Ciliary-associated proteome has been analysed and most actin-binding proteins has been found in cilia (Kohli, Höhne et al. 2017). The transport of proteins into the primary cilium involves two phases: transportation of ciliary proteins through the cytoplasm to the ciliary base and translocation inside the ciliary shaft via IFT (Fig.13). Ciliary base is a selective barrier that prevent movement of unnecessary proteins inside the organelle (Malicki and Avidor-Reiss 2014).
Transmembrane proteins are embedded in specific exocytotic vesicles which are transported into the cilium. At the ciliary base these vesicles fuse together and proteins rapidly translocate into the ciliary membrane. Thus, to travel from the Golgi network to the ciliary tip, transmembrane proteins switch their motors at the ciliary base. Cytosolic proteins transportation does not require any vesicle formation and fusion. Inside the ciliary compartment these proteins are actively transported by the ITF machinery (Malicki and Avidor-Reiss 2014). Both transmembrane and cytosolic proteins must cross selectively a permeable barrier at the cilium base, which regulates trafficking in both directions to keep some proteins outside and others inside the cilium. Dysfunction of the ciliary barrier may cause cell death (Fliegauf, Benzing et al. 2007, Benmerah, Durand et al. 2015).
The base of the cilium is a hub for protein transport and is localized in the edged cytoplasm. It includes a centriole, the septin ring, the transition fibers and the transition zone (membrane associated components) as well as a specialized membrane area (ciliary pocket) (Mirvis, Stearns et al. 2018, Wang, Fei et al. 2018). The centriole serves as a start point for ciliary axoneme growth and gives rise to transition fibers. Other components are involved in the ciliary transport regulation. Septins are
self-21 polymerizing GTPases that form membrane barriers. In yeast bud neck, septins separate membrane compartments of the mother cell and the budding daughter cell. In primary cilium septins limit protein transport between ciliary membrane and plasma membrane (Malicki and Avidor-Reiss 2014).
1.9 Identification of ciliary proteins by the IFT machinery
One can speculate that transmembrane proteins are generally targeted to the primary cilium if they do not contain a signal directing them somewhere else (Fig.12). However, several specific signal sequences, commonly referred to as ciliary targeting sequences (CTSs), have been identified and guide proteins to the primary cilium compartment (Follit, Li et al. 2010, Madugula and Lu 2016, Han, Xiong et al. 2017). The CTSs mostly localize to the C-terminal protein end, less often it can be found to N-terminus or intracellular loops (Nakata, Shiba et al. 2012). VxP motif is the most common feature of CTSs and mutations in this motif often abolishes protein localization to the cilium. This sequence is found in G protein-coupled receptors (GPCRs), polycystins, CNG channels and retinol dehydrogenase. Other motifs known to be important for ciliary targeting are FR motif of ODR-10 and Smoothened as well as AxxxQ motif of SSTR3 (Brear, Yoon et al.
2014). Another abundant ciliary protein, peripherin, does not display classical CTS, therefor its translocation to the cilium should be achieved via a different pathway. The CTSs of cytoplasmic proteins that may mediate interaction with IFT apparatus remain to be determined.
22 Figure 12. Ciliary targeting sequences (Malicki and Avidor-Reiss 2014)
23 1.10 Cilium assembly and disassembly
Cilium assembly starts in response to mitogen deprivation or differentiation hints, although several types of differentiated cells (lymphocytes, hepatocytes, mature adipocytes, renal intercalated cells and skeletal muscle cells) do not display primary cilium. Ciliogenesis is a very dynamic and tightly regulated process; it can be divided into several phases that contain all the events that take place before and after the basal body docks into plasma membrane (Scholey 2013, Sanchez and Dynlacht 2016). Internal components of the basal body undergo dramatic remodelling at early stages of ciliogenesis. For instance, asymmetric destruction of proteins essential for centriole duplication CP110 and CEP97 on mother centriole is an obligate event that initiate cilium assembly (Tsang and Dynlacht 2013, Walentek, Quigley et al. 2016). The first visible sign of centriole-to-basal-body transition appears after 10-15 minutes of mitogen withdrawal. This stimulates accumulation of small cytoplasmic vesicles, originated from Golgi and called distal appendages vesicles (DAVs), around distal appendage of mother centriole. These vesicles fuse into a membranous cap/sheath, called primary cilia vesicle. Centriolar microtubules extend at its distal tip under this cap which also enlarges by increased vesicular trafficking until the whole structure docks into the plasma membrane (Mirvis, Stearns et al. 2018). Ciliary sheath then fuse with plasma membrane forming united compartment. After anchoring elongation of the ciliary axoneme occurs and distal appendages assemble on mother centriole. At this stage orchestrated recruitment of five proteins (CEP83, CEP89, CEP164, SCLT1 and FBF1) is required (Bhogaraju, Engel et al. 2013, Tsang and Dynlacht 2013, Sanchez and Dynlacht 2016). They are involved in recruitment and docking into membrane and serve for distal appendage assembly, which is a recruitment point for IFT proteins and other ciliary components (Fig. 13).
