D RNAi; UAS-Dicer/+) and homozygous Klp64D^kj353 mutant (Endo-Orco:GFP; Klp64D^kj353) backgrounds at 12 hours AE (a). The histograms depict total fluorescence intensity (mean ± S.D.) of Endo-Orco:GFP in control, the kinesin-2 knock downs, and homozygous Klp64D^kj353 mutant backgrounds at 12 hours AE (b). The pairwise significance of difference was estimated using the one-way ANOVA test, and p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are indicated on the plots. (F) The recovery of Endo-Orco:GFP fluorescence in the bleached region of the cilia at 60 minutes after the photobleaching as compared to the unbleached region in the Control, Klp64D RNAi, Klp68D RNAi and homozygous Klp64D^kj353 mutant backgrounds (also see S6 Fig). The cilia were photobleached during 4–5 hours AE. Examples of prebelach and postbleach images are shown in panel a, and the corresponding quantification is presented in panel b. The pairwise significance of difference as compared to the wild type control was estimated using Mann-Whitney U-test, and the p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are indicated on the box plots. All images are shown in the false colour intensity heat map (FIRE, ImageJ). Scale for images—2 μm (A-D), 10 μm (E-a), and 2 μm (F-a). Number of sensillae (n) quantified is indicated on the graphs.

https://doi.org/10.1371/journal.pgen.1009752.g004

After the Orco transfer to the ciliary membrane, it could potentially diffuse throughout the cilium. In such a scenario, one would expect a uniform distribution of the protein along the cilium. However, like Smo [14] and the salt-sensing receptor-type guanylate cyclase (GCY-22) [34], Orco is distinctly enriched in the ciliary OS. Therefore, to explore a kinesin-2 role in the Orco movement inside the cilia, we used the Endo-Orco:GFP fluorescence recovery after photobleaching (FRAP) at the distal half of the ciliary OS during 4–5 hours AE, which coincided with the second Orco enrichment episode. In a majority of the wild-type cilia (n = 8), the fast recovery stage completed within 20 minutes (S6B Fig) and in some cilia, the FRAP profiles had multiple episodic increments (n = 3), each lasting for 10–20 minutes (S6B Fig). The episodic nature of the recovery indicated that the Orco movement occurs inside the cilia through active transport. The recovery pattern appeared divergent because it was difficult to synchronise the photobleaching time with the ageing process.

In comparison, the recovery profiles in the kinesin-2 RNAi backgrounds were visibly slower, with no clearly discernible episodic profiles (S6B Fig). Although some episodic increments were observed in homozygous Klp64D^kj353 background (n = 2), the extent of recovery did not exceed 30%, which is significantly below the minimum recovery level observed in the wild-type control (Figs 4E and S6B). Together, these data suggested that Orco movement inside the cilium would require kinesin-2. Furthermore, the relatively slow rate of initial recovery in the kinesin-2 mutants might indicate that Orco diffusion in the cilium would be highly restricted.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. IFT172/Oseg2 is not required for Orco enrichment in the olfactory cilia.

(A) The patterns of GFP:Orco, Oseg2:GFP, KLP64:GFP, and Endo-Orco:GFP localizations along the olfactory cilia during 0–6 hour AE in the wild-type (cha>) and Klp64D^kj353 mutant backgrounds. Fine arrows indicate relatively uniform localisation of KLP64D:GFP, Endo-Orco:GFP and GFP:Orco and the arrowheads indicate clearly discernible Oseg2:GFP puctae in the inner segment. (B) Endo-Orco:GFP localisation in ab1-type s. basiconica from the control (orcoGal4, Endo-Orco:GFP/+; UAS-Dicer/+), Oseg2 RNAi (orcoGal4, Endo-Orco:GFP/UAS-Oseg2 RNAi; UAS-Dicer/+) and NompB RNAi (orcoGal4, Endo-Orco:GFP/UAS-NompB RNAi; UAS-Dicer/+) backgrounds, respectively, at 6 hours AE (a). Histograms indicate Endo-Orco:GFP fluorescence intensity (mean ± S.D.) in the cilia in the control, Oseg2 and NompB RNAi backgrounds at 6 hours AE (b). The pairwise significance of difference was estimated using one-way ANOVA, and the p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots. (C, D) Oseg2:GFP and Oseg4:GFP localisations at the basal body (arrowhead) and cilia (arrow) in the control and Oseg-2 RNAi (driven by orcoGal4) backgrounds. Images are shown in a false colour intensity heat map (FIRE, ImageJ), arrowheads indicate the dendritic knob, arrows indicate the OS and scale bars indicate 10 μm. Number of sensillae (n) quantified is indicated on the graphs.

