The olfactory system in animals and humans is optimally tuned to recognize a diverse set of chemical cues and odorants in the environment. In mammals, chemical cues are detected by specialized multi-ciliated olfactory sensory neurons (OSNs) embedded in the olfactory epithelium (OE), transmitting sensory information through action potentials to the olfactory bulb (Firestein, 2001). In most mammalian OSNs, signal transduction is mediated by a canonical cAMP-dependent signaling pathway (Bradley et al., 2005; Kaupp, 2010). Initial binding of an odorant to an olfactory receptor activates a G-protein, Gαolf-coupled cascade that triggers the catalytic activity of adenylyl cyclase 3 (AC3, also known as ADCY3) (Brunet et al., 1996; Jones and Reed, 1989) to generate cAMP. When transiently elevated inside cilia, cAMP opens cyclic nucleotide-gated channels (CNGCs) leading to the influx of Ca2+ ions, which, in turn, activates the ADCY3-dependent chloride channels (TMEM16B, also known as ANO2) as a secondary amplification cascade (Kaupp and Seifert, 2002; Reisert et al., 2005; Stephan et al., 2009). Importantly, all proteins controlling effective recovery from the transient excitation and overwhelming elevation of intraciliary Ca2+, including cAMP hydrolyzing phosphodiesterase 1C, K+-dependent Na+/Ca2+ exchanger and the Ca2+ pump, are localized in the ciliary membrane (Cygnar and Zhao, 2009; Mayer et al., 2009; Saidu et al., 2009; Stephan et al., 2012). Despite all the studies that have dissected the main components of this cascade, much less is understood about how the transduction is tuned and regulated within the cilia microenvironment to support optimal sensitivity and resolution of the incoming sensory information.

It is well known that the constituents and composition of the cell membranes act as regulators of signaling proteins that reside in them. Emerging evidence indicates that the lipid composition of cilia may differ from the bulk of the plasma membrane (Lechtreck et al., 2013; Zhao et al., 2012). Surprisingly, until recently very little attention was given to the organization of olfactory cilia, in particular, to the lipid membrane ensheathing the axoneme and harboring both polytopic and peripheral olfactory signaling proteins. A gradually building body of evidence suggests some organizational complexity to the olfactory ciliary bilayer. Our previous work demonstrated a differential partitioning of various lipid-anchored GFP probes that bind to the inner leaflet of the olfactory cilia membrane (Williams et al., 2014). This suggested the presence of ciliary membrane domains with distinct lipid compositions. In addition, the cholesterol-binding protein caveolin-1 (CAV-1) has been implicated as a scaffold to localize proteins in the odor detection pathway to lipid raft domains (Schreiber et al., 2000). In line with these findings, the olfactory CNGA2 channel has been shown not only to have a spatially restricted localization in primary cilium (PC) but also to be functionally regulated by cholesterol (Brady et al., 2004; Jenkins et al., 2006). Another cholesterol binding protein, stomatin-like protein 3 (SLP3; also known as STOML3), was identified in OSNs and localized to the transition zone (TZ) of olfactory cilia (Kobayakawa et al., 2002; Tadenev et al., 2011). Intriguingly, SLP3 co-immunoprecipitated with AC3 and CAV-1 from olfactory cilia isolates (Kobayakawa et al., 2002). Indeed, CAV-1 is not only localized to the PC in other cells types but is also implicated in the regulation of cilia length and sonic hedgehog signaling via a polyphosphoinositide (PI)-dependent pathway (Maerz et al., 2019; Rangel et al., 2019; Schou et al., 2017).

