Abstract
Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. Here, we report a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in TZ maturation in the Drosophila melanogaster male germline. We show that reduction of cellular PIP2 levels by ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. We also report that the onion rings (onr) allele of Drosophila exo84 decouples TZ hyperelongation from loss of cilium-plasma membrane tethering. Our results reveal a requirement for PIP2 in supporting ciliogenesis by promoting proper TZ maturation.
Brief summary statement The authors show that the membrane phospholipid PIP2, and the kinase that produces PIP2 called Skittles, are needed for normal ciliary transition zone morphology and function in the Drosophila male germline.
Introduction
Cilia are sensory organelles that are important for signalling in response to extracellular cues, and for cellular and extracellular fluid motility [1, 2, 3, 4]. Consistent with their importance, defects in cilium formation (i.e. ciliogenesis) are associated with genetic disorders known as ciliopathies, which can display neurological, skeletal and fertility defects, in addition to other phenotypes [5, 6, 7, 8]. Many ciliopathies are associated with mutations in proteins that localize to the transition zone (TZ), the proximal-most region of the cilium that functions as a diffusion barrier and regulates the bidirectional transport of protein cargo at the cilium base [9, 10]. For example, the conserved TZ protein CEP290 is mutated in at least six different ciliopathies [11] and is important for cilium formation and function in humans [12, 13] and Drosophila [14]. Although the protein composition of TZs has been investigated in various studies [15], the process of TZ maturation, through which it is converted from an immature form to one competent at supporting cilium assembly, is relatively understudied.
Ciliogenesis begins with assembly of a nascent TZ at the tip of the basal body (BB) [9]. During TZ maturation, its structure and protein constituents change, allowing for establishment of a compartmentalized space, bounded by the ciliary membrane and the TZ, where assembly of the axoneme, a microtubule-based structure that forms the ciliary core, and signalling can occur. In Drosophila, nascent TZs first assemble on BBs during early G2 phase in primary spermatocytes [16]. This occurs concomitantly with anchoring of cilia to the plasma membrane (PM), microtubule remodelling within the TZ [17, 18], and establishment of a ciliary membrane that will persist through meiosis [16] (Figure 1A). TZ maturation has been described in Paramecium [19], Caenorhabditis elegans [20] and Drosophila [18], and is most readily observed by an increase in TZ length in the Drosophila male germline.
We previously showed that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for formation of the axoneme in the Drosophila male germline [21, 22]. PIP2, which is one of seven different phosphoinositides (PIPs) present in eukaryotes, localizes primarily to the PM, where it is required for vesicle trafficking, among other processes [23]. PIP2 has recently been linked to cilium function. Although the ciliary membrane contains very little PIP2 due to the action of the cilium resident PIP phosphatase INPP5E, the base of the cilium is enriched in PIP2 [24]. Inactivation of INPP5E causes a build up of intraciliary PIP2, which disrupts transport of Hedgehog signalling proteins in vertebrates [25, 26, 27] and ion channels involved in mechanotransduction in Drosophila [28]. In light of the current understanding of PIP2 as a modulator of cilium function, we sought to investigate the cause of defects we had observed in axoneme assembly in Drosophila male germ cells with reduced levels of PIP2 [21, 22].