https://doi.org/10.1371/journal.pgen.1009752.g005

Orco localises in the cilia independent of IFT172/Oseg2 and IFT88/NompB

Several receptors, for example, GCY-22, move inside the cilium through IFT [34], and Oseg2/IFT172 is particularly implicated in the transmembrane receptor transport into the cilia [35]. IFT movements are most prominent in the middle segment of the amphid and AWC cilia {Scheidel, 2018 #115}. The episodic Orco recovery during 0-6-h AE suggested a similar mode of movement. To explore whether Orco indeed moves in the cilia in an IFT-like manner, we compared the Endo-Orco:GFP and GFP:Orco localisation patterns with that of the IFT-B component Oseg2:GFP and KLP64D:GFP at the ciliary inner-segment (IS) in this period. We found prominent Oseg2:GFP puncta representing the IFT trains in the ciliary IS (arrowheads, Fig 5A, N = 4, n = 7), whereas the KLP64D:GFP distribution was relatively more uniform (arrows, Fig 5A, N = 5, n = 7). No such GFP:Orco puncta were detected in the IS region in the overexpressed GFP:Orco (N = 6, n = 9), as well as the EndoOrco:GFP in wild-type (N = 5, n = 8) and homozygous Klp64D^kj353 mutant (N = 4, n = 9) backgrounds (Fig 5A). Furthermore, the adult OSN-specific RNAi of NompB/IFT88 and Oseg2/IFT172 did not affect the Endo-Orco:GFP localisation in the cilia (Fig 5B). The RNAi, however, eliminated both the Oseg2:GFP (Fig 5C) and an IFT-A complex component Oseg4/WDR35:GFP (Fig 5D) from the adult olfactory cilia. Together, these results suggested that the Orco entry through the transition zone and movement inside the cilium is unlikely to involve IFT.

Orco associates with the tail domains of kinesin-2 motor subunits

To supplement the genetic evidence, we then used copurification assay to test whether the heterotrimeric kinesin-2 motor (Fig 6A) could directly engage with Orco/ORx. The N-terminal domains of kinesin-2 motor subunits bind to the microtubule and generate the force [29]. The coiled-coil stalk domains in the middle are involved in heterodimerisation [32] and bind to the accessory subunit, kinesin associated protein (KAP) [36], and an IFT complex subunit [37]. Both the KLP68D (kinesin-2β) and DmKAP (Fig 6B) were pulled down from the head extracts of chaGal4>UAS-GFP:Orco adults using agarose beads conjugated with GFP-nanobody (chromotek, USA), suggesting that Orco interacts with the heterotrimeric kinesin-2. Further, a relatively more prominent level of GFP:Orco (Fig 6C) and OR47b, respectively, were pulled-down using the GST-KLP68D-Tail fragment (Fig 6D), which identified the potential Orco/ORx binding region at the tail domain of KLP68D (2β) motor subunit. Together, these results suggested that Orco/ORx associates with the heterotrimeric kinesin-2 motor, which subsequently transports it into the cilium.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Orco associates with the C-terminal ’tail’ domain of the kinesin-2 motor subunits.

