Ciliary structure

All cilia are covered by the plasma membrane (ciliary membrane) and have an axoneme at their core that extends from the centriole-derived basal bodies (figure ​(figure2).2). The arrangement of the microtubules and associated proteins is quite heterogeneous throughout the axoneme (Mohri et al. 2012). These differences attribute stability yet flexibility to the organelle. Out of the three (A, B, and C) tubules present in a centriole (discussed below), the C-tubule terminates within a structure known as the transition zone (TZ) that lies between the basal body and the axonemal shaft. TZ has Y-shaped linkers between the ciliary membrane and the microtubules, and a structure called the ciliary necklace, one that is conspicuously absent from the sperm flagellum (Reiter et al. 2012; reviewed by Gonçalves and Pelletier 2017). It seems to be the selective gate preventing the free entry of soluble proteins (Takao and Verhey 2016). Using a combination of cryo-electron tomography and expansion microscopy techniques, Hoek et al. (2022) have shown the molecular architecture of the ciliary base in the green alga Chlamydomonas reinhardtii. Using cryo-ET and subtomogram averaging, the group has demonstrated that membrane binding is not a prerequisite for Y-link formation. Distal to the axonemal structure, a previously unseen helical ‘sleeve’ was observed decorating the microtubules, which the authors predict might designate sites of axoneme severing.

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Figure 2

Overall structure of the 9+2 cilium and its cross-section. The cilium is made up of the axoneme that is formed by nine microtubule doublets surrounded by the ciliary membrane. The other substructures (see the adjacent illustration of the cross-section) include the central pair, radial spokes, and nexin–dynein regulatory complex, typical of the 9+2 axoneme. Kinesin present along the axoneme moves the IFT complex and its cargo towards the ciliary tip (anterograde transport), whereas dynein moves the IFT complex and its cargo towards the cell body (retrograde transport). The transition zone present at the base of the cilium provides selective entry of proteins into the axoneme. Y-shaped linkers are occupied in the transition zone and connect the axoneme to the base of the ciliary membrane. The cilium is anchored on to the basal body (in gray). The ciliary pocket programs ciliary-associated vesicular trafficking. Note the A, B (axonemal doublets), and C (centriolar) microtubules.

Each of the A-tubules in the outer doublet is connected to nine lollipop-like structures called the radial spokes that project toward a structure called the central pair of singlet microtubule apparatus and its proteinaceous projections, which together are believed to regulate ciliary beating (figure ​(figure2;2; Satir et al. 2014; Teves et al. 2016). An excellent cryo-EM structure of the active central pair has recently been solved (Han et al. 2022). Cilia are classified based on the presence of this central pair (designated 9+2) and its absence (designated 9 + 0). Both 9+2- and 9+0-containing cilia are either motile or immotile, depending on the structural differences present in the axoneme (Satir and Christensen 2007). Extensive work on this ciliary architecture has revealed that the arrangement of the sub-ciliary structures like central pair, radial spokes, and dynein arms may be responsible for the observed differences among the types of motilities (figures ​(figures11 and ​and2;2; Teves et al. 2016; Kempeneers and Chilvers 2018; Kempeneers et al. 2019). This makes it important to characterize cilia in several organisms so that the scope of cross-species differences is evident.