We now know that PIs are involved in specific aspects of sensory function. For example, elevation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) within olfactory cilia can inhibit the CNGCs (Brady et al., 2006; Spehr et al., 2002), whereas odorant stimulation may induce dynamic redistribution of phosphatidylinositol (4,5)-bisphosphate (PIP2) in the dendritic knob of OSNs (Ukhanov et al., 2016). Recently, PIs were discovered to play a role in ciliogenesis and regulation of ciliary function (Garcia-Gonzalo et al., 2015; Phua et al., 2017). The interplay between the two PIs, PIP2 and phosphatidylinositol 4-phosphate (PI4P), is crucial to the organization of the cilia TZ, and controls protein trafficking and signaling within the PC (Garcia-Gonzalo et al., 2015; Garcia et al., 2018; Phua et al., 2017; Xu et al., 2016). The localization and relative abundance of these two PIs, were found to be in dynamic reciprocity to each other and under the tight control of INPP5E, a phosphoinositide 5′-phosphatase that hydrolyzes PIP2 and PIP3 (Bielas et al., 2009; Hasegawa et al., 2016; Kisseleva et al., 2000). Each of the PIs, hydrolyzed by INPP5E, represents a small fraction of all membrane-associated lipids, but plays an indispensable role in regulating many aspects of cellular physiology including cell division, vesicle trafficking and control of transmembrane ionic transport (Balla, 2013; Di Paolo and De Camilli, 2006; Hilgemann et al., 2018; Logothetis et al., 2015). Importantly, mutations in the Inpp5e gene cause its loss-of-function due to mislocalization or impairment in catalytic activity and manifest in a ciliopathy termed Joubert syndrome (JBTS).

To better understand the role of lipids and specifically PIs in the cell biology of olfactory cilia, we sought to investigate the localization and relative abundance of several lipids by utilizing a conditional Inpp5e-deficient mouse mutant. A panel of highly selective probes to several important classes of lipids were used in live mouse OSNs in situ. Using this approach and mouse model allowed us, for the first time, to analyze the distribution and functional implication of JBTS ciliopathy-related changes to the phospholipid composition of cilia in terminally differentiated mammalian sensory neurons.

INPP5E hydrolyzes two phosphoinositide species PIP2 and PIP3 with high affinity generating PI4P and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2], respectively (Kisseleva et al., 2000; Kong et al., 2000). Distribution of PIP2 in mature OSNs was measured using en face confocal microscopy of intact olfactory epithelium transduced with adenovirus encoding the PLCδ1-PH (PLCPH) domain tagged with GFP (Fig. 1). In wild-type (WT) littermate control mice, 52.7±10.8% (n=318, 4 mice) of cells infected with the PLCPH probe had an extremely polarized distribution of PIP2 with an accumulation in the OSN knob and no ciliary localization (Fig. 1A,C; Fig. S1B; note, all results presented in the main text are given as mean±s.e.m.). In those cells, PIP2 was uniformly distributed in the plasma membrane of the knob and adjacent dendrite and extended all the way to the axons (Fig. 1A, right panel; Fig. S1B,C). The total number of cilia (21.8±0.5 cilia per OSN; n=37, 4 mice) and cilia length (29.5±0.5 µm; n=753, 4 mice) was measured by co-expression of an inert lipid-anchored probe MyrPalm fused to mCherry (MP-mCherry) (Fig. 1A, middle panel). In a fraction of OSNs, however, we detected PIP2 in a small subset of cilia ranging from one to five cilia per neuron, but this allocation was much fewer than the total number of cilia (Fig. 1C; Fig. S1B). The distribution of PIP2 along the length of a given cilium was highly variable and ranged from a short segment to the full length (Fig. 1A,D). A full-length distribution of PIP2 was rare and often seen in only a single cilium. Overall distribution of PIP2 and MP-mCherry in the WT cilia resulted in non-overlapping histograms, as summarized in Fig. 1D.

Fig. 1.

