Role of the primary cilium in transepithelial fluid and electrolyte transport control

Thesis

Reference

Role of the primary cilium in transepithelial fluid and electrolyte transport control

KOMARYNETS, Olga

Abstract

Ce travail de thèse a eu pour objet l'étude de liens potentiels entre les cils primaires et la régulation de la tubulogénèse. Grâce à un essai in vitro, nous avons montré que les cils primaires participent à l'expansion du lumen et à la maturation des tubules. En revanche, nous n'avons pas pu mettre en évidence le rôle des cils primaires dans les stades précoces de la tubulogénèse branchiale. Enfin, nous avons démontré que les cils primaires n'ont pas d'influence sur la mobilité et la prolifération cellulaires. De plus, nous avons étudié la relation entre les cils primaires et le transport trans-épithélial de Na+ par les cellules épithéliales du tubule rénal. Nous avons trouvé qu'une augmentation du transport de sodium induit un allongement des cils primaires. En approfondissant les mécanismes impliqués dans ce processus, nous avons identifié ses principaux composants, qui comprennent notamment l'axe minéralocorticoïde (aldostérone, récepteur minéralocorticoïde, ENaC) ainsi que la protéine de machinerie de transport intra-flagellaire IFT88. L'allongement des cils est engendré à la [...]

KOMARYNETS, Olga. Role of the primary cilium in transepithelial fluid and electrolyte transport control. Thèse de doctorat : Univ. Genève, 2019, no. Sc. 5341

DOI : 10.13097/archive-ouverte/unige:119062 URN : urn:nbn:ch:unige-1190628

Available at:

http://archive-ouverte.unige.ch/unige:119062

Disclaimer: layout of this document may differ from the published version.

1 / 1

UNIVERSITÉ DE GENÈVE

Département de Département de biologie cellulaire FACULTÉ DES SCIENCES Professeur Sandra Citi

Département de PHYM FACULTÉ DE MÉDECINE Professeur Eric Feraille ___________________________________________________________________

ROLE OF THE PRIMARY CILIUM IN TRANSEPITHELIAL FLUID AND ELECTROLYTE TRANSPORT CONTROL

THÈSE

présentée à la Faculté des sciences de l`Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Olga KOMARYNETS de

Moscou, Russie

Thèse n° 5341 Genève

2019

UNIVERSITÉ DE cENÈvr

rncurrÉ DEs scrENcEs

DOCTORAT ÈS SCIENCES, MENTION BIOLOGIE Thèse de Madame Olga KOMARYNETS

intitulée:

<<Role

of the Primary Gilium ln Transepithelial Fluid and Electrolyte Transport Control>

La Faculté des sciences, sur le préavis de Monsieur E. FERAILLE, professeur associé et directeur

de

thèse (Faculté

de

médecine, Département

de

physiologie cellulaire et métabolisme), Madame

S. ClTl,

professeure associée

et

codirectrice

de

thèse (Département

de

biologie cellulaire), Madame

C.

DIBNER, docteure (Faculté de médecine, Département

de

médecine interne

-

spécialités), Monsieur

J.

^LOFFING,

professeur (lnstitute of Anatomy, University of Zurich, Switzerland), autorise I'impression de la présente thèse, sans exprimer d'opinion sur les propositions qui y sont énoncées.

Genève, le 8 mai 2019

Thèse

-5341 -

Le Doyen

N.B.

-

^La thèse doit porter la déclaration précédente et remplir les conditions énumérées dans les "lnformations relatives aux thèses de doctorat à I'Université de Genève".

't

1 I ABSTRACT

Ce travail de thèse a pour objet l’étude du rôle physiologique des cils primaires des cellules épithéliales du rein. Nous avons tout d’abord étudié le rôle des cils primaires dans la régulation de la tubulogénèse. Grâce à un essai in vitro, nous avons montré que les cils primaires participent à l’expansion de la lumière et à la maturation des tubules.

En revanche, nous n’avons pas pu mettre en évidence de rôle des cils primaires dans les stades précoces de la tubulogénèse et le nombre de branchements. Enfin, nous avons démontré que les cils primaires n’ont pas d’influence sur la mobilité et la prolifération cellulaires.

De plus, nous avons étudié la relation entre les cils primaires et le transport trans- épithélial de sodium par les cellules du tubule rénal. Nous avons trouvé qu’une augmentation du transport de sodium induit un allongement des cils primaires. En approfondissant les mécanismes impliqués dans ce processus, nous avons identifié ses principaux composants, qui comprennent notamment l’axe minéralocorticoïde (aldostérone, récepteur minéralocorticoïde, ENaC) ainsi que la protéine de machinerie de transport intra-flagellaire IFT88. L’allongement des cils est engendré à la fois par une diminution de la dégradation de la protéine IFT88 et par l’inhibition de l’autophagie consécutive à l’activation du récepteur aux minéralocorticoïdes via une voie de signalisation indépendante de mTORC1. Nous avons également découvert que ENaC, qui est nécessaire au transport trans-épithélial du sodium dans le tube collecteur, joue également un rôle dans le contrôle de la longueur des cils. Le « silencing » ou le blocage pharmacologique d’ENaC engendrent en effet un rétrécissement des cils primaires. En revanche, la modulation de la longueur des cils primaires n’altère ni le transport de sodium ENaC-dépendant ni l’expression de ses sous-unités. Grâce au modèle d’étude C.