Based on the function and axonemal format, the human body can generate four types of cilia: 9+0 motile, 9+0 non-motile, 9+2 motile, and 9+2 non-motile (figure ​(figure1).1). Of the four axonemal formats, the 9+2 motile cilia in vertebrates are present in the respiratory airways, the reproductive system, and the ependymal cells, whose ciliary motility renders fluidic movement (figure ​(figure1).1). Also known as primary cilia, 9+0 non-motile cilia are usually solitary and are deprived of the dynein arms, radial spokes, and central pair. These cilia are present on most differentiated cells in humans relying on the solitary cilium to monitor the environment. Recent studies on the sensory role of primary cilia have revealed their importance in development and tissue homeostasis. It has been found that some tissue-specific cell types also harbour 9+2 non-motile cilia lacking dynein arms; for example, olfactory neurons with multiple long chemoreceptive cilia, the vestibular and cochlear mechano-sensory cilia present in the inner ear of the embryo (figure ​(figure1;1; Satir and Christensen 2007; Kempeneers and Chilvers 2018; Kempeneers et al. 2019). Primary cilia present on sensory cells of mammalian olfactory and vision systems transduce extracellular signals that are chemical, physical (smell and visual), or electrical in nature (Falk et al. 2015). In particular, the specialized kinocilia present in the auditory hair cells of the inner ear use their sensory feature for responding to sound. Unlike non-motile cilia, the 9+2 motile cilia are usually (although not always) present as clusters (figure ​(figure1).1). The 9+0 motile nodal monocilia found at the embryonic node are solitary and show unique rotatory motion. They function in determining the left/right (L/R) body symmetry during early development by generating the leftward fluid flow (nodal flow) followed by the differential patterning of lateral plate mesoderm and then asymmetric organogenesis (McGrath et al. 2003; Tabin and Vogan 2003). The clockwise movement of this cilium present at the centre of the ventral node is also known as the left–right organizer (LRO). It acts to break the L/R symmetry in organisms such as zebrafish, Xenopus, and mouse (Nonaka et al. 1998; Essner et al. 2019; Schweickert et al. 2007). This cilium is different from the airway cilia that show planar beating. But, it is the 9+0 immotile sensory cilium present at the periphery of the ventral node that can sense the flow-dependent signal coming from calcium (Pennekamp et al. 2002; Field et al. 2011; Yoshiba et al. 2012). FOXJ1, a master regulator of the multiciliogenesis program (Yu et al. 2008), and NOTO, a homeodomain transcription factor expressed in the ventral node of the mouse embryo, play a crucial role in determining L/R patterning (Abdelkhalek et al. 2004; Didon et al. 2013). Microarray analysis of the NOTO targets identified about 716 candidate genes of which 59 overlap with lung candidate genes (Stauber et al. 2017). Mutational and rescue studies in mouse embryos showed that both function together in the localization of cilia at the posterior pole. NOTO acts upstream of FOXJ1, and mutation in FOXJ1 causes randomization of left/right body asymmetry (Beckers et al. 2007; Alten et al. 2012; Wallmeier et al. 2019). However, studies in urochordate and sea urchin embryos show the involvement of the 9+2 motile cilia in establishing L/R patterning (Nishide et al. 2012; Tisler et al. 2016). Further, the molecular mechanism of motility is central to understanding physiological processes carried out by the respiratory airways for clearing mucus, fertilization rendered by sperm, movement of the ovum in the fallopian tubes, and the directional flow of cerebrospinal fluid (CSF) generated by the ciliated ependymal cells (Banizs et al. 2005). In contrast, the 9+2 motile sperm flagellum and other multiciliated epithelial cells exhibit sigmoidal, i.e. symmetric waveform and planar waveform, respectively (figures ​(figures11 and ​and2;2; Hirokawa et al. 2006). Taken together, a detailed understanding of cilia will provide the foundation  in determining the cause and effect of ciliopathies.

Centriole production (centriole genesis, or centriologenesis) and duplication (copy number)

Cell division requires centriole duplication that begins during the G1–S phase transition in cycling cells. This duplication is marked by the appearance of a new procentriole (a short daughter centriole) at the proximal end of each existing centriole in an orthogonal orientation. Polo-like kinase (PLK4) is known as the earliest marker that depicts the site for procentriole emergence (figure ​(figure4A;4A; Bettencourt-Dias et al. 2005; Habedanck et al. 2005; Eckerdt et al. 2011). The recruitment of PLK4 is mediated by CEP152 and CEP192, which co-operate in promoting this process (Kim et al. 2013). During the G1–S phase transition, PLK4 further interacts with SCL/TAL1 interrupting locus (STIL, where SCL is stem cell leukaemia and TAL is T-cell acute lymphoblastic leukaemia), which initiates trans-autophosphorylation and activation of PLK4. This active PLK4 phosphorylates STIL which in turn triggers the recruitment of spindle assembly abnormal-6 (SAS-6; Arquint et al. 2015), leading to the formation of the cartwheel structure serving as a building block for the procentriole (Dzhindzhev et al. 2014). Another protein centrosomal P4.1-associated protein (CPAP), with its interacting partners CEP135, CEP120, and spindle and centriole-associated protein 1 (SPICE1), further elongates the procentriole by forming microtubules (figure ​(figure4A;4A; Tang et al. 2009). The procentriole elongates during the S and G2 phases, finally detaching from the mother centriole in the late G2 phase. After mitosis, each cell inherits a pair consisting of the mother centriole and a newly duplicated daughter centriole. At this stage, the mother centriole acquires its unique fibrous DAs and SDAs. This basal body finally serves as a nucleation centre for a cilium.