24 Figure 13. Key proteins that mediate and maintain transport into the primary cilium (Malicki and Avidor-Reiss 2014). Transmembrane proteins are transported from the Golgi apparatus in vesicles. Proteins and pathways that function in this process are shown in grey. Diffusion is thought to be the driving force that brings soluble proteins toward the cilium base. UNC119 enable solubilization of lipidated proteins in the cytoplasm. Despite their hydrophobicity, these proteins also move via the IFT mechanism inside the ciliary shaft.
Ciliary membrane assembly and trafficking process include Rab GTPases which control vesicularization of the donor membrane and its fusion with the acceptor membrane.
These are regulated by GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins), which convert RABs into an active (GTP-bound) or inactive form (GDP-bound) (Li and Hu 2011, Agbu, Liang et al. 2018).
25 Cilia disassembly is a biphasic process that includes ubiquitin-mediated protein degradation. First phase starts in G1 after mitogen stimulation of quiescent cells, second phase prior to mitosis. Key regulators of the process are HEF1 (aka NEDD9), Kif24 and Kif2a as well as calcium/calmodulin activated Aurora A kinase. In early G1 cilium disassembly is triggered by serum growth factors through action of two kinesins: Kif-2A and Kif-24 (Kim, Lee et al. 2015, Miyamoto, Hosoba et al. 2015). Activated by Nek2 and Plk1 kinases, kif proteins promote de-polymerization of ciliary microtubules and therefore inhibition of axoneme extension. Aurora A, activated by trihoplein and Pitchfork (Pifo), phosphorylates histone deacetylase HDAC6 which activity leads to deacetylation of tubulin and axoneme disassembly (Pugacheva, Jablonski et al. 2007, Kinzel, Boldt et al. 2010).
1.11 Cilium length and its regulation
Once cell has developed a primary cilium, it needs to be maintained in order to function properly. Ciliary length reflects ciliary function. This parameter is defined by the cell type (table1) and when adaptive aberrations occur, they are normally caused by cell malfunctioning due to a pathological process or a stress (Verghese, Ricardo et al. 2009, Besschetnova, Kolpakova-Hart et al. 2010, Avasthi and Marshall 2012, Prodromou, Thompson et al. 2012, Kim, Kim et al. 2013, Canterini, Dragotto et al. 2017, Han, Jang et al. 2017, Park 2018). The ciliary length is maintained by co-regulated anterograde and retrograde IFT velocities, the ciliary components availability and other factors (Fig.9) (Armour, Carson et al. 2012, Avasthi and Marshall 2012, Ying, Avasthi et al. 2014, Lechtreck, Van De Weghe et al. 2017).
Table1. Primary cilium length in different cell types (modified from (Dummer, Poelma et al. 2016))
26
Cell type Primary cilium length
Vascular endothelial cells 1–5 μm
Kidney epithelial cells 4–6 μm
Neurons 4–9 μm
Osteoblasts 3–4 μm
Chondrocytes 2 μm
1.12 Autophagy and ciliogenesis co-regulation paradigm
Macroautophagy (hereafter referred as “autophagy”) is a catabolic process by which cell recycles its own constituents (Mizushima, Levine et al. 2008, Lindqvist, Simon et al.
2015). It plays an important role in cellular adaptation to stress situations by optimising protein recycling and quality control as well as cellular energetic balance via degradation of cellular components through the lysosomal system (Mizushima, Levine et al. 2008, Wang and Levine 2010, Pampliega, Orhon et al. 2013, Lindqvist, Simon et al.
2015). The ciliary axoneme is a localization site of five key ATG proteins in serum starved cells and another nine ciliary proteins were found associated with the basal body under different conditions (Pampliega, Orhon et al. 2013). A functional primary cilium is required for activation of autophagy upon starvation and, consecutively, when autophagy is downregulated the cilium grows longer (primary cilium modulated autophagy, Fig.14A) (Pampliega, Orhon et al. 2013). Slower velocities of the anterograde transport components IFT88 and IFT20 lead to impaired ciliogenesis and failure to fully activate starvation-mediated autophagy (Pampliega, Orhon et al. 2013). It has been recently shown that in kidney cells, primary cilia and autophagy display mutual co-regulation through mTOR signalling pathway and ubiquitin-proteasome system (Wang, Livingston et al. 2015).