Loss of INPP5E causes redistribution of PIP2 and elongation of cilia in mouse OSNs. (A) PLCPH–GFP, a probe for PIP2, is mostly restricted to the knob of WT OSNs. In a small percentage of OSNs, a ciliary segment of varying length up to the full length (black arrows) is also enriched in PIP2. The inert membrane-bound lipid probe MP–mCherry was used as a counterstain to label the full length of axoneme, and does not have the highly restricted localization of PLCPH-GFP resulting in overlapping colors (middle panel, white). PIP2 was evenly distributed in the plasma membrane of the knob as shown in z-stack view (right panel). Yellow lines denote z-stack projection shown at the bottom and right side of the image. (B) In contrast to what is seen in the WT, in Inpp5eosnKO (INPP5E-KO) PLCPH–GFP decorated the entire length of every cilium. A color-shifted image is shown to accentuate the equal distribution of PLCPH–GFP and MP–mCherry labeling (B, middle panel). The PIP2 redistribution is evident also in the z-stack side view showing substantial enrichment at the base of cilia and along the proximal segment (PS, red arrows) whereby PIP2 level in the knob periciliary plasma membrane was not changed (right panel). (C) More than 50% of WT OSNs showed no PIP2 in their cilia. 18% of OSNs had only a single PIP2-positive cilium whereas three other groups of neurons equally represented the remaining 30%. Conversely, PIP2 was detected in 100% of OSNs in Inpp5eosnKO (KO, green bar). A total of 318 cells in 4 mice were analyzed in the WT group and 36 cells in 3 mice were analyzed in the KO group. (D) Length distribution within the same sets of cells of PLCPH–GFP-positive aspects of cilia (PIP2 domain) in WT was substantially shifted to shorter values compared to the full cilia length measured with MP-mCherry, yielding 29.5±0.5 µm (n=753, 4 mice). (E) Distribution of both PLCPH–GFP and MP–mCherry length values showed a complete overlap in Inpp5eosnKO OSNs. The average full ciliary length in the KO OSNs, 35.3±0.6 µm (n=495, 3 mice) was significantly longer than in the WT (unpaired t-test, t=7.363, d.f.=1246, P<0.0001). Data shown as mean±s.e.m.

Fig. 1.

Loss of INPP5E causes redistribution of PIP2 and elongation of cilia in mouse OSNs. (A) PLCPH–GFP, a probe for PIP2, is mostly restricted to the knob of WT OSNs. In a small percentage of OSNs, a ciliary segment of varying length up to the full length (black arrows) is also enriched in PIP2. The inert membrane-bound lipid probe MP–mCherry was used as a counterstain to label the full length of axoneme, and does not have the highly restricted localization of PLCPH-GFP resulting in overlapping colors (middle panel, white). PIP2 was evenly distributed in the plasma membrane of the knob as shown in z-stack view (right panel). Yellow lines denote z-stack projection shown at the bottom and right side of the image. (B) In contrast to what is seen in the WT, in Inpp5eosnKO (INPP5E-KO) PLCPH–GFP decorated the entire length of every cilium. A color-shifted image is shown to accentuate the equal distribution of PLCPH–GFP and MP–mCherry labeling (B, middle panel). The PIP2 redistribution is evident also in the z-stack side view showing substantial enrichment at the base of cilia and along the proximal segment (PS, red arrows) whereby PIP2 level in the knob periciliary plasma membrane was not changed (right panel). (C) More than 50% of WT OSNs showed no PIP2 in their cilia. 18% of OSNs had only a single PIP2-positive cilium whereas three other groups of neurons equally represented the remaining 30%. Conversely, PIP2 was detected in 100% of OSNs in Inpp5eosnKO (KO, green bar). A total of 318 cells in 4 mice were analyzed in the WT group and 36 cells in 3 mice were analyzed in the KO group. (D) Length distribution within the same sets of cells of PLCPH–GFP-positive aspects of cilia (PIP2 domain) in WT was substantially shifted to shorter values compared to the full cilia length measured with MP-mCherry, yielding 29.5±0.5 µm (n=753, 4 mice). (E) Distribution of both PLCPH–GFP and MP–mCherry length values showed a complete overlap in Inpp5eosnKO OSNs. The average full ciliary length in the KO OSNs, 35.3±0.6 µm (n=495, 3 mice) was significantly longer than in the WT (unpaired t-test, t=7.363, d.f.=1246, P<0.0001). Data shown as mean±s.e.m.