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Figure 4

(A) Molecules important for centriole duplication and assembly in humans. The flow diagram shows the molecular pathway responsible for centriole production. The solid lines represent a physical association between the molecules and the arrow heads indicate the recruitment/regulation of the next molecule in the pathway. The pathways can be separated in three major parts. First, the PLK4-STIL association is required for the initiation of centriolar duplication. CEP192/CEP152/CEP63 is required for the recruitment of PLK4 to the parent centriole. This association results in PLK4 activation, which further phosphorylates STIL and itself, as represented by blue arrows. Second, the association with HsSAS-6 is required for the initiation of cartwheel assembly in the centriole. The black arrow indicates self-polymerization of HsSAS-6 after its activation. The third step includes the recruitment of CEP135 which further recruits a set of molecules required for centriole elongation. CPAP, in collaboration with CEP135, CEP120, and SPICE1, facilitates the formation of the procentriole’s microtubule wall. γ-tubulin helps in the microtubule stabilization. (B) Pathways for centriole migration and docking at plasma membrane (formation of basal body and cilium assembly). The mature mother centriole serves as a precursor for the basal body formation and cilium assembly. There are two main pathways known in mammals for centriole migration and/or docking at the plasma membrane: (1) In the extracellular pathway (top), the parent centriole migrates towards the plasma membrane and the axonemal elongation starts only after it has docked at the plasma membrane on the cell surface. Loss of CP110 from distal end of the parent centriole marks the initiation of cilium assembly. At similar time points, IFT particles are also recruited to the centriole for initiating the formation of the TZ. The TZ specific complexes are then localized to TZ. (2) The intracellular pathway (bottom) starts with the recruitment of pre-ciliary vesicles to the distal appendages of the mature centriole with the help of MYO5A. These are called distal appendage vesicles (DAVs), and they contain Rab-11 GTPase associated with Rabin8 that helps in the recruitment of Rab8. Protein EHD1 helps in fusion of these DAVs to form the ciliary vesicle (CV), and Rab8 is responsible for the growth of CV. The loss of CP110 happens at the same time marking the initiation of TZ formation. This results in maturation of ciliary vesicles forming the distinct domains of ciliary sheath and ciliary membrane. Axonemal extension takes place inside the ciliary sheath until the fusion of ciliary sheath with plasma membrane exposes the axoneme to the external environment.