27 Fugure14. Autophagy regulates ciliogenesis. A. Basal autophagy mediated degradation of the ciliary proteins leads to shortening of the primary cilium. B. Extracellular signalling via ciliary receptors leads to specific degradation of the ODF1 protein which blocks IFT transport. This enables cilium elongation (Cianfanelli and Cecconi 2013).
By contrast to previously discussed, induction of autophagy in mouse kidney proximal cells was associated with cilium elongation (Fig.14B) and inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine (CQ) as well as bafilomycin A1 (Baf) led to shorter cilia. Primary cilium length could be normalized by blocking mTORC signalling.
Cultured Atg5-knockout (KO) cells and in Atg7-KO kidney proximal tubular cells also displayed shorter cilia (Wang, Livingston et al. 2015). Basal autophagy promotes ciliogenesis by removal ODF1 from centriolar satellites. Interestingly, OFD1 depletion promotes cilia formation in both MEF cells and breast cancer MCF7 cells, which normally form no cilia (Tang, Lin et al. 2013). These studies show that the relationships between
28 cilia and autophagy highly depends on the cellular context and compartmentation. Basal and cilia-mediated autophagy play different roles. It has been reported that free ubiquitin and the ubiquitin-conjugating enzyme CrUbc13 are present in C.reinhardtii flagella (Huang, Diener et al. 2009). Also, experiments in isolated flagella exposed to exogenous ubiquitin and adenosine triphosphatase resulted in ubiquitination of several proteins, pointing that the ubiquitin conjugation system operates inside of the organelle. A negative regulator of ciliogenesis at the initiation stage, trichoplein, is degraded by UPS (Kasahara, Kawakami et al. 2014).
1.13 Regulation of ciliogenesis
1.13.1 Protein kinases regulate ciliogenesis
Initial steps of ciliogenesis are controlled by microtubule associated/affinity regulating kinase 4 (MARK4) and tau-tubulin kinase 2 (TTBK2). It has been shown that MARK4 and TTBK2 are both required to initiate axoneme extension and to remove the inhibitory protein complex CP110/Cep97 (Carvalho, Wang et al. 2015). Cell Cycle Related kinase (CCRK) mice mutants display abnormalities in ciliary morphology, delayed anterograde transport velocity and delayed enrichment of cilia with key Hh pathway components:
Smo and Gli2 (Snouffer, Brown et al. 2017). NimA-related protein kinase 10 (NEK10) is localized to the centriole satellites and required for ciliogenesis in both mammals and lower vertebrates (Porpora, Sauchella et al. 2018). NEK10 phosphorylated by PKA degrades via ubiquitin proteasome system resulting in dramatic cilia abortion. The kinases never in mitosis-kinase 2 (Nek2) and Aurora A (AurA) are essential for depolymerisation of the cilia when cells enter the cell cycle from G0. AurA and Nek2 individually are able to induce cilia shortening only when cilia are assembling. Presence of both of them is required for cilia resorption.
1.13.2 Cytoskeleton modifications in the primary cilium
The ciliary maintenance and length is controlled by diverse functional relationships of the IFT proteins with the cytoskeleton as well as post-translational modifications (PTM)
29 of the microtubules (Mirvis, Stearns et al. 2018). Axonemal microtubules carry several PTM including acetylation and glycylation.
Acetylation of α-tubulin was discovered in Chlamydomonas reinhardtii and linked to Acetyl-K40 which marks long-lived microtubules found in the axonemes and basal bodies of primary cilia (L'Hernault and Rosenbaum 1983, Piperno and Fuller 1985).
Ciliary elongation requires α-tubulin acetylation and in contrast, the amount of histone deacetylase 6 (HDAC6) negatively correlates with primary cilium length (Sanchez de Diego, Alonso Guerrero et al. 2014, Nakakura, Asano-Hoshino et al. 2015, Ran, Yang et al. 2015). Ciliary Hh (Hedgehog) signalling activity stimulates α-tubulin acetylation via DYRK1B-dependent deactivation of HDAC6 (Singh, Holz et al. 2018). Cilia loss in the absence of Ift88 and Kif3a leads to hyper‐acetylation of microtubules resulting from increased α‐tubulin acetyl‐transferase activity.(Berbari, Sharma et al. 2013)
Tubulin glycylation has been shown in both motile and primary cilia where it participates in axoneme stabilization. It has been shown that glycylated tubulin accumulates in primary cilia in a length-dependent manner. Reduction of the protein level of glycylating enzymes TTLL3 and TTLL8 affects the primary cilia length and leads to its abortion in cultured fibroblasts (Gadadhar, Dadi et al. 2017). Glycilation of the primary cilia happens after ciliary assembly and is important for cilia length control and maintenance (Gadadhar, Dadi et al. 2017).