(A) Schematic illustrates the organisation of Drosophila kinesin-2 and the tail domains of the motor subunits. (B) Co-immunoprecipitation of the Drosophila kinesin-2 subunits from the head extracts of flies expressing GFP:Orco (driven by chaGal4) using agarose beads conjugated with GFP-nanobody. (C, D) Affinity copurification of GFP:Orco (C), and OR47b (D), respectively, from the adult head extracts by using the GST tagged kinesin2 tail fragments, KLP64D-T and KLP68D-T.

https://doi.org/10.1371/journal.pgen.1009752.g006

Orco enriches in a characteristic distal domain at the ciliary OS

Next, we examined how Orco localises inside the cilium after crossing the transition zone. The time-resolved plot profile analysis revealed that Orco is mostly enriched in the distal OS, in an approximately 4 μm band with a stereotypic peak at the distal OS (Fig 7A and 7B). This portion of the cilia, housed within the porous shaft of basiconica, consists of predominantly singlet microtubule-bearing branches [20,25]. Similar Orco localisation, distinct from that of the tubulin, was observed in the Or42b cilium (Fig 7C and 7D). The distribution of Orco was maintained throughout 0–6 hours AE, and it was distinct from that of the membrane marked by Bodipy-FL-C₁₂ (Fig 7E and 7F). The cytoskeleton marked by chaGal4>UAS-GFP:tubulin84B and endogenous Jupiter:GFP (MAP) (S7B Fig), as well as the cytoplasm labelled by GFP (S7C Fig), also elicited a relatively uniform distribution profile throughout the OS (Figs 7F and S7). Thus, the enrichment domain appeared exclusive for Orco. Interestingly, the mCD8:GFP, a non-specific transmembrane protein, was also found to have a distribution profile similar to that of Endo-Orco:GFP (S7C Fig), suggesting that transmembrane receptor proteins are likely to be enriched in the distal OS domain of the olfactory cilia.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. Orco enrichment domain in the cilia.

(A-B) Fluorescence micrographs (A) and plot profiles (B) of Endo-Orco:GFP distribution (mean ± S.E.M.) along the ciliary OS inside ab1-type s. basiconica during 0–6 hours AE. (C, D) GFP:Orco and GFP:Tubulin84B localisation in single cilium expressed on OR42b neuron inside ab1-type sensilla (C) and plot profile of corresponding intensities along the cilia (D). (E, F) Fluorescence micrographs (E) and relative distribution profiles (F) of Endo-Orco:GFP, KLP64D:GFP, Jupiter:GFP and BODIPY FL C₁₂ along the ciliary OS inside ab1-type s. basiconica during 0–6 hours AE. The broken line marks cuticle contour. The green, red, and cerulean arrows indicate peaks of Endo-Orco:GFP, KLP64D:GFP and Jupiter:GFP profiles, respectively, along the cilia. The separations between the peak Endo-Orco:GFP with KLP64D:GFP and Endo-Orco:GFP with Jupiter:GFP are indicated by red and cerulean numerals, respectively. The pairwise significance of difference was estimated using two-tailed Student’s T-test, and p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots. (G) Endo-Orco:GFP, KLP68D:YFP colocalisation in the ciliary OS. The GFP and YFP images were obtained by the 445 nm (for GFP) and 515 nm (for YFP) excitations, respectively (see Materials and Methods for details). Scale bars indicate 10 μm. (H) Plot profiles of Endo-Orco:GFP and KLP68D:YFP distribution (mean ± S.E.M.) along the ciliary OS inside ab1-type s. basiconica. All images are shown in a false colour intensity heat map (FIRE, ImageJ). Scale for all images—10 μm. Number of sensillae (n) quantified is indicated on the graphs.

https://doi.org/10.1371/journal.pgen.1009752.g007

At 6 hours AE, the Endo-Orco:GFP domain partly overlapped with the kinesin-2 subunits KLP64D:GFP (Fig 7E and 7F) and KLP68D:YFP (Fig 7G and 7H). Surprisingly, both the KLP64D:GFP (S7D Fig) and the KLP68D:YFP (Fig 7H) peaks were shifted towards the tip of the ciliary OS. Analysis of the full-width-half-maxima (FWHM) separation among the Endo-Orco:GFP, Klp64D:GFP and Jupiter:GFP profiles indicated that the Orco band partly coincides with that of the kinesin-2 subunit KLP64D:GFP at different stages during 0–6 hours AE (Fig 7E and 7F). Also, separation between peaks of their distribution revealed that the Endo-Orco:GFP accumulates much closer to the KLP64D:GFP as compared to the Jupiter:GFP. Together with the FRAP data described above, these observations may suggest that a fraction of ciliary kinesin-2 is engaged in delivering Orco to its parking zone at the distal OS. The anterograde motors are known to inactivate after reaching the distal ends of the cilia and flagella, which may account for the increased accumulation of the kinesin-2 motor subunits at the distal tips. The tip accumulation of KLP64D:GFP and KLP68D:YFP helped to clearly distinguish the tip compartment as separate from that of the Orco domain.

