Regulation and Function of Calcium in the Cilium

Abstract

The cilium is a cell surface organelle with unique composition and shape. Although it has now been well appreciated as a signaling compartment for the vertebrate cell, the regulation and function of intraciliary calcium is less clear and sometimes controversial.

This review focuses on publications regarding calcium in the cilium and the potential interactions between intraciliary calcium and signaling pathways mediated by cilia.

Unresolved questions and future directions in the field are also discussed.

I. introduction

The cilium is a cell surface organelle widely distributed on vertebrate cells. Motile cilia beat to propel cell movement or extracellular fluid flow. Cilia also function as a privileged signaling hub. Dynamic localization of receptors and downstream effectors to the cilium has been shown to be essential for the sonic hedgehog (SHH) pathway and signaling by multiple G-protein coupled receptors (GPCR) [1–5]. The second messenger Ca²⁺ plays a significant role in regulating the beating frequency and pattern of motile cilia [6–8]. By contrast, even though immotile cilia show an elevated concentration of free Ca²⁺ ions ([Ca²⁺]) in comparison to the cytosol, the role of Ca²⁺ in cilia-mediated signaling remains murky and controversial [9–11]. Here we review publications that highlights the unique features of cilia and discuss their potential impact on Ca²⁺ and cilia-mediated signaling.

II. Cilia as a signaling hub:

Protruding from the cell surface into the environment, the cilium is uniquely positioned to function as a cellular antenna that detects environmental signals and couple these signals to cellular responses. Ciliary defects can lead to a variety of human diseases, ranging from polycystic kidney disease, laterality defects, obesity, to mental retardation and retinal degeneration (for a review, see [12]).

Currently the best understood aspect of cilia-mediated signaling centers on HH signaling. Many components of the pathway are dynamically trafficked to the cilium [13–15]. In the absence of the ligand, the HH receptor Patched is localized on the cilium. There it prevents the trafficking of Smoothened into the cilium and keeps the pathway in an “off” state. Upon ligand binding, Patched is removed from the cilium and Smoothened enters, leading to the activation of the pathway and in turn the expression of target genes. Recent studies also suggest that a subset of GPCRs are enriched on the cilium. The activation of these receptors leads to increased levels of cAMP, resulting in downstream regulations, including inhibition of the HH pathway [1, 4, 5, 16–22].

To dissect ciliary signaling, it is essential to understand the unique composition and shape of the cilium. Although the ciliary membrane is contiguous with the cytoplasmic membrane, leaving a direct passage between the cilium and the cytosol at the ciliary base, there exist specialized structures called the transition zone and transition fibers. While the diffusion of soluble proteins into the cilium is limited by a size exclusion barrier at the ciliary base [23, 24], the entrance of large protein complexes such as axonemal dynein arms into the cilium is facilitated by intraflagellar transport (IFT) powered by motor proteins [25, 26]. The trafficking of membrane proteins into and out of cilia is also under strict control. For example, the tubby like protein Tulp3 connects IFT-A complex and membrane phosphoinisotides to promote the trafficking of a subset of GPCRs and Polycystins to the cilium [19, 27]; and polymerized BBSome trains are required for removing activated GPCRs from the cilium through the transition zone and a second periciliary barrier [28, 29]. In addition to retrieval via the ciliary base, ciliary components can be shed into the environment via ciliary ectosomes, fine tuning signaling within the cilium and mediating non-cell autonomous communication [30–34].

Compared to protein components, the lipid composition of the cilium and its impact on ciliary signaling has only recently received increasing attention. It was shown that Inpp5e, a cilia-targeted inositol-5-phosphatase, creates a unique phosphoinositol composition for the ciliary membrane characterized by a high PI4(P) and minimal PI(4,5)P2 level; and this composition regulates the localization of GPCRs in the cilium and HH signaling [35, 36]. Cholesterol is another lipid that shows distinct localization pattern on the cilium [37, 38]. A recent seminal work demonstrated that a number of oxysterols are enriched in the ciliary membrane, bind to and stimulate Smoothened [39]. It was also shown that the ciliary cholesterol level regulates the mobility of the HH receptor Patched 1 in the ciliary membrane [40]. Moreover, through a lipid-focused CRISPR screen in NIH/3T3 cells, it was found that whereas enzymes involved in cholesterol biosynthesis enhances HH signaling, enzymes in sphingolipid biogenesis play an opposite role, revealing a role of accessible cholesterol in HH signaling [41].