1.14 Primary cilium-dependent signalling mechanisms
The major function of a cilium is to transduce signals from the cellular milieu into intracellular responses (Praetorius and Leipziger 2013, Pluznick and Caplan 2015, Han, Xiong et al. 2017, Malicki and Johnson 2017, Pala, Alomari et al. 2017). Particularly, the primary cilium contains receptors and potentially controls calcium signalling, Hedgehog, Wnt, PDGFR, Notch, TGF-β, mTOR, OFD1 autophagy, and some other GPCR-associated pathways (Pala, Alomari et al. 2017).
30 Figure 15. A. Cilia-dependent calcium and Wnt signalling. Fluid flow bends the cilium and triggers intracellular Ca2+ to entry through the ciliary channels. Intracellular signalling cascades are activated by the Ca2+ influx. This stimulates gene expression. In another recently proposed model, ciliary bending does not open Ca2+ channels. Ca2+
influx is due to its diffusion from the cell body or caused by a damage in the cilium. B. In the absence of fluid flow Wnt ligand binds to the co-receptors frizzled and DSH is recruited to frizzled but GSK3 is inactivated. β-catenin translocates to the nucleus, where it acts as a transcriptional co-activator in tandem with LEF and TCF family proteins to induce transcription of Wnt target genes. In noncanonical Wnt signalling fluid flow causes intracellular Ca2+ increase and an upregulates inversin expression. Inversin
31 targets cytoplasmic DSH for ubiquitylation and degradation, making it unavailable for canonical Wnt signaling. Figures adapted from Pala, et al. 2017.
1.14.1 Calcium signalling
Primary cilia were thought to regulate calcium signalling via polycystins channels, polycystin1 (PC1) and polycystin 2 (PC2) encoded by PKD1 and PKD2 genes, respectively (Fig.14A). In kidney epithelial cells, these proteins are co-localised and are thought to function as a mechanosensory unit (Nauli, Alenghat et al. 2003, AbouAlaiwi, Takahashi et al. 2009, Raghavan and Weisz 2016). PC2 is activated upon cilium bending by a fluid flow and lead to calcium entry into the cell through the cilium. This calcium signalling would alter gene expression and regulate cellular functions (Nauli, Alenghat et al. 2003, Praetorius and Spring 2003, AbouAlaiwi, Takahashi et al. 2009, Besschetnova, Kolpakova-Hart et al. 2010, Nauli, Jin et al. 2013, Jin, Mohieldin et al. 2014, Praetorius 2015). In opposition to these early experimental results, Delling et al. engineered mice expressing a sensor protein that fluoresces in response to increased Ca2+ influx in primary cilia and measured Ca2+ signals following application of a mechanical force.
Using this model, they found no evidence of mechanical force-driven Ca2+ influx and therefore conclude that the primary cilia are not involved in calcium-based mechanosensation (Delling, Indzhykulian et al. 2016, Norris and Jackson 2016). An alternative theory is that the urinary flow brings metabolic signalling molecules that are recognized by other ciliary receptors to control water and salt reabsorption.
Transient receptor potential (TRP) channels are candidates for this role that was recently liked with calcium signalling (Minke 2006, Hasan and Zhang 2018). The key regulatory mechanism of ciliary signalling in collecting ducts is calcium-dependent but how the signal affects physiology remains to be determined (Norris and Jackson 2016).
1.14.2 Hedgehog Signalling
32 Hedgehog (Hh) signalling pathway regulates homeostasis, tissue patterning and embryonic development in many organisms (Davenport and Yoder 2005, Malicki and Johnson 2017, Pala, Alomari et al. 2017). The primary cilium is thought to be a Hh transduction hub (Goetz, Ocbina et al. 2009). This theory stands on results obtained in experiments with conditional genetic deletion of Ift88 or Kif3a which demonstrated that the primary cilium is essential for Hh signalling responses (Bangs and Anderson 2017).