Kinesin-2 is required for packing Orco in the distal domain

We also considered an additional possibility that the interaction with the kinesin-2 motor could actively maintain Orco at the distal part of the ciliary OS. For this purpose, we estimated the total as well as relative intensity distribution of Endo-Orco:GFP along the cilia in control, Klp64D^kj353 mutant and kinesin-2 RNAi backgrounds during 0–6 hours AE. The surface plots show that the levels of Endo-Orco:GFP is reduced in the homozygous Klp64D^kj353 mutant background (Fig 8A). In the kinesin-2 RNAi backgrounds, the Endo-Orco:GFP fluorescence was highly depleted from the OS domain, particularly at the 3 hours and 6 hours AE, intervening the active transport spurts at 1–2 hours and 4–5 hours AE, respectively (Fig 8A). Also, the FWHM analysis of the length-normalised profile of Endo-Orco:GFP distribution suggested a proximal spreading of the domain (Fig 8B). We also noticed that initially, during 0–3 hours AE, the Endo-Orco:GFP distribution was proximally shifted by a significant margin in the Klp64D^kj353 background (S8Ba-d Fig), which was prominent in the kinesin-2 RNAi backgrounds at 6 and 12 hours AE (Figs 8C-b and S8-g), suggesting a comparatively higher penetrance of the Klp64D and Klp68D RNAi as the time progressed.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. Endo-Orco:GFP distribution in the kinesin-2 mutant and RNAi backgrounds.

(A-B) Surface plots for total (A) and normalised intensity (B) distributions of Endo-Orco:GFP along the ciliary OS during 0–6 hours AE in the wild-type control, homozygous Klp64D^kj353 mutant, as well as Klp64D and Klp68D RNAi backgrounds. Dotted lines demarcate the full-width-at-half-maxima (FWHMs) of the Endo-Orco:GFP localisation domains. Note that FWHMs in the mutant backgrounds expands beyond the wild-type domain boundary (4 μm position). (C) Images of Endo-Orco:GFP location in the cilia inside ab1-type sensilla from different genetic backgrounds are shown in the false colour heat map (FIRE, ImageJ) with scale bars indicating 10 μm (a). Plot profiles indicates the Endo-Orco:GFP (b and d) and Jupiter:GFP (c) distribution (mean ± S.E.M.) along the ciliary OS at 6 hours AE in different genetic backgrounds. The pairwise significance of difference was estimated using one-way ANOVA test, p-values (*p < 0.05, **p < 0.01, and ***p<0.001) are shown on the plots. Number of sensillae (n) quantified is indicated on the panels.

https://doi.org/10.1371/journal.pgen.1009752.g008

To further investigate whether the kinesin-2 activity is essential for compacting the Orco domain, we assessed the Endo-Orco:GFP distribution in the heterozygous Klp64D^k5 mutant background. Klp64D^k5 is an amorphic allele, which carries a missense mutation (Glu551-Lys) at the C-terminal stalk domain of KLP64D. The ciliary OS contained fewer branches bearing singlet microtubules in the Klp64D^k5 homozygous background [20]. The EAG responses were, however, not significantly reduced in the Klp64D^k5/+ heterozygous background [20]. The KLP64D(E551K) mutation restricts the stalk dynamics [38] and zips the N-terminal stalk in the presence of KAP [39], and thereby, it is expected to disrupt the motor function. Accordingly, one could designate Klp64D^k5 as a dominant-negative allele. In Klp64D^k5/+ background, the Endo-Orco:GFP distribution spread further towards the proximal domain as compared to the control, Klp64D^kj353 and kinesin-2 dsRNA backgrounds (Fig 8C-d), indicating that the compaction of the Orco domain is highly sensitive to the kinesin-2 dose and function. Together, these observations further suggested that interaction between Orco/ORx and kinesin-2 could maintain them in the designated domain at the ciliary OS.