The spatial, numerical, and temporal regulation of this process serves importantly to ensure copy number control where one mother centriole can only give rise to a single new procentriole. Studies in human somatic cells have shown that experimental depletion of existing centrioles triggers de novo centriole genesis (Uetake et al. 2007). An example of de novo centriole production is seen in terminally differentiated multiciliated cells (MCCs). These have completely different molecular dynamics as compared with monociliated cells. Being multiciliated, and to ensure multiciliogenesis, MCCs require several centrioles to be docked at the plasma membrane after exiting the cell cycle (at a point where normal cells suppress centriole duplication). It was in 1971 that Anderson and Brenner distinguished two pathways for centriologenesis in MCCs, namely, centriolar and acentriolar (major pathway) (figure ​(figure4B;4B; Anderson and Brenner 1971). In the centriolar pathway, an existing centriole acts as a scaffold to produce multiple new procentrioles around it. The origin of the fundamental centriole that produces these new procentrioles is yet to be elucidated. In the acentriolar pathway, clusters of dense granules (intermediary structures) that may or may not be associated with the existing centriole spontaneously arise in the cytoplasm. New procentrioles are produced around these rather than the existing centriole. These dense granules further condense and give rise to novel structures called deuterosomes triggered by the expression of the Deup1 protein. Deup1 is a paralog (37% identical) of CEP63 (a human microcephaly protein) that regulates mother centriole-dependent centriole duplication (Zhao et al. 2013). Another protein, multicilin (encoded by the gene Mcidas), is largely expressed during multiciliated cell differentiation and positively regulates the expression of Deup1, Plk4, Cep152, Stil, and Sas-6 (figure ​(figure4A;4A; Kim et al. 2018). High expression of Deup1 initiates deuterosome formation, thus making a scaffold for supporting multiple de novo centriole duplication. Deup1 binds to the CEP152 protein which in turn activates PLK4 resulting in the initiation of the process of centriolar assembly. These centrioles further mature to form basal bodies. Most of the key regulators (Plk4, Cep152, Stil, and Sas-6) are similar in both centriolar and acentriolar duplication pathways (figure ​(figure4B).4B). Another example where the copy number of centrioles deviates is seen in the sexual reproduction of mammals wherein the oocytes lose the centrioles and the centrosome of the zygote comes entirely from sperm cells (Pimenta-Marques et al. 2016) (table ​(table11).

Centrioles in flagella (Chlamydomonas reinhardtii)

Centrioles in Chlamydomonas play the role of basal bodies after attachment of DAs and ciliary vesicles docking to it. Here too, the basal bodies serve as the main nucleation centre for flagellar assembly. The 9+2 flagella are anchored onto the basal bodies and are formed in three cycles of semi-conservative division. Out of the three generations of basal bodies present during the interphase, a flagellum is extended from the first- (mother) and the second-generation (daughter) basal body, while that of the third generation – the pro-basal body – is associated with each of the two at the proximal end. The two pairs of basal bodies duplicate during the late prophase/early metaphase that follows and form spindle poles (Wingfield et al. 2018). A class of mutants in C. reinhardtii called uniflagellate have a mutation in the uni3 gene that encodes for δ-tubulin (makes the C-tubules). These cells preferentially assemble the flagella on the mother basal body; this means that more than one cycle of division is required for a basal body to mature for flagellar transformation (Beech et al. 1991). Another mutant called bld2 shows no presence of flagella. The gene BLD2 codes for ε-tubulin, which is required for forming the incomplete B-tubules (Dutcher and Trabuco 1998). Mutation in this gene results in the defective formation of the whole cartwheel structure of the basal body. The normal formation of the ring structure is controlled by the SAS-6 complex encoded by the bld12 gene. The Sas-6 spokes harbour CEP135 at their tips, which is encoded by the gene bld10. Bld12 mutants show defective ring structure in basal bodies, but bld10 mutants completely lack basal bodies (Hiraki et al. 2007). This shows that these two proteins are of utmost importance for the normal formation of the 9-fold symmetry of the basic cartwheel structure. These proteins are recruited to their location by CEP70, a protein present in the ring structure in pro-basal bodies. An amorphous ring structure serves as a precursor for the cartwheel formation and harbours CEP70. This protein is lost during the maturation of basal bodies (Shiratsuchi et al. 2011). Along with proper formation, the correct positioning of these basal bodies is also very important for flagellar assembly. The attached distal and proximal fibres with the basal body ensure that docking happens at the correct site. Distal fibres contain a protein called centrin that provides elastic properties to these fibres, ensuring correct spatial positioning of basal bodies. A mutant with a mutation in the VLF2 gene that encodes for centrin has defective basal body positioning with varied numbers of flagella (Wright et al. 1985; Silflow and Lefebvre 2001). The basal bodies also harbour proteins required for the formation of the TZ. The two major protein components of TZ are NPHP4 and CEP290. In Chlamydomonas, mutants for these two proteins show altered protein content in the axoneme resulting in defective biochemical and structural composition of the whole axoneme (Craige et al. 2010; Awata et al. 2014). The proteins destined for the axoneme are first recruited to the basal body and then allowed to enter the axoneme through the TZ. These proteins are carried towards the axoneme through the IFT system (discussed in section 3.5) (table ​(table11).