The Hh signalling comprises serial inhibitory reactions. Under basal condition (Fig15B), in the absence of Hh ligand, the Hh receptor Patched (Ptch) inhibits the activity of smoothened (Smo). Afterwards the suppressor of fused (SUFU) binds to the transcription factor GLI and prevents its activation. After Hh ligand binding to Ptch, Smo migrates to the cilia which results in conversion of full-length GLI/Ci into transcriptional activator form. GLI-activator translocates from the cilium to the nucleus and activates the GLI target gene expression (Goetz, Ocbina et al. 2009, Nachury 2014, Bangs and Anderson 2017). SUFU/KIF7 isolate Gli2 and Gli3 repressors in the ciliary tip where they will be phosphorylated by PKA and Ck1 (Li, Nieuwenhuis et al. 2012). This PTM is recognised by the elements of the ubiquitin proteasome degradation system where Gli2 and, partially, Gli3 are being degraded (Pan, Wang et al. 2009, Snouffer, Brown et al.
2017).
1.14.3 Wnt signalling
Wnt signalling is involved in regulation of cell migration, healing process, neural patterning, planar cell polarity, skeletal development and organogenesis (Gao 2012, Barker, Thomas et al. 2014, Pala, Alomari et al. 2017, Meyer and Leuschner 2018).
Dysregulation of the canonical Wnt signalling (Fig.15A) shown in cancer development is suspected in polycystic kidney disease (Benzing, Simons et al. 2007, Lancaster and Gleeson 2010). Wnt/β-catenin signalling is hyperactive in 70% of the aldosterone producing adenomas (APA) where it controls aldosterone production and cell proliferation acting in cooperation with miRNA-203 (Berthon, Drelon et al. 2014, Peng, Chang et al. 2018).
33 Wnt signalling comprises canonical (β-catenin dependent) and non-canonical pathways;
either of them can be activated by Wnt binding to the membrane receptor frizzled.
When fluid flow is absent, canonical Wnt signalling predominates. Soluble Wnt molecules bind to Frizzled receptors which results in recruitment of the dishevelled (DSH) co-receptors and glycogen synthase kinase-3 (GSK3) becomes inactive. Beta-catenin (β-cat) is no longer degraded and translocates to the nucleus to initiate TCF-dependent transcription of its target genes (Lancaster, Louie et al. 2009). Non-canonical Wnt signalling functions under fluid flow conditions and the generation of calcium signalling upon cilium bending is required. This stimulates Inversin expression and localization in the ciliary base and other cellular locations. Inversin has been proposed to be a switch between canonical and non-canonicat Wnt signalling activity. It targets the cytoplasmic fraction of DSH for ubiquitination and degradation which results in suppression of the β-cat activity (Lienkamp, Ganner et al. 2012).
1.14.4 Notch signalling
Notch signalling is required for various aspects of biogenesis including patterning and differentiation decision of progenitor cells during neurogenesis as well as adult tissue growth and development (Liu, Kiseleva et al. 2018). There are four Notch receptors (Notch1-4) distinguished by a transmembrane domain associated with a calcium ion. It is thought that Notch signalling is related to the primary cilium (Pala, Alomari et al.
2017). Regulation of the left-right asymmetry via cilium length modulation is controlled by notch signalling (Lopes, Lourenço et al. 2010). Notch also facilitates transition of the Shh mediators to the primary cilium subsequently enhancing Hh signalling response (Pala, Alomari et al. 2017)
1.14.5 mTOR signalling
Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, nucleates a major eukaryotic signalling cascade which coordinates cell growth and metabolism, cell cycle as well as organismal survival (Malicki and Johnson 2016, Pala, Alomari et al. 2017).
34 mTOR signalling in kidney is associated with cyst formation, hyper-proliferation of renal cells and hypercalcemia (Huber, Walz et al. 2011, Armour, Carson et al. 2012). Fluid flow bend the cilium which results in mTOR inhibition. This effect is coordinated by LKB1-AMPK-mTOR regulatory network, which is also required for regulation of the cell size by autophagy (Boehlke, Kotsis et al. 2010). Tumor suppressor kinase LKB1 and AMP dependent protein kinase are localized to the primary cilium and its basal body. Upon cilium bending, LKB1 becomes active and translocates to the basal body region and activates AMPK. AMPK in turn phosphorylates tuberin (TSC2) which recruits hamartin (TSC1). TSC1/TSC2 complex stimulates the Pheb GTPase activity which becomes sequestered away from mTOR and therefore cannot activate it (Huber, Walz et al. 2011, Armour, Carson et al. 2012, Pala, Alomari et al. 2017).
1.14.6 Other signalling mechanisms
Platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase receptor, localized to the primary cilium, which regulates cell growth, proliferation and migration. It also plays an important role in embryonic development and tissue growth. When activated, it can induce a cellular response via MEK/ERK signalling cascades (Pala, Alomari et al.
2017). Another receptor protein, localized to the ciliary membrane is transforming
2017). Another receptor protein, localized to the ciliary membrane is transforming