To get insight in the regulation of phospholipids, in particular PIP2, in olfactory cilia and OSNs, we utilized an olfactory-specific conditional knockout mouse Inpp5eosnKO. The mutant was generated by crossing Inpp5eloxP founder described previously (Jacoby et al., 2009) with a mouse carrying Cre-recombinase under the promoter of the olfactory marker protein (OMP), which is expressed exclusively in mature OSNs (Green et al., 2018). Consistent with previous transcriptomic and proteomic data in OSNs (Kuhlmann et al., 2014; Nickell et al., 2012), western blot data (representative images and densitometry) of OE extracts show protein expression of a doublet at ∼72 kDa, corresponding to the WT INPP5E and a splice variant (Jacoby et al., 2009), that is decreased in the Inpp5eosnKO mouse (Fig. S1A). The remaining signal likely reflects the presences of multiple cell types in the OE. The loss of INPP5E in OSNs of Inpp5eosnKO mouse severely impacted ciliary PIP2 distribution resulting in its homogenous redistribution along the entire axoneme (Fig. 1B). Remarkably, this deficiency affected every cilium (Fig. 1C, KO) shifting distribution of PIP2 domain length to a complete overlap with that of the ciliary length marker MP-mCherry (Fig. 1E). Another salient feature of the PIP2 localization in the KO cilia was its abundance within the proximal segment of each cilium, overlapping with the TZ (Fig. 1B, right panel, red arrows). Notably, the mean cilia length was significantly increased from 29.5±0.5 µm in WT littermates to 35.3±0.6 µm in the Inpp5eosnKO mice (n=495, 3 mice, unpaired t-test, t=7.363, d.f.=1246, P<0.0001). Cilia length is controlled by an evolutionarily conserved process of intraflagellar transport (IFT) (Rosenbaum and Witman, 2002). The loss of INPP5E impacts IFT in primary cilia, resulting in the selective accrual of IFT-A particles (Chávez et al., 2015; Garcia-Gonzalo et al., 2015). Surprisingly, we did not find any abnormality in the velocity of IFT-A-dependent transport of IFT122 particles or its accumulation inside olfactory cilia of Inpp5eosnKO mice (Fig. S2A,B, Movie 1). IFT-B-related trafficking of IFT88 was also unaltered, with a similar particle velocity to that published previously for the wild-type OSNs (Uytingco et al., 2019; Williams et al., 2014) (Fig. S2C–E, Movie 2).

To address the potential of virally assisted therapy of the JBTS ciliopathy model in vivo, we used a rescue adenoviral vector carrying the full-length sequence of human INPP5E (NM_019892) fused with GFP on the N-terminal, GFP–INPP5E-FL (Chávez et al., 2015). Ectopically expressed GFP–INPP5E-FL was enriched in the OSN knobs and localized to the full length of cilia in the WT (Fig. S1D) and KO mouse (Fig. 2A). As shown in Fig. 2, full-length WT INPP5E was necessary for restoration of normal PIP2 distribution in OSNs. Ectopic expression of GFP–INPP5E-FL in Inpp5eosnKO OSNs resulted in a significant decrease of PIP2 ciliary domain length as measured with PLCPH-mCherry (Fig. 2B,C). The average length of the PIP2 domain in WT cilia was 4.9±0.27 µm (n=110, 16 cells, 3 mice), in Inpp5eosnKO cilia 28.5±1.37 µm (n=54, 5 cells, 3 mice) and in rescued KO cilia 4.2±0.3 µm (n=122, 17 cells, 3 mice) [P<0.0001, one-way ANOVA, F(DFn, DFd) 86.73 (2283)] (Fig. 2D). As a negative control we used a catalytically inactive point mutant GFP–INPP5E-D477N (Chávez et al., 2015), which failed to change localization of PIP2 when co-expressed with PLCPH–mCherry in HEK293 cells (Fig. S3). Co-expression of PLCPH–mCherry with GFP–INPP5E-D477N resulted in a significantly larger number of OSNs having a complement of PIP2-decorated cilia, 61.2±0.05% (D477N, n=61, 3 mice) compared to INPP5E-WT, 17.6±0.09% (INPP5E-WT, n=83, 3 mice) (P=0.0001, unpaired t-test, t=4.536, d.f.=24) (Fig. 2E–H). Together, these data indicate that the catalytic activity of INPP5E is required for restricting the distribution of PIP2 in olfactory cilia.