Centriole migration and acquisition

After the entire centriole is formed, it is positioned correctly and is ready to function, a process known as centriolar maturation. This sets the stage for the initiation of cilium assembly. Sorokin (1962) described two different pathways for cilium assembly. One of the pathways elucidated in the fibroblast and smooth muscle cells includes small vesicles most likely derived from the Golgi bodies directly binding to the DAs of the mother centriole thereby forming a large membranous ciliary vesicle at the distal end. These small (pre-ciliary) vesicles are directed to the centriole by the coordinated action of dynein and myosin-Va (Wu et al. 2018). Later, it was demonstrated that one of the DA proteins, CEP164, is responsible for the docking of vesicles at the centriole (figure ​(figure4;4; Schmidt et al. 2012). With the help of the Eps15 homology domain (EHD) family of proteins, these pre-ciliary vesicles (PCVs) then fuse together to form the ciliary vesicles (CVs; Lu et al. 2015). In turn, these vesicles act as a protective sheath for the growth of nascent cilium inside the cell. Following the growth of this nascent cilium, CVs fuse with the plasma membrane, exposing the cilium for the very first time to the extracellular environment. This fusion results in the formation of the ciliary membrane and the adjacent ciliary pocket (a specific membrane domain found at the base of cilia). The other pathway has been reported in epithelial cells, where the distal appendage protein CEP164 facilitates the attachment of DAs to cell membrane without the involvement of CVs (Graser et al. 2007; Schmidt et al. 2012). This migration is facilitated by microtubule and actin cytoskeleton (Pitaval et al. 2010, 2017). It has been reported that since actin inhibitors can promote ciliogenesis, actin is absent from the site of docking (Kim et al. 2010; Francis et al. 2011). After migration, the fully mature mother centriole that docks at the plasma membrane and nucleates the axoneme serves as basal bodies for ciliary elongation. The basal body acquires additional structures like transition fibres and basal feet (like DAs and SDAs, respectively), as well as ciliary rootlets for support (Anderson and Brenner 1971). The DAs play a role in the docking of the centriole to the apical plasma membrane, while the SDAs help in microtubule anchoring (Tanos et al. 2013; Mazo et al. 2016; Breslow and Holland 2019).

How a cell decides which pathway to undertake remains elusive. It can be dependent on the molecular requirements for different pathways or on the functional aspects of the cilia that are formed. The cells harbouring the motile cilia are mostly seen to undergo the extracellular pathway where the cilium extends beyond the cell surface. This helps sensory cilia sense and regulate the extracellular fluid flow (Sorokin 1968), extending very little of the cilium beyond the apical surface. These cilia are found emerging near the nucleus and Golgi body deep inside the cell (Sorokin 1962). Experimental alteration in the positioning of these cilia can result in the dysregulation of their function. A study wherein the submerged cilia were converted to apical cilia although showed poor sensory functions, did promote the regulation of fluid flow (Mazo et al. 2016). This implies that the cell type specificity for cilium positioning is correlated with the functional properties of the cell.

Transportation and compartmentalization of ciliary components to the site of assembly (protein trafficking)