Results
PIP2 is essential for transition zone maturation
To investigate how reduction of cellular PIP2 affects ciliogenesis in the Drosophila male germline, we used transgenic flies expressing the Salmonella PIP phosphatase SigD under control of spermatocyte-specific β2-tubulin promoter (hereafter β2t-SigD) [21]. To examine whether axoneme defects in β2t-SigD [21] were caused by aberrant TZ function, we examined localization of fluorescently-tagged versions of the core centriolar/BB protein Ana1 (CEP295 homolog) [29, 30] and the conserved TZ protein Cep290 [14] during early steps of cilium assembly. Cep290 distribution appeared similar in control and β2t-SigD in early G2 phase, when TZs are still immature. In contrast, Cep290-labelled TZs were significantly longer in β2t-SigD compared to controls by late G2 phase, following the period of TZ maturation (Figure 1B and 1C). Unlike Drosophila cep290 mutants, which contain longer than normal BBs [14], Ana1 length was not affected in β2t-SigD, and we did not observe a strong correlation between Cep290 and Ana1 lengths (Figure 1D). Consistent with this result, the ultrastructure of BBs in β2t-SigD is normal, and localization of the centriolar marker GFP-PACT [31] is similar, in controls and β2t-SigD [21]. In contrast, TZ proteins Chibby (Cby) [32] and Mks1 [33, 34] exhibited hyperelongation in β2t-SigD (Figure 1E), indicating that this phenotype is not unique to Cep290. TZ hyperelongation was a highly penetrant phenotype (>70%) and showed high correlation (>0.95) within syncytial germ cell cysts, suggestive of a dosage-based response to a shared cellular factor, presumably SigD. Despite persistence of hyperelongated TZs through meiosis, axonemes were able to elongate normally in post-meiotic cells (Figure 1F). Nonetheless, the ultrastructure of these axonemes is frequently aberrant, either lacking nine-fold symmetry or containing triplet microtubules in addition to the usual doublets [21].
The type I PIP kinase Skittles regulates TZ length
Although PIP2 is its major substrate in eukaryotic cells in vivo [35, 36, 37], SigD can de-phosphorylate multiple PIPs in vitro [38]. To address whether TZ hyperelongation observed in β2t-SigD represented a physiologically relevant phenotype due to decreased PIP2, we at-tempted to rescue this phenotype by co-expressing β2t-SigD with fluorescently-tagged Skittles (Sktl) under control of β2-tubulin promoter. We found that Sktl expression was able to suppress TZ hyperelongation in a cilium-autonomous manner (Figure 2A and 2B). Furthermore, the BB/TZ protein Unc-GFP [39, 21], exhibited TZ hyperelongation at a low penetrance in sktl2.3 mutant clones (Figure 2C), indicating that Sktl is important for TZ maturation.
Vertebrate type I PIP kinase PIPKIγ has previously been shown to be important for cilium formation in cultured cells [40]. The two Drosophila PIPKIs, Sktl and PIP5K59B, arose from recent duplication of the ancestral PIPKI gene, and are not orthologous to specific vertebrate PIPKI isoforms (Figure 2D). Sktl has diverged more than its paralog PIP5K59B and seems to be functionally related to PIPKIγ and the C. elegans PPK-1 in having roles at cilia [41]. However, unlike the human PIPKIγ, which licenses TZ assembly by promoting CP110 removal from BBs [40], our results suggest that Sktl functions in regulating TZ length but not TZ assembly. Notably, neither inactivation nor overexpression of cp110 affects cilium formation in Drosophila, and Cp110 is removed from BBs in early primary spermatocytes [42].
Hyperelongated transition zones exhibit functional defects
We next sought to examine whether TZ hyperelongation due to SigD expression affected TZ function. Following meiosis in the Drosophila male germline, TZs detach from the BB and migrate along the growing axoneme, maintaining a ciliary compartment at the distal-most ~5μm where tubulin is incorporated into the axoneme [14, 43]. As shown by Unc and Cep290 localization, TZs in β2t-SigD were frequently incapable of detaching from BBs and migrating along axonemes despite axoneme and cell elongation (Figures 1E, 3A and 3B). Indeed, the previously reported “comet-shaped” Unc-GFP localization in β2t-SigD [21] persists during cell elongation after meiosis (Figure 3A, bottommost panel) despite elongation of the axoneme (Figure 1E).