In addition to protein and lipid compositions, the unique slender shape of the cilium has significant implications for signaling within this compartment. Immotile cilia are typically a few microns in length and about 250 nanometers in width, resulting in a volume less than 1/10,000 of the cell and a higher surface area to volume ratio in comparison to the cell proper. Combined, the diffusion barriers, targeted trafficking mechanisms, high surface to volume ratio and small volume of the cilium allow a concentrated and dynamic regulation of signaling within this compartment.

III. Cilia as a distinct Ca²⁺ signaling compartment

Ca²⁺ ion, by changing protein charge and conformation, is a potent second messenger for many signaling pathways [42, 43]. However, traditional Ca²⁺ dyes cannot be easily targeted to specific cellular compartments. The exceedingly small size of the cilium makes the visualization of intraciliary Ca²⁺ distinct from the large signal generated by cytosolic Ca²⁺ very challenging. In order to evaluate intraciliary Ca²⁺, reporters need to be loaded specifically into the cilium or the cytoplasm, which was made possible by the development of genetically encoded Ca²⁺ indicators (GECIs) [44]. We and others [9, 11, 45–47] have successfully targeted and imaged GECIs in cilia by fusing them to ciliary proteins such as the small GTPase Arl13b. We detected significantly higher [Ca²⁺] in the cilium (around 210 nM) when compared to the cytosol (around 110 nM) [11]. An independent work reported resting intraciliary [Ca²⁺] as high as 580 nm [9], almost in the range of activated cytosolic [Ca²⁺]. This discrepancy of intraciliary [Ca²⁺] could be caused by difference in cell lines and reagents used. However, both studies revealed that at the resting state, the cilium contains higher [Ca²⁺] than the cytosol, suggesting that the cilium is a distinct compartment with respect to Ca²⁺ composition. Currently, the level of activated intraciliary [Ca²⁺] remains to be determined. The elevated resting intraciliary [Ca²⁺] may elicit distinct calcium-mediated signaling, for example by engaging calcium binding proteins with lower Ca²⁺ affinities, in comparison to that in the cytosol.

Polycystins have been hypothesized as regulators of intraciliary Ca²⁺, although this hypothesis is being actively debated. Polycystins are a family of proteins composed of Polycystin 1 (Pc1), Polycystin 2 (Pc2), encoded by the autosomal dominant polycystic kidney disease genes PKD1 and PKD2 respectively, and their homologs; and they belong to the transient receptor potential superfamily of cation channels. Pc2 can form homomeric complexes or hetromers with Pc1 that display cation channel activities [48–50]. In addition, Pc1 and Pc2 are specifically targeted to cilia and this specific trafficking highly correlates with their in vivo function [51–55], making them attractive candidates as ciliary cation channels. By patch-clamping isolated primary cilia and reconstituted ciliary membrane from cultured kidney epithelial cells, Raychowdhury et al identified single channel currents across the ciliary membrane [56]. This line of research was reinvigorated by new tools that fluorescently label cilia in live cells, enabling direct patch clamping of primary cilia displayed on cultured cells [45]. A non-selective cation current across the ciliary membrane was detected [45]. Although Pc2 was initially reported as dispensable, later studies provided evidence that it is essential for the detected ciliary cation channel activity, at least in renal epithelial cells [45, 57, 58].

Since cilia, Pc2 and cytosolic Ca²⁺ are essential for the establishment of the left-right (LR) asymmetry of the vertebrate body plan, we investigated intraciliary Ca²⁺ in vivo in live zebrafish embryos. We discovered novel intraciliary Ca²⁺ oscillations (ICOs) at the zebrafish left-right organizer (LRO) that are left-biased and precede known molecular asymmetry [11]. In addition, ICOs depend on Pc2. Finally by developing a novel cilia-targeted Ca²⁺ sink, we demonstrated that intraciliary Ca²⁺ at the LRO is required for normal LR development [11]. These results establish that intraciliary Ca²⁺ is dynamic and highly regulated, that it is functionally significant and that Pc2 is essential for ICOs, at least in LR development in zebrafish. In mouse LR development, although a previous study failed to detect ICOs [10], a very recent study convincingly demonstrated Pc2 dependent and left-biased ICOs at the mouse LRO and provided evidence that technical issues of mouse embryo culture could account for the discrepancy [59]. Combined, these results suggest that Pc2 dependent ICOs is required for normal LR development in vertebrates. Although important in the LRO and renal epithelial cells, Polycystins are not the only calcium conducting channels on the ciliary membrane. In olfactory cilia, cyclic nucleotide-gated channels open in response to cAMP [60] and the resultant increase of intraciliary Ca²⁺ regulates both excitation and adaptation in odor response (for a review, see [61]).