Several proteins are required to build the cilium. Since cilia do not contain ribosomes, and therefore lack the protein synthesis machinery, proteins required for the elongation of cilia have to be imported from the cytoplasm (Rosenbaum and Child 1967; Long and Huang 2020). For the continuous assembly of cilia, these proteins must be transported to their site of assembly. This transport can either happen by diffusion or by a special translocation system called the intraflagellar transport (IFT), the pathways that contribute towards their formation being highly conserved across ciliated organisms (Kozminski et al. 1993; Rosenbaum and Witman 2002; Lechtreck 2015). Interestingly, soluble proteins smaller than 50 kDa and certain transmembrane proteins can freely diffuse into cilia (Kee et al. 2012; Breslow et al. 2013; Lin et al. 2013). Several studies have demonstrated the presence of a gate that serves as a barrier with a lower limit of the Stokes radius (~4.5 nm) for free diffusion into the cilia (Lin et al. 2013). For proteins with higher molecular weights such as outer dynein arms (~2 MDa), the TZ acts as a molecular barrier, therefore making IFT an important part of cilium assembly. It has been reported that for ciliary transmembrane-associated proteins, a targeting sequence known as ciliary localization signal (CLS) is required for the transport of these proteins into cilia (Berbari et al. 2008). However, in contrast to the nuclear localization signal (NLS), no unique consensus sequence is found for the transport of ciliary proteins, suggesting that other pathways are involved in the transport of these proteins. A rare exception was found in Plasmodium falciparum where there is no requirement of IFT for ciliogenesis as the axoneme assembles in the cytoplasm and directly transports proteins from cytoplasm to the tip of the growing cilium (Sinden et al. 1976; Briggs et al. 2004; Absalon et al. 2008). Another exception was found in Drosophila sperm flagella where necessary proteins for the formation of cilia are independent of the IFT (Han et al. 2003; Hao and Scholey 2009). First observed in the green alga C. reinhardtii, IFT provides the bidirectional movement of multimolecular protein complexes formed by strings of many IFT particles encompassing molecular motor proteins (Kozminski et al. 1993; Hao and Scholey 2009; Thomas et al. 2010). To be precise, its IFT system consists of 22 IFT protein particles and IFT motors (kinesin-2 for anterograde and dynein for retrograde movements) that come together to make the multisubunit long filamentous chains/strings called IFT trains for the transport of cargo to and fro from the axonemal tip. Particles that constitute the IFT are essential for flagellar assembly and maintenance. Jordan and Pigino (2019) used of in situ cryo-electron tomography (cryo-ET) and subtomogram averaging to investigate the structure and mechanism of the IFT depicting the movement of IFT trains along the microtubule doublets while transporting ciliary building blocks between the cell body and the ciliary tip. A comparison of the spatial arrangement of the various IFT components revealed that trains are assembled in a sequential manner (Hoek et al. 2022). The trains or strings that were complete were adjacent to the TZ at one end. The other end extended into the cytosol, and as it extends into the cytosol, the densities of dynein-1b and IFT-A keep reducing in a sequential order. This suggests that the IFT-B backbone is built first, followed by IFT-A and dynein-2 recruitment. Using cryo-ET and expansion microscopy techniques, the relatively small and flexible kinesin-2 was identified as the final component that is loaded onto the IFT trains close to the TZ (Hoek et al. 2022). These studies also indicated drastic differences between assembling and mature IFT trains. Assembling IFT trains were found to be more flexible than their mature axonemal counterparts and contained an extra density on IFT-B near the kinesin-2 binding site of unknown identity (Hoek et al. 2022). The importance of this process is evident wherein mutations in the motors DHC1B (a member of dynein superfamily) and kinesin-II cause defective or no flagella assembly, respectively (Silflow and Lefebvre 2001). The IFT protein subunits are formed in the cell body and then recruited by the basal body, where they start the construction of the IFT particles (Wingfield et al. 2017).

Further, the BBSome, a complex composed of eight proteins, works as an adapter in association with the IFT trains, thereby providing stabilization and maintenance. Mutation in these proteins causes Bardet-Biedl syndrome, a multi-organ disease specified by the defects in the sensory function of cilia (Wei et al. 2012; Williams et al. 2014). The axonemal assembly is normal in these mutants and so suggests the main role for the BBSome in ciliary signalling mechanisms (Lechtreck et al. 2009; Weihbrecht et al. 2017). Certain ciliary signalling proteins including G protein-coupled receptors (GPCRs), protein kinases, and ion channels have been found to transmit signals from the extracellular environment to the cell body. The BBS3 protein is thought to have a role in the interaction of ciliary signalling cargoes like phospholipase D (PLD). It has been shown that the BBSome relies on the BBS3 protein to enter the basal body via direct interaction with the BBS1 subunit of the BBSome in mammalian cells (Jin et al. 2010; Zhang et al. 2012; Mourão et al. 2014). However, in C. reinhardtii, GTPases ARL6/BBS3 recruit BBsomes from the cell body to the basal body through IFT22/RABL5 (Xue et al. 2020; Liu et al. 2021). Further, the BBSome splits from BBS3 and loads on to the anterograde IFT train and enters the cilium (Xue et al. 2020). The GTP-bound BBS3 then binds to the cell membrane, allowing it to laterally diffuse into the cilium (Liu and Lechtreck 2018). Through BBSome/IFT train remodelling, the BBSome is liberated from anterograde IFT trains at the ciliary tip. BBS3 binds to GTP and attracts the BBSome to the ciliary membrane, where it interacts with PLD. PLD-laden BBSomes are subsequently loaded onto retrograde IFT trains for ciliary escape by connecting with IFT-B1 (Liu and Lechtreck 2018; Liu et al. 2021; Xue et al. 2020). The entire protein transportation by the IFT/BBSome complex is divided into (i) IFT binding to cargo, (ii) anterograde movement, and (iii) retrograde movement (sections 3.5.1 to 3.5.3) (table ​(table22).