Kinesin-2 maintains Orco in a distinct domain at the ciliary outer-segment

The Orco/ORx localisation into the olfactory cilia is critical for the sensory reception [24,40]. Here, we show that the Orco is specifically enriched in the part of the branched OS that is housed inside the porous cuticle shaft. Nano-pores of the cuticle shaft, which houses the ciliary OS containing the branched extensions [20,25], is essential for odour sensing [41]. The branching also increases the surface area of the cilia and accommodates a larger number of the receptors. In addition, packing a large number of branches, typically between 100–140, inside an LB-type shaft would increase the receptor density and facilitate the trapping of cognate odorant molecules at low concentrations. Therefore, the branching of the outer segment and Orco localisation along the branches are crucial for optimising the olfactory responses. We found that the heterotrimeric kinesin-2 (kinesin-2α/β & KAP) motor associates with Orco/ORx complex, helps transfer the protein through the transition zone, and plays a crucial role in localising Orco to this ciliary subdomain in the adult stage (Fig 9). The cytoskeleton of olfactory cilia develops in the pupal stages and remains stable during the early hours after eclosion requiring limited support from the IFT and kinesin-2. Thus, the adult-specific knockdown of kinesin-2 helped identify an independent role of the motor in the Orco transport.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 9. Model for Orco transport by kinesin-2 motor protein.

Targeting of vesicles carrying receptor proteins from the trans-Golgi network to the ciliary base is brought about by dynein motor and other accessory ciliary trafficking proteins like Rab-GTPases (Rab11, Rab8) along with their GEFS, and also BBSome protein complex. Our results indicate that once the receptor proteins (Orco) are targeted to the ciliary base, heterotrimeric kinesin-2 interacts with Orco and facilitates the transition of the Orco across the ciliary barrier/transition zone into the ciliary OS. Kinesin-2 also helps localise Orco in a defined domain in the distal region of the ciliary OS.

https://doi.org/10.1371/journal.pgen.1009752.g009

The Orco compartment appeared to be distinct from that of the receptor signalling domains organised at the tips of the primary cilia [14] and that of the cilia on ASER neuron of C. elegans [34]. In both these cases, IFT is required for receptor movement. In comparison, the genetic analysis indicated that kinesin-2 might independently control the anterograde transport of Orco and maintain them in an approximately 4 μm domain in the distal OS. The orco domain is separated from that of the kinesin-2 enrichment at the tips with approximately 1 μm offset. The colocalisation study only indicated a partial overlap between the Orco and the tip domain marked by kinesin-2. Therefore, the motor is likely to disengage from Orco once it reaches the designated parking slot in the ciliary membrane.

Further, the FRAP analysis in the kinesin-2 mutant background suggested that Orco diffusion is highly restricted in the cilia. The lipid composition in the ciliary membrane can alter the diffusion coefficient of the transmembrane receptor, such as the Patched-1 [42]. Therefore, altered lipid composition at the distal OS could impede the Orco movement and disengage it from the motor.

The heterotrimeric kinesin-2 is implicated in transporting Rhodopsin [30,43] and Transient Receptor Potential Vanilloid (TRPV) channels [44], via the IFT, although a recent study has contested the claims and suggested that kinesin-2 and IFT function is majorly restricted to the ciliary axoneme assembly [45]. Also, the behaviour analysis in homozygous Ceklp11 and Cekap1 mutant C. elegans suggested that the heterotrimeric kinesin-2 could maintain the chemosensory functioning of the AWB and AWC cilia [6,19] through IFT and BBS/Rab8 transports [7]. Cilia maintenance is also IFT dependent [29]. Hence, it has been difficult to distinguish the independent roles of kinesin-2 and IFT in receptor transport. Consistent with this inference, substantial sensory defects were associated with significant cilia growth defects in the recessive, lethal Klp64D mutants [20]. The isolation of homozygous viable Klp64D mutants with selective olfaction defects and mostly intact cilia further helped identify an independent role of kinesin-2 in the Orco/ORx transport Drosophila.