Fig. 2.

Virally induced ectopic expression of full-length WT human INPP5E tagged with GFP completelyreversed mislocalization of PIP2 in Inpp5eosnKO mouse cilia. (A,B) Inpp5eosnKO mice were infected at P8–P14 with a triple dose of Ad-GFP-INPP5E-WT and tested 8–10 days later. GFP–INPP5E-WT is enriched in OSN knobs and also localizes to cilia. The KO mice were co-infected with PLCPH–mCherry to measure rescue of the PIP2 localization. Several knobs of co-infected OSNs are indicated with arrowheads. (C) Magnified dual-color view of the area marked with a square in B shows several knobs of OSNs co-infected with both viruses (arrowheads) resulting in a complete loss of ciliary PIP2 (magenta). (D) Rescue was quantified by measuring length of PIP2 positive ciliary aspect in the WT littermates and KO mice. The KO OSNs were identified within the same preparation by a strong ciliary distribution of PLCPH–mCherry, and also lacking any detectable GFP–INPP5E-WT fluorescence. Rescue completely reversed Inpp5eosnKO deficiency [PIP2 domain length 4.9±0.27 µm (n=110, 16 cells, 3 mice), WT; 28.5±1.37 µm (n=54, 5 cells, 3 mice), KO; 4.2±0.3 µm (n=122, 17 cells, 3 mice), Rescue, one-way ANOVA, F(DFn, DFd) 86.73 (2283), ****P<0.0001]. ns, not significant. (E,G) Inpp5eosnKO KO mice in a different group were infected with Ad-PLCPH-mCherry and Ad-GFP-INPP5E-D477N encoding for catalytically inactive phosphatase. The GFP–NPP5E-D477N mutant was localized to the full cilia length (E). Knobs of co-infected OSNs showing no change in PLCPH ciliary localization are marked with solid arrows. Some knobs had less PLCPH probe localized to cilia (open arrows) reminiscent of the KO phenotype. (G,H) Expression of GFP–INPP5E-D477N resulted in a significantly smaller number of OSNs having a complement of PIP2-decorated cilia. This reduction was quantified in H, 17.6±0.09% (D477N, n=61, 3 mice), compared to GFP–INPP5E-WT, 61.2±0.05% (INPP5E-WT, n=83 cells, 3 mice), unpaired t-test, t=4.536, d.f.=24, ***P=0.001). Data shown as mean±s.e.m.

Fig. 2.