Role of Sonic Hedgehog signalling in primary cilia

Sonic Hedgehog (SHH) signalling is the major pathway that regulates crucial events during the development process. It is noted that abnormal activation of this process has a significant association with glioma, basal cell carcinoma (BCC), prostate, and lung cancer (Goetz et al. 2009; Hassounah et al. 2012). The presence of the canonical SHH receptor has therefore a connection with the primary cilia. Almost 90% of BCC and 30% of medulloblastoma have a mutation in the genes involved in SHH signalling (Hassounah et al. 2012; Pugh et al. 2012). When the ligand SHH binds to the receptor patched (PTCH1) present on the plasma membrane, there is an accumulation of SMO (smoothened, a seven-pass transmembrane protein) and SUFU (suppressor of fused) on the primary ciliary membrane. Out of the three Gli transcription factors (Gli1, Gli2, and Gli3), Gli2 and Gli3 are converted by proteolytic cleavage to perform roles of transcriptional activation or repression. Active forms of SMO and SUFU recruit Gli1 to form a complex that together targets Gli2 and Gli3 to act as transcriptional activators upon translocation to the nucleus and mediate SHH signalling. In the absence of SHH, Gli2 and Gli3 act as transcriptional repressors resulting in the suppression of SHH signalling genes (Rohatgi et al. 2007; Wong et al. 2009). Depending on the initiating carcinogenic factor, SHH signalling has a dual role in promoting and preventing carcinoma (Han and Alvarez-Buylla 2010; Archer et al. 2012; Hassounah et al. 2012; Gerhardt et al. 2016). This was observed in BCC (a skin cancer) wherein cells with an activated SMO possess primary cilia, and removal of primary cilia inhibits the progression of BCC (Wong et al. 2009; Athar et al. 2014; Basset-Seguin et al. 2015). In contrast, however, cells with the active form of Gli2 possess primary cilia and the removal of primary cilia accelerates the progression of BCC (Wong et al. 2009). Similarly, in the case of medulloblastoma (malignant brain tumour), cilia are present when there is a loss of function in PTCH1 and SUFU. On the other hand, a gain of function in SMO and Gli causes an enhanced activity of SHH, thereby promoting medulloblastoma. In non-ciliated cells, amplification/mutation of Gli transcription factors without the activation of PTCH1 and SMO causes an activation of Gli2-R which acts as an antagonist of Gli2-A, causing the suppression of SHH medulloblastoma (Han and Alvarez-Buylla 2010).