Role of kinesin-2 in Orco movement across the transition zone

How does the same kinesin-2 complex independently engage in both IFT-based maintenance and Orco/ORx transport? We show that it happens through a time-gated process. The basiconic cilia fully develop during the pupal stages [20]. A previous study showed that compared to the kinesin-2 motor, the ciliary tubulin is highly stable with very low turnover in the adult stage [33], which is confirmed by the low GFP:Tubulin84B enrichment in the cilia when expressed in the adult OSNs using the Orco promoter. Whereas the bulk of the Orco is transported into the cilia at the adult stage immediately after eclosion. Hence, the adult-specific RNAi of kinesin-2 in OSNs had a selectively identifiable impact on Orco transport. The Orco transport in the cilia appears to take place independent of the IFT. The following observations support this assumption. First, an inspection of a large number of images obtained from the orco^fTRG, chaGal4>GFP:Orco and or42bGal4>GFP:Orco backgrounds during the 0–6 hours AE failed to identify instances of punctate Orco localisation in the ciliary IS as observed easily in the chaGal4>Oseg2:GFP antennae. Also, the loss of Oseg2/IFT172 at the adult stage failed to disrupt the Orco localisation. However, more extensive experiments would be needed to entirely exclude the role of IFT in the Orco transport process.

Further, the Orco/ORx entry is specific to the cilia type, and it is tightly regulated during development. What might be the mechanism of such selectivity? Recent studies suggested that post-translational modifications such as SUMOylation trigger the receptor entry into the cilia [46]. Also, concerted activation of tubby-like protein, exocyst complex and small GTPases like Rab8 regulates transmembrane protein delivery to the ciliary pocket. The Bardet-Biedel Syndrome (BBSome) proteins and the Rab-family of small GTPases plays a critical role in the second stage of this transport [13,47]. The entry and exit through the transition zone are also regulated by cell signalling [48,49]. Therefore, a combination of these functions could deliver Orco/ORx at the base of the cilia. The ciliary membrane is isolated from that of the plasma membrane through an elaborate structure, called ciliary sheet and ciliary necklace [10], at the base of the cilia. Therefore, after the Orco/ORx carrying vesicles fuse at the ciliary pocket, the receptor complex must be actively taken across the barrier. We showed that interaction with the heterotrimeric kinesin-2 motor would be responsible for this movement. It will now be interesting to probe how Orco engages kinesin-2 at the ciliary base.

Drosophila culture and stocks

All the fly stocks used in this study are listed in S3 Table. Flies were reared on standard cornmeal agar medium at 25°C for all the studies. All the experiments were done on dissected antennae of adult flies of different ages as per the experimental requirements.

Jump assay

Jump tests were performed using a previously established technique [50] with additional modifications described later [51]. Briefly, 2–4 days old, adult flies were anaesthetised on ice and then collected in batches of five in a glass testing tube (length, 14 cm and diameter, 2 cm). The jump tubes were kept vertically with a cotton plug at the bottom, and the tapered end was connected to a chamber containing concentrated odour. As flies walk up the tube wall due to negative geotaxis, a brief (5 seconds) air puff was bubbled through the odour solution and passed into the jump tube using an aquarium pump. The odour puff causes a startling jump response, and the number of flies jumped within 5 seconds of the stimulus was counted as one response. An average of four such jump responses consisting of 20 flies was calculated as one experimental data point, and this was expressed as the percentage jump response. Five of the six kj lines (353, 925, 1072, 1070, and 429) reported significantly reduced jump response by this assay (Fig 2B). All odorants were diluted 1:2 in paraffin oil.

Ethylmethanesulphonate (EMS) mutagenesis screen (Fig 2A)