The molecular mechanism underlying ICOs remains to be defined. Since the small size of the cilium, a limited number of Ca²⁺ will lead to a significant change of [Ca²⁺]. The high surface to volume ratio may also contribute to dynamic changes of ciliary [Ca²⁺]. Conceptually, the concentration of free Ca²⁺ in the cilium is determined by multiple factors, including Ca²⁺ influx through ion channels on the ciliary membrane and from the cytosol through the ciliary base, buffering by calcium binding proteins and efflux through the ciliary base. In addition, Ca²⁺ extrusion from the ciliary membrane could also play a role. Both Na⁺/Ca²⁺ exchanger and plasma membrane calcium ATPase have been found in olfactory cilia and may contribute to calcium clearance from this organelle during odor response [62, 63].

IV. Remaining questions, challenges and future directions

The mechanisms by which intraciliary Ca²⁺ regulates cellular response remains murky. Due to the small volume of the cilium, the total amount of Ca²⁺ molecules in the ciliary compartment is minuscule compared to the amount of cytosolic Ca²⁺. Therefore, it is unlikely that the cilium serves simply to transport large amounts of Ca²⁺ from the extracellular surroundings into the cytosol. Rather, ciliary Ca²⁺ could potentially regulate ciliary signaling, which subsequently controls downstream responses (Fig. 1). One attractive target is the second messenger cAMP. Adenylyl cyclase 3, 5 and 6 (AC3, AC5 and 6) are present in the cilium and are regulated by Ca²⁺ [64–69]. AC5 and AC6 have been shown to negatively regulate the HH pathway via cAMP production and PKA activation [68]. AC3 was also implicated in the repression of the HH pathway during neural tube patterning very recently [70]. However, it remains to be tested whether these components are localized at the right time and cell type to allow direct cross-talks between intraciliary Ca²⁺ and HH signaling. Another potential target of ciliary calcium signaling is Inversin (Inv)/Nphp2 (Fig. 1). Inv contains two Calmodulin binding IQ domains and is required for ciliary functions, including LR development [71, 72]. In addition, given that Ca²⁺ plays a critical role in regulated exocytosis, it will be interesting to investigate whether intraciliary Ca²⁺ is involved in ciliary exocytosis.

Figure 1. A model for intraciliary calcium signaling.

Intraciliary Ca²⁺ regulates adenylyl cyclases (ACs) and thereby influences the level of the second messenger cAMP. Intraciliary Ca²⁺ can also modulate CamKII and Inv through Calmodulin (CaM). The Pc1/2 complex, attached to large glycoproteins/domains outside of the cilium and axonemal microtubules through a linker, is a candidate Ca²⁺ channel on the ciliary membrane.

Reciprocally, activation of signaling pathways could regulate intraciliary Ca²⁺. It was shown that sustained activation of the HH pathway leads to an elevated level of intraciliary Ca²⁺ possibly through altered trafficking of ion channels into the cilium [9]. In addition, ciliary components could regulate ion channel activity directly. For example, membrane lipids play a critical role in regulating the activity of TRP channels. A recent study identified PIP and cholesterol binding sites in Pc2 by combining molecular dynamics simulation and cryoelectron microscopy, although whether the channel activity of Pc2 is regulated by these lipids remains to be investigated [73].

An additional outstanding question is how ciliary Pc1/2 is regulated. Both mechano- and chemo-regulation have been proposed, and the two models are not necessarily mutually exclusive. Intriguingly, a very recent study in the green alga Chlamydomonas showed that the Pc2 homolog anchors an array of fiber like glycoprotein polymers called mastigonemes on cilia in a plane perpendicular to the plane of ciliary beating [74]. This unique position puts Pc2 at an ideal location to sense mechano-signal. In addition, cryo-electron tomography in the same study revealed a thin linker between Pc2 and the outer microtubule doublets [74]. The lipid composition, anchoring of glycoproteins outside of the ciliary membrane and interaction with the microtubule axoneme may all influence the native structure and ion conductance of the Pc1/2 complex on the cilium (Fig. 1). Experimental approaches that allow for the preservation of these physical and biochemical interactions will be key to resolve the native function of Pc1 and Pc2.

In short, we are only starting to tease out the role of intraciliary Ca²⁺, how it is regulated and how it regulates downstream responses. Given the importance of the cilium in human diseases, understanding these questions will be significant. New tools, including biosensors, optically controlled modulators and imaging modalities are breaking the technical barriers in studying these questions.