Apart from the primary cilium, motile cilia also contain some components of SHH signalling, but in a non-canonical manner (Mao et al. 2018). Studies on human airway epithelia showed that SHH (Cigna et al. 2012; Peng et al. 2015; Henkin et al. 2017) is present in the basolateral side of epithelia. During early development, the primary cilium present on the airway cell produces SHH for its normal functioning. After completion of development, i.e., in mature airway epithelia, the primary cilium disappears and numerous motile cilia appear on the apical portion of cells (Sorokin 1968; Jain et al. 2010). Several efforts have been carried out to identify the components of SHH signalling based on arrayed siRNA libraries (Roosing et al. 2015). Recently, a whole-genome-wide CRISPR screen technology was used to identify the factors required for ciliary signalling, revealing the relation of certain disorders caused by ciliary signalling (Breslow et al. 2018; Pusapati et al. 2018). The novel ciliary components (FAM92A, TTC23, TXNDC15, and TEDC proteins) that were candidate ciliopathy genes came to light after the CRISPR-based screening. For FAM92A, a mutant cell pool was generated to identify the precise role in ciliogenesis, and it has been shown that the formation of cilia is drastically reduced in Fam92A-knockout pools (Breslow et al. 2018). Further, localization studies using FAM92A antibodies found it to be present in the TZ (Breslow et al. 2018). FAM92A was found to co-localize with its partners, viz., CBY1 and DZIP1L (Glazer et al. 2010; Burke et al. 2014; Li et al. 2021). TTC23, on the other hand, has been found to be associated with IQCE (IQ domain-containing protein E) and EFCAB7 (EF-hand calcium-binding domain-containing protein 7) previously shown to localize to the Ellis-van Creveld (EvC) zone (proximal region of cilium; Ruiz-Perez and Goodship 2009; Dorn et al. 2012; Pusapati et al. 2014). Although knockout of TTC23 does not show any defect in the cilium assembly, it certainly reduces the localization of IQCE and EFCAB7 proteins to the EvC zone (Breslow et al. 2018). Thioredoxin-domain containing 15 (TXNDC15) has been shown to be associated with the Meckel syndrome (Shaheen et al. 2016). Knockout studies of TXNDC15 have shown decreased levels of the ciliary GTPase ARL13B, indicating a clear defect in SHH signalling (Breslow et al. 2018). Tubulin ε and δ complex 1 and 2 (TEDC1 and 2) proteins have been found to associate with ε-tubulin, δ-tubulin, α/β-tubulin, and CENPJ (centriolar regulator of microtubule dynamics). Mutation in the cenpj gene disrupts the triplet microtubules present in the flagella of Drosophila spermatocytes (Zheng et al. 2016). Knockout of both TedC1 and TedC2 showed complete loss of the centrioles. These mutants lack cilia and show strong defects in the SHH signalling (Breslow et al. 2018). Thus, the CRISPR screening platform has led to the identification of genes involved in ciliary signalling with direct relevance to human health and diseases.

Role of cilia in neurodegenerative diseases

LRRK2 (leucine-rich repeat kinase 2), a large multifunctional kinase, is responsible for neurodegeneration such as autosomal-dominant Parkinson’s disease. It has been shown that in cultured cells, the pathogenic form of LRRK2 phosphorylates the switch II domain of Rab GTPases which are considered master regulators of intracellular vesicle trafficking (Steger et al. 2017). Rab-interacting lysosomal protein-like proteins 1 and 2 (RILPL1 and 2) localize in both primary cilium and centrosome (mother centriole) and are known to regulate the protein concentration of the primary ciliary membrane. Depletion of these two proteins causes an imbalance in the concentration of signalling molecules in the ciliary membrane and prevents the proper organization of epithelial cells in 3D (ALI) cultures (Schaub and Stearns 2013). RILPL1 and RILPL2 are the primary ciliogenesis regulators that help in the formation of cilia; the phosphorylated form of Rab GTPases interacts with RILPL1 and RILPL2 and thus blocks the formation of primary cilia (Schaub and Stearns 2013). This was the first study to show the link between Parkinson’s disease and cilia. Experimental analysis has shown that phosphorylation of Rab GTPase proteins and the dephosphorylation by a phosphatase, viz., protein phosphatase Mg²⁺/Mn²⁺-dependent1H (PPM1H) is crucial for ciliary functioning. Precisely, PPM1H, a family of phosphatase enzymes, has been shown to counter this process by specifically dephosphorylation of the subset of Rab proteins, namely, Rab8A, Rab8B, Rab10, and Rab35 (Steger et al. 2017; Dhekne et al. 2018; Schmidt et al. 2022). In conclusion, cells expressing hyperactive LRRK2 kinase mutants cause depletion of PPM1H and subsequently block the formation of primary cilia. Hence, this needs to be further ascertained with greater insight into these processes so that one can apply these as therapeutics to treat Parkinson’s disease (Berndsen et al. 2019).