Approximately 200 two-day-old isogenised eye colour mutant males (w¹¹¹⁸) were desiccated for 6–8 hours and then left overnight (~16 hours) on tissue paper soaked with 1% sucrose solution containing 26 mM ethyl methanesulphonate (EMS, Sigma Chemical Co., MO, USA). Subsequently, the EMS-fed males were resuscitated on fresh food for one day and crossed to w/w, klp64D^k5 h/TM3 Sb e virgins in 1:4 ratios. The resultant F1 male offspring of the genotype w; */+; */Klp64D^k5 h (*- indicates mutagenised chromosome) were subjected to jump test with n-butanol. Those of which failed to jump were collected and individually crossed to four w/w, Klp64D^k5 h/TM3 Sb e virgins in separate vials. A total of 8000 such lines were set in the F1. The resultant F2 males of the genotype w; */Klp64D^k5 h obtained from each line were further subjected to jump tests for enrichment. Only 1400 lines had greater than 30% jump defects. The w; */Klp64D^k5 h males from these lines were further outcrossed to w/w, Klp64D^k5 h/TM3 Sb e virgins. The process was repeated for six successive generations to remove the mutated first and second chromosomes. In the end, it yielded seven enriched jump defect lines (Fig 2A). All lines were labelled as kj, post-fixed with an isolation identity number, homozygosed through sibling crosses, and tested for jump response defects to various odours. Of these, five were homozygous viable and consistently failed to jump in response to a multiple different odours (Fig 2B).

Drosophila sample preparation

For live imaging of adult Drosophila heads were dissected and mounted in a drop of grade 700 Halocarbon oil (Sigma Chemical Co., MO, USA). For imaging of aged pupal antennae, late third instar larvae were monitored every 30 minutes until they became stationary. It was considered the beginning of the white pupae stage, marked as 0 hours APF, and maintained at 25°C afterwards. The pupal case was opened, and the head was dissected in Phosphate Buffered Saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4 and pH 7.4) at designated times after the 0 hours APF. The dissected heads were fixed in 4% formaldehyde solution (4% Paraformaldehyde in PBS, pH 7.4) for 30 minutes at room temperature, followed by five washes with PBS. The antennae bearing the second and third segments were then pinched off from the head and mounted in a drop of Vectashield (Vector Laboratories Inc., USA) on a glass slide under a 0.17 mm coverslip.

Immunostaining

For Orco immunostaining, 1-day old adult Drosophila heads were dissected, sectioned in a cryo-microtome (Leica, GmBh) and fixed following standardised protocols. 10 μm sections were laid on a poly-D-Lysine coated slide and fixed in 4% formaldehyde solution for 30 minutes at room temperature. Then, they were stained using 1:50 dilutions of MAb-Orco (Abcam, USA) and Alexa 647 conjugated goat anti-mouse (Molecular Probes Inc., USA) following the published method [52].

Dyes microinjection

Both Alexa 546-Dextran (Invitrogen, USA) and BODIPY FL C₁₂ (Invitrogen, USA) dyes were injected into the head capsule of anesthetised adults and incubated for 10 minutes to allow the dyes to permeate the third antennal segment through hemolymph. The exclusion of Alexa 546-Dextran from the sensilla confirmed that the ciliary lymph is isolated from the hemolymph (S9B Fig) and the BODIPY label in the sensillum shaft most likely marks the ciliary membrane.

Image acquisition and analysis

All fluorescence images were obtained under constant acquisition conditions in Olympus (Olympus Imaging Corp., Japan) confocal microscope FV1000SPD using a 63x oil 1.4 NA objective lens or FV3000 using a 60x oil 1.4 NA objective lens. Subsequently, all images were processed using ImageJ (rsweb.nih.gov/1 IJ). All images presented using the false-colour heat map across different time points or genetic backgrounds as placed within a figure panel were collected using identical laser power and gain setting to maintain comparability. Detailed analysis for volume and intensity measurements on 3-D volume-rendered image stacks was done using Imaris 6.1.5 graphics package (Bitplane Scientific Solutions AG, Switzerland). All Figures containing images and line arts were composed in Illustrator (Adobe Inc., USA). The cilia of the OSNs innervating a single sensillum are projected together into the sensory shafts. They are tightly intertwined and could not be resolved using confocal microscopy. Therefore, all the length and volume measurements indicate the combined value of the cilia inside a shaft.

FRAP assay

Adult Drosophila heads, severed at the neck region inside a drop of grade 700 halocarbon oil (RI 1.5), were mounted on a coverslip bottom Petri dish with the antennae pressed against the cover glass. The antennae were imaged using 60x Oil 1.4 NA objective and at 3.5x digital zoom using Olympus FV3000 confocal microscope. Multiple, Z-slices (0.25 μm each) were collected to cover the entire ciliary outer segment at 5-minute intervals with 2 μ-seconds pixel-dwell-time. A rectangular ROI (2 μm x 5 μm) at the distal part of the ciliary OS was photobleached for 2 seconds using 400 μW laser power, and the post bleach images of the entire frame were collected at 5-minute intervals for up to 60 minutes. The frame rate was optimised to avoid excessive photobleaching during the post-bleach imaging.