elegans, nous avons analysé les réponses comportementales à différentes concentrations en NaCl chez des mutants de différents membres de la famille de protéines ENaC/DEG. Nous avons montré que les canaux ioniques localisés dans les cils

2 primaires, dont MEC-10, UNC105 et UNC-8, jouent un rôle sensoriel dans la voie de signalisation, ce qui indique la réponse comportementale chimiotactique au NaCl.

Nos résultats, appuyés par la littérature scientifique à propos de la régulation des cils primaires, suggèrent que la modification de la longueur des cils constitue une réponse adaptative aux changements physiologiques de la fonction rénale. La longueur des cils est affectée en réponse à une exposition hormonale et à la variation de la quantité d’ions délivrée ou du débit de fluides. D’autre part, les cils envoient des signaux aux cellules afin de contrôler leur taille et donc leur capacité de transport. L’intensité de cette signalisation est modulée par la longueur du cil primaire.

ABSTRACT

In this PhD thesis we explored a potential link between the primary cilium and regulation of tubulogenesis. Using an in vitro assay, we found that primary cilia participate in lumen expansion and tubule maturation. In contrast, we failed to detect any contribution of primary cilia to the early stages of branching tubulogenesis. Finally, primary cilia did not influence cell migration or proliferation.

In addition, we investigated the interplay between primary cilia and transepithelial sodium transport by kidney tubule epithelial cells. We found that increased sodium transport induces primary cilia lengthening. Delving into the mechanism of this effect, we discovered that major players are the mineralocorticoid axis (aldosterone, mineralocorticoid receptor, ENaC) and the intraflagelar transport machinery protein IFT88. Cilium lengthening relies on decreased IFT88 degradation and inhibition of autophagy by MR via a mTORC1-independent pathway. We also found that ENaC which enables transepithelial Na⁺ transport in the collecting duct, plays a role in cilium length control. ENaC silencing or blocking results in shortening of primary cilium. Alternatively, modulation of primary cilium length did not alter ENaC-dependent sodium transport or subunit expression. Taking advantage of C.elegans model, we analysed the behavioural

3 responses to different concentrations of NaCl in mutants of several ENaC/DEG protein family members. Ion channels localized to the primary cilia including MEC-10, UNC105 and UNC-8 were shown to play a sensory role in the signalling pathway which underlines NaCl chemotaxis behavioural response.

Our data supported by literature review on cilium length modulations suggest that the cilium length response occurs as an adaptive mechanism to physiological changes of renal function. In response to alterations in hormone levels, GFR or ion delivery, cilium length changes and modulates cell size to cope with ion homeostasis.