Virally induced ectopic expression of full-length WT human INPP5E tagged with GFP completelyreversed mislocalization of PIP2 in Inpp5eosnKO mouse cilia. (A,B) Inpp5eosnKO mice were infected at P8–P14 with a triple dose of Ad-GFP-INPP5E-WT and tested 8–10 days later. GFP–INPP5E-WT is enriched in OSN knobs and also localizes to cilia. The KO mice were co-infected with PLCPH–mCherry to measure rescue of the PIP2 localization. Several knobs of co-infected OSNs are indicated with arrowheads. (C) Magnified dual-color view of the area marked with a square in B shows several knobs of OSNs co-infected with both viruses (arrowheads) resulting in a complete loss of ciliary PIP2 (magenta). (D) Rescue was quantified by measuring length of PIP2 positive ciliary aspect in the WT littermates and KO mice. The KO OSNs were identified within the same preparation by a strong ciliary distribution of PLCPH–mCherry, and also lacking any detectable GFP–INPP5E-WT fluorescence. Rescue completely reversed Inpp5eosnKO deficiency [PIP2 domain length 4.9±0.27 µm (n=110, 16 cells, 3 mice), WT; 28.5±1.37 µm (n=54, 5 cells, 3 mice), KO; 4.2±0.3 µm (n=122, 17 cells, 3 mice), Rescue, one-way ANOVA, F(DFn, DFd) 86.73 (2283), ****P<0.0001]. ns, not significant. (E,G) Inpp5eosnKO KO mice in a different group were infected with Ad-PLCPH-mCherry and Ad-GFP-INPP5E-D477N encoding for catalytically inactive phosphatase. The GFP–NPP5E-D477N mutant was localized to the full cilia length (E). Knobs of co-infected OSNs showing no change in PLCPH ciliary localization are marked with solid arrows. Some knobs had less PLCPH probe localized to cilia (open arrows) reminiscent of the KO phenotype. (G,H) Expression of GFP–INPP5E-D477N resulted in a significantly smaller number of OSNs having a complement of PIP2-decorated cilia. This reduction was quantified in H, 17.6±0.09% (D477N, n=61, 3 mice), compared to GFP–INPP5E-WT, 61.2±0.05% (INPP5E-WT, n=83 cells, 3 mice), unpaired t-test, t=4.536, d.f.=24, ***P=0.001). Data shown as mean±s.e.m.

One of the main routes of PIP2 synthesis is thought to be by PI5K and PI4K-dependent phosphorylation of PI4P and PI(5)P, respectively (Schramp et al., 2015). PI4P was shown to be highly enriched in PC of several cell types (Chávez et al., 2015; Garcia-Gonzalo et al., 2015) and under the tight control of INPP5E which seems not to use PI5P as a substrate (Conduit et al., 2017; Kisseleva et al., 2000; Madhivanan et al., 2015; Schramp et al., 2015). Adenoviral expression of a probe specific for PI4P, P4M-SidM (Hammond et al., 2014) tagged with mCherry showed low abundance in the olfactory cilia of the control WT mice (Fig. 3A, top panel). Conversely, in most OSNs, PI4P was highly enriched in the knob (Fig. 3A). We directly compared levels of PI4P in the knobs of WT and Inpp5eosnKO by measuring absolute fluorescence intensity. In the Inpp5eosnKO OSNs the mean level of PI4P showed a slight but not significant decrease (Fig. 3D; 179±26 units, WT, n=94, 3 mice and 143±17 units, KO, n=54, 3 mice; t-test, t=0.9777, d.f.=146, P=0.3298). Besides PIP2, INPP5E also dephosphorylates PIP3 at even higher efficiency than PIP2, generating PI(3,4)P2 (Conduit et al., 2012). PI(3,4)P2 was measured using ectopic expression of the Tapp1-PH domain (Fukumoto et al., 2017) and was found to be mostly restricted to the knobs with a low level in cilia. Its distribution pattern was not changed by the loss of INPP5E (Fig. 3B). However, quantitative analysis of PI(3,4)P2 revealed significant depletion in the OSN knobs of Inpp5eosnKO mice (Fig. 3E, 280±11 units, n=830, 3 mice, WT; 174±7, n=858, 3 mice, KO; t-test, t=8.453, d.f.=1686, P<0.0001). Finally, to assay the distribution of PIP3, we used a GFP-tagged PH domain of Bruxton tyrosine kinase (Btk–GFP), a well-characterized highly selective PIP3 lipid probe (Balla, 2013). Similar to both PI4P and PI(3,4)P2, PIP3 was highly enriched in the OSN knob, with relatively low quantities in cilia (Fig. 3C, upper panel). Several other probes selective for PIP3 based on the PH domains of ARNO, Akt and Grp1 proteins showed an identical distribution to Btk–GFP in OSNs (data not shown). Intriguingly, quantitative analysis of PIP3 in OSN knobs of Inpp5eosnKO showed a significant increase, by nearly 3-fold (Fig. 3F; 668±64 units, n=60, 3 mice, WT; 1495±185, n=91, 3 mice, KO; unpaired t-test, t=3.536, d.f.=149, P=0.0005) with very little if any build-up in cilia (Fig. 3C, KO bottom panel). An inert membrane lipid anchor probe MP-mCherry did not show any preferred partitioning in the membrane in OSN knobs in the WT and the KO (Fig. 3G, MP-mCherry, 340±31 units, n=46, 3 mice, WT; 378±23 units, n=70, 3 mice, KO; t-test, t=1.001, d.f.=114, P=0.3188).