Estimation of Endo-Orco:GFP and KLP68D:YFP colocalisation

Antennae from Endo-Orco:GFP and chaGal4>KLP68D:YFP adults were separately excited in sequence using 445 nm and 514 nm, respectively, and the emissions were collected in 20 nm bands encompassing 495 to 615 nm. KLP68D:YFP emission was negligible in 495–515 nm band when excited using 445 nm. The Endo-Orco:GFP emission was negligible in the 545–565 nm band when excited by 514nm. Therefore, antennae from Endo-Orco:GFP/chaGal4>KLP68D:YFP stock was sequentially excited at 445 nm and 514 nm, respectively, and corresponding images were collected in sequence at 495–515 nm and 545–565 nm bands, respectively, for the colocalisation study. We used Olympus FV3000 confocal microscope and 60x 1.4 NA Oil objective with 3.5x digital zoom.

Drosophila head extracts

Fly heads were manually separated by razor blades, and homogenised in 250 μl of ice-cold, high KCl buffer [19 mM KH2PO4, 81 mM K2HPO4, 400 mM KCl and 1 mM DTT (pH 7.4)], containing the protease inhibitor cocktail (Roche GmbH). Motorised plastic homogeniser and specially designed microfuge tubes were used for this purpose (Pellet Pestle, Kontes, USA). The homogenate was first centrifuged at 17,000 x g for 30 minutes in a refrigerated micro-centrifuge (Biofuge Fresco, Heraeus AG, Germany) to clear the tissue debris and then the supernatant was further centrifuged at 100,000 x g for 1 hour at 4°C to separate the soluble (supernatant) and membrane-associated proteins (pellet). The pellet was homogenised in 250 μl high KCl buffer containing 1% Triton-X-100 and 1% Sarkosyl, then centrifuged at 100,000 x g for 1 hour at 4 C to extract the membrane-associated proteins, and the resultant supernatant was used as the detergent-extracted fraction for subsequent pulldown experiments.

Affinity copurification

100 μl aliquots of the soluble and the detergent extracted fractions of the Drosophila head homogenates were separately mixed with fixed amounts of purified recombinant proteins in the high KCl buffer. The mixtures containing GST-tagged recombinant proteins were incubated with an equal quantity of Glutathione Sepharose beads for 2 hours at 4°C. Following this, the Glutathione SepharoseTM beads were washed with several changes of the ice-cold, high KCl buffer containing 0.1 mM Glutathione. Finally, the bound proteins were eluted by incubating the beads in 100 μl proportions of the high KCl buffer containing 1 mM Glutathione. 0.3 ml of the eluted fractions was further incubated with 0.3 ml Glutathione SepharoseTM beads and washed and eluted with equal bed volumes of TMN-D containing 1 mM Glutathione. The buffer conditions and salt concentrations were maintained constant throughout this process. The eluates were boiled in sample loading buffer for SDS-PAGE and western analysis.

Western blots and immunostaining

The protein mixtures were separated on SDS-PAGE and transferred to PVDF membrane (Hybond-P, Amersham Biosciences Plc. UK) following the supplier’s protocol and incubated in different primary antisera solutions (1:1000 dilutions) in 20 mM Tris-buffered saline (TBS, pH 7.4) containing 0.1% TweenR 20. Subsequently, they were incubated either in Rabbit anti-mouse:HRP (dilution, 1:20000; Sigma Chemicals Co. MO, USA), or Goat anti-rabbit:HRP (dilution, 1:20000; Sigma Chemicals Co, MO, USA), in the TBS-T, and developed by using ECLR chemiluminescence detection kit (GE Healthcare Ltd. USA).

Statistical analysis

All statistical comparisons were carried out using either student’s T-test or one way ANOVA with p values calculated according to Bonferroni Test in Origin. P values for data sets in each figure are indicated in the respective figure legends.