In Drosophila and humans, BBs consist of microtubule triplets [44, 45], whereas axonemes contain microtubule doublets due to obstruction of C-tubules at the TZ [18]. Consistent with a defect in this barrier and the presence of microtubule triplets in axonemes in β2t-SigD [21], a subset of cilia (<5%) in β2t-SigD contained puncta of Ana1 at the distal tips of TZs (Figure 3C). Treatment of germ cells with the microtubule-stabilizing drug Taxol increased the penetrance of this phenotype from <5% in untreated cells to >25% in cells treated with 4 μM Taxol (arrowheads in Figure 3D) without significantly affecting Cep290 length (Figure 3E). Taxol-treated controls did not exhibit TZ-distal Ana1 puncta (p <0.01 at 5% penetrance). Fluorescently-tagged Asterless (CEP152 homolog), a pericentriolar protein [46, 47], did not localize to TZ-distal puncta in β2t-SigD (p <0.01) suggesting that these TZ-distal sites are not fully centriolar in protein composition. Taxol has been hypothesized to disrupt TZ maturation by inhibiting microtubule remodelling in the Drosophila male germline [17]. Similar to β2t-SigD, Taxol-treated male germ cells assemble extremely long axonemes that contain triplet microtubules [17], further supporting a functional relationship between PIP2 and microtubule reorganization in TZ maturation.
The onion rings (onr) mutant decouples defects found in cells with reduced levels of PIP2
Male flies homozygous for the onion rings (onr) mutant of Drosophila exo84 are sterile and exhibit defects in cell elongation and polarity similar to β2t-SigD [21]. Exo84 is a component of the octameric exocyst complex, which binds PIP2 and regulates membrane trafficking at the PM [48]. To investigate whether defects in TZ hyperelongation could be explained by defective Exo84 function, we examined TZs in onr mutants. Unlike β2t-SigD, onr did not display hyperelongated TZs (Figure 4A), suggesting that Exo84 is dispensable for TZ maturation.
Due to involvement of the exocyst in trafficking at the PM, we examined whether cilium-associated membranes were affected in β2t-SigD or onr mutants in a manner similar to dilatory; cby mutants [33]. Dilatory (Dila), a conserved TZ protein, cooperates with Cby to assemble TZs in the Drosophila male germline [33]. Whereas TZs in β2t-SigD and onr cells were able to dock at the PM initially, they were unable to maintain membrane connections, and were rendered cytoplasmic (Figure 4B and C), similar to TZs in dila; cby mutants. We found that fluorescently-tagged Exo70, a PIP2-binding exocyst subunit, localized to BBs (Figure 4D). Our results suggest that the exocyst, and Exo84 in particular, regulates cilium-PM associations, similar to PIP2, and that TZ hyperelongation and loss of cilium-PM association are genetically separable phenotypes.
Discussion
The process of maturation of a TZ from a nascent form to a fully functional state, leading ultimately to axoneme assembly and ciliary signalling, requires orchestration of various pro-teins and cellular pathways [9, 15]. Our results indicate that normal execution of this process requires PIP2 and that depletion of PIP2 induces TZs to grow longer than normal. Similar to β2t-SigD, Drosophila dila; cby and cby mutants display hyperelongated TZs [32, 33], whereas mks1 mutants have shorter TZs [34]. Because both Cby and Mks1 are hyperelongated in β2t-SigD cells, PIP2 regulates TZ length independently of an effect on Cby or Mks1 recruitment.
We also show that hyperelongated TZs are dysfunctional. Similar to dila; cby [33] and cep290 [14] mutants, axonemes can assemble in β2t-SigD, albeit with aberrant ultrastructure [21], despite the lack of functional TZs or membrane association. The presence of TZ-distal Ana1 puncta in β2t-SigD cells, without the increase in BB length seen in cep290 mutants lacking a functional TZ barrier, suggests that β2t-SigD expression selectively disrupts the ability of TZs to restrict C-tubules and Ana1 without abolishing the TZ barrier entirely. CEP295, the human Ana1 ortholog, regulates post-translational modification of centriolar microtubules [49], which may explain the presence of Ana1 along with supernumerary microtubules in β2t-SigD cells. Asterless (Asl), a pericentriolar protein important for centrosome formation and centriole duplication [46, 47], did not exhibit this TZ-distal localization, possibly due to differences dynamics of Ana1 and Asl loading onto centrioles [50, 51] or the more peripheral nature of Asl within the centriole [46].