Fig. 3.

Other phosphoinositides than PIP2in mouse OSNs are almost exclusively restricted to the knobs and changed their level in an INPP5E-dependent manner. (A,D) The location of the PI(4)P probe mCherry–P4M-SidM was not significantly affected by loss of INPP5E showing only insignificant trending decrease in the knobs (179±26 relative units, WT, n=94, 3 mice; 143±17 relative units, KO, n=54, 3 mice; unpaired t-test, t=0.9777, d.f.=146, P=0.3298). (B,E) A tandem PH domain, Tapp1 tagged with GFP, was used to specifically label membrane PI(3,4)P2, which was found to only be enriched in the knobs and in cilia in a small fraction of OSNs. Importantly, the overall pattern of PI(3,4)P2 distribution did not change in Inpp5eosnKO. Fluorescence intensity, however, measured in OSN knobs showed a significant decrease in the KO compared to WT mice (280±11 relative units, n=830, 3 mice, WT; 174±7 relative units, n=858, 3 mice, KO; unpaired t-test, t=8.453, d.f.=1686, P<0.0001). (C,F) PIP3 detected with a Btk-PH domain tagged with GFP, was restricted mostly to the knobs with a relatively low presence in cilia of the WT and KO. Quantitative analysis of fluorescence showed increase of the intensity in the knobs of the KO (668±64 relative units, n=60, 3 mice, WT; 1495±185 relative units, n=91, 3 mice, KO; unpaired t-test, t=3.536, d.f.=149, ***P=0.0005). (G) Fluorescence intensity of MP–mCherry, used as a negative control, was not significantly different in the OSN knobs of WT and KO mice (340±31 relative units, n=46, 3 mice, WT; 378±23 relative units, n=70, 3 mice, KO; unpaired t-test, t=1.001, d.f.=114, P=0.3188). Data shown as mean±s.e.m.

Fig. 3.

Other phosphoinositides than PIP2in mouse OSNs are almost exclusively restricted to the knobs and changed their level in an INPP5E-dependent manner. (A,D) The location of the PI(4)P probe mCherry–P4M-SidM was not significantly affected by loss of INPP5E showing only insignificant trending decrease in the knobs (179±26 relative units, WT, n=94, 3 mice; 143±17 relative units, KO, n=54, 3 mice; unpaired t-test, t=0.9777, d.f.=146, P=0.3298). (B,E) A tandem PH domain, Tapp1 tagged with GFP, was used to specifically label membrane PI(3,4)P2, which was found to only be enriched in the knobs and in cilia in a small fraction of OSNs. Importantly, the overall pattern of PI(3,4)P2 distribution did not change in Inpp5eosnKO. Fluorescence intensity, however, measured in OSN knobs showed a significant decrease in the KO compared to WT mice (280±11 relative units, n=830, 3 mice, WT; 174±7 relative units, n=858, 3 mice, KO; unpaired t-test, t=8.453, d.f.=1686, P<0.0001). (C,F) PIP