The majority of PIP2 at the PM is produced by PIPKIs [23, 52]. In this study, we showed that mutation of the PIPKI Sktl induced hyperelongated TZs and that expression of Sktl could suppress TZ hyperelongation in β2t-SigD, with some cells showing cilium-autonomous suppression, suggesting Sktl might function in situ to regulate TZ length. In humans, PIPKIγ is linked to lethal congenital contractural syndrome type 3 (LCCS3), which has been suggested to represent a ciliopathy [40]. The recent discovery of a role for LCCS1-associated GLE1 protein in cilium function [53] corroborates this hypothesis. Our data support the idea that PIPKIs might represent ciliopathy-associated genes or genetic modifiers of disease.
Members of the exocyst complex such as Sec10 and Sec8 are important for cilium formation in cultured cell lines and zebrafish [54, 55, 56], but their precise roles in ciliogenesis are not well understood. The subunits Sec3 and Exo70 regulate exocyst targeting to the plasma membrane through a direct interaction with PIP2 [48, 57]. We previously showed that the onr allele of Drosophila exo84 phenocopies defects in cell polarity and elongation observed in β2t-SigD [22]. Here, we show that the onr mutation phenocopies the loss of cilium-membrane contacts in β2t-SigD, similar to dila; cby mutants [33], but not TZ hyperelongation. Thus, TZ hyperelongation is not a prerequisite for the failure of cilium-PM association in male germ cells, and Exo84 uniquely regulates the latter process, potentially by supplying membrane required to maintain cilium-PM association. This result is supported by the Drosophila cep290 mutant, which lacks a functional TZ but retains cilium-PM association [14]. Notably, EXOC8, which encodes the human Exo84, has been linked to the ciliopathy Joubert syndrome [58], and a similar process might underlie defects in humans with mutations in EXOC8.
Methods
Transgenic flies
Drosophila stocks were cultured on cornmeal molasses agar medium at 25°C and 50% humidity. Stocks expressing β2t::Styp\SigD (chromosome 3) and β2t::YFP-Sktl (chromosome 2) were described previously [21, 59]. GFP-Exo70 was cloned into the low-level expression vector tv3 [59] and transgenic flies were generated using standard P element-mediated transformation. Ana1-tdTomato (chromosome 2) and Cep290-GFP (chromosome 3) were provided by T. Avidor-Reiss [14]. Sp/CyO; Unc-GFP was originally provided by M. Kernan [39]. Stocks expressing GFP-tagged Chibby and Mks1 were provided by B. Durand [32, 33]. The onr mutant was described previously [60]. Stocks for generating sktl2.3 clones were originally provided by A. Guichet [61]. Clones were induced by heat shock for two hours on days 3, 4 and 5 after egg laying. w1118 was used as the wild-type control.
Antibodies
The following primary antibodies were used for immunofluorescence at the indicated concen-trations: chicken anti-GFP IgY (abcam), 1:1000; rat anti-RFP IgG (5F8, ChromoTek), 1:1000; rabbit anti-Centrin (C7736, Sigma-Aldrich), 1:500; mouse anti-acetylated α-tubulin 6-11-B (Sigma-Aldrich), 1:1000. Secondary antibodies were Alexa 488- and Alexa 568-conjugated anti-mouse, anti-rabbit and anti-chicken IgG (Molecular Probes) at 1:1000. DAPI at 1:1000 was used to stain for DNA.
Fluorescence microscopy
For live imaging, testes were dissected in phosphate buffered saline (PBS). To stain for DNA, intact testes were incubated in PBS with Hoechst 33342 (1:5000) for 5 minutes. Testes were transferred to a polylysine-coated glass slide (Thermo Fisher Scientific) in a drop of PBS, ruptured using a syringe needle and squashed under a glass coverslip using Kimwipes. The edges of the coverslip were sealed with nail polish and the specimen was visualized using an epifluorescence microscope (Zeiss Axioplan 2) with an Axiocam CCD camera. Cells were examined live whenever possible to avoid artefacts from immunostaining.
For Taxol treatments, testes from larvae or pupae expressing Ana1-tdTomato; Cep290-GFP were dissected into Shields and Sang M3 medium (Sigma-Aldrich) supplemented with a predefined concentration of Taxol (Sigma-Aldrich) in DMSO and incubated overnight in a humidified sterile chamber in the dark at room temperature. These were then squashed in PBS and imaged live.
For CellMask staining, cells were spilled from testes in M3 medium onto a sterilized glass-bottom dish pre-treated with sterile polylysine solution to enable cells to adhere. CellMask Deep Red (Invitrogen) solution (20 μg/mL) was added to the medium dropwise immediately before visualization under a confocal microscope.
For immunocytochemistry, testes were dissected in PBS, transferred to a polylysine-coated glass slide in a drop of PBS, ruptured with a needle, squashed and frozen in liquid nitrogen for 5 minutes. Slides were transferred to ice-cold methanol for 5-10 minutes for fixation. Samples were then permeabilized and blocked in PBS with 0.1% Triton-X and 0.3% bovine serum albumin, and incubated with primary antibodies overnight at 4°C, followed by three 5-minute washes with PBS, 1 hour incubation with secondary antibodies, and three 5-minute washes with PBS. Samples were mounted in Dako (Agilent) and imaged with a Zeiss Axioplan 2 epifluorescence microscope or a Nikon A1R scanning confocal microscope (SickKids imaging facility).
Statistical methods
Statistical analysis and graphing was performed using R (version 3.4). A Gaussian jitter was applied when plotting results in Figures 1 and 2 for clearer visualization of trends, but raw data was used for all analyses. Statistical tests for “absence of phenotype” were computed using a binomial test under the assumption that the probability of the phenotype occuring was fixed. All t-tests were unpaired and two-sided with Welch’s correction for unequal variances. n represents the pooled number of samples (individual cilia) from multiple flies. A significance level of 0.01 was fixed a priori for all classical analyses. All raw data and code for analysis and plotting can be found online at http://www.github.com/alindgupta/germline-paper/.
Phylogenetic analysis
Candidate orthologs of Skittles and PIP5K9B were queried from Inparanoid (version 8.0) and FlyBase (version FB2017_05). Poorly annotated protein sequences were confirmed to encode type I phosphatidylinositol phosphate kinases using reciprocal BLAST search. Phylogeny.fr (http://www.phylogeny.fr) [62] was used for phylogenetic reconstruction with T-Coffee for multiple alignment and MrBayes for tree construction. The output was converted to a vector image in Illustrator and colours were added for the purpose of illustration.
Author contributions
A.G. and J.A.B. conceived the project. A.G. performed all the experiments and analyses, and wrote the manuscript. L.F. generated GFP-Exo70 flies. J.A.B. edited the manuscript and supervised the project.
Declaration of interests
The authors declare no competing interests.
Acknowledgements
We are grateful to Brian Ciruna for insightful discussions, to Bénédicte Durand, Tomer Avidor-Reiss, Antoine Guichet and Maurice Kernan for providing fly stocks, and to Bénédicte Durand and Bill Trimble for critical comments on the manuscript. This research was supported by a grant from the Canadian Institutes for Health and Research to J.A.B (MOP #130437). A.G. was supported by a University of Toronto Open Fellowship and an Ontario Graduate Scholarship.
Footnotes
List of Abbreviations
- β2t-SigD
- SigD driven by the male germline-specific β2-tubulin promoter
- PIP2
- phosphatidylinositol 4,5-bisphosphate
- PIP
- phosphoinositide, also known as phosphatidylinositol phosphate
- TZ
- transition zone
- BB
- basal body
- PM
- plasma membrane
- onr
- onion rings (an allele of exo84)