4 II–TABLEOFCONTENTS

IABSTRACT ... 1

IITABLEOFCONTENTS ... 4

IIIACKNOWLEDGEMENTS ... 6

IVINTRODUCTION ... 7

1.1THE KIDNEY: STRUCTURE AND MAIN FUNCTIONS ... 7

1.2STRUCTURE OF THE DISTAL NEPHRON ... 9

1.3 NA⁺ AND CL^-TRANSPORT BY THE NEPHRON ... 11

1.4THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS) ... 12

1.5ROLE OF ENAC IN ALDOSTERONE ESCAPE ... 15

1.6 CILIARY STRUCTURE AND CILIOGENESIS ... 16

1.7 CILIARY MEMBRANE BIOGENESIS AND ORGANIZATION ... 18

1.8 TRANSPORT OF THE CILIARY COMPONENTS INTO THE CILIOPLASM ... 20

1.9 CILIARY PROTEINS IDENTIFICATION BY THE IFT MACHINERY ... 21

1.10 CILIUM ASSEMBLY AND DISASSEMBLY ... 23

1.11 CILIUM LENGTH AND ITS REGULATION ... 25

1.12 AUTOPHAGY AND CILIOGENESIS CO-REGULATION PARADIGM ... 26

1.13 REGULATION OF CILIOGENESIS ... 28

1.13.1 PROTEIN KINASES REGULATE CILIOGENESIS ... 28

1.13.2 CYTOSKELETON MODIFICATIONS IN THE PRIMARY CILIUM ... 29

1.14 PRIMARY CILIUM-DEPENDENT SIGNALLING MECHANISMS ... 29

1.14.1 CALCIUM SIGNALLING ... 30

1.14.2 HEDGEHOG SIGNALLING ... 31

5

1.14.3 WNT SIGNALLING ... 32

1.14.4 NOTCH SIGNALLING ... 33

1.14.6 MTORC SIGNALLING ... 33

1.14.3 OTHER SIGNALLING MECHANISMS ... 34

1.15 CILIOPATHIES: CLASSIFICATION, GENERAL SYMPTOMS AND KIDNEY PHENOTYPE 34 1.16 CYSTIC KIDNEY DISORDERS ... 36

1.17C ELEGANS - A MODEL ORGANISM FOR KIDNEY RESEARCH ... 38

1.18 SALT SENSING AND SENSORY TRANSDUCTION IN C. ELEGANS ... 40

CONCLUSION ... 42

V HYPOTHESIS AND PROJECT AIMS ... 43

VIRESULTS ... 44

2.1 PUBLISHED RESULTS (INSERTED WITH NO PAGE NUMBER) ... 44

2.2 UNPUBLISHED RESULTS ... 45

VIIGENERALDISCUSSION ... 55

X LIST OF ABBREVIATIONS ... 65

XIREFERENCES ... 68

6 III ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Prof. Eric Feraille for the continuous support of my PhD study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis.

Besides my advisor, I would like to thank my PhD good parents Prof. Monica Gotta and Prof. Patrick Meraldi as well as their lab members. I am grateful not only for professional support, insightful comments and encouragement but also for the hard question which helped me to widen my research from various perspectives.

My sincere thanks also go to Prof. Johannes Loffing who gave access to the laboratory and research facilities and MDr. Jan Czogalla for sharing his professional experience.

Many thanks to Charna Dibner and Emi Nagoshi for reviewing my Horse-these on circadian clock and their feedback which hopefully made me a better writer.

I thank my labmates and Sophie de Seigneux `s laboratory for sharing the lab with me and for stimulating discussions of my research project over the the last four years. In particular, I am grateful to my friends Vasiliki Delitsikou and Aderonke Sofoluwe who have provided me through moral and emotional support in my PhD-student-life.

Last but not the least, I would like to thank my family for supporting me spiritually throughout writing this thesis and my life in general.

7 IV INTRODUCTION

1.10 The kidney: structure and main functions

The kidneys are, in humans, paired bean shaped structures that filter blood and remove waste metabolic products of our organism, regulate body fluid homeostasis and produce hormones involved in the regulation of blood pressure, i.e. renin, red blood cell number, i.e. erythropoietin, and mineral metabolism, i.e. 1-25OH-Vitamin D3 (Boron and Boulpaep 2009). A human kidney section (Fig.1) can be divided into two layers: the cortex (outer layer) and the medulla (inner region).

Figure 1. Kidney section (left) and its respective diagram (right). From antranik.org The cortex is characterized by the presence of glomeruli (a small capillary knots) and convoluted epithelial tubules. The medullary level comprises parallel epithelial tubules and blood vessels; it can be divided into 8-18 renal pyramids with a base located at the border between the cortex and the medulla. Each human kidney contains about

8 1.000.000 individual filtration units called nephrons (Fig.2). A nephron consists of a renal corpuscule (glomerulus + Bowman’s capsule) assembled with an epithelial tubule where the filtrate converts into the urine. The kidney tubule can be divided into several functional segments: the proximal tubule (PT) with three successive portion (S1, S2 and S3), the thin descending limb (tDL), the thin ascending limb (tAL) of the loop of Henle, the medullary and cortical thick ascending limb (mTAL and cTAL), the distal convoluted tubule (DCT), the connecting tubule (CNT) and the cortical, outer medullary and inner medullary collecting duct (CCD, OMCD and IMCD).

Figure 2. Scheme of the nephron structure (left). Glomerulus and kidney epithelial tubule segment are shown. Segment-specific cell structure (right) shows major specific characteristics of different kidney epithelial cell types. From antranik.org

9 1.2 Structure of the aldosterone-sensitive distal nephron

The aldosterone-sensitive distal nephron (ASDN) is subdivided into three segments:

DCT, CNT and CD. The DCT starts from the macula densa and ends at the transition to the CNT. CNT and CD are made of principal and intercalated cells which are morphologically and functionally different. Most cells comprising the collecting system (CNT and CD) are considered principal cells (Fig.2 right side, Fig3A). The principal cells have less mitochondria and less developed basolateral membrane invaginations than intercalated cells and a central cilium that sticks out of the apical membrane while intercalated cells are non-ciliated (Fig. 4B).

Figure 4. Primary cilia in the kidney. (A) The nephron structure. Primary cilia are found on epithelial cells along the nephron except intercalated cells. (B) A scanning electron microscopy imaging of primary cilia (shown with arrows) in a mouse nephron. Scale bar 4μm (Deane and Ricardo 2012).

Principal cells express the epithelial sodium channel (ENaC) on their apical membrane and the Na-K-ATPase on the basolateral membrane. The Na-K-ATPase extrudes intracellular Na⁺ and provides the electrochemical gradient for the apical Na⁺ entry.

These cells also express apical and basolateral water channels (aquaporins) involved in water reabsorption and apical K-channels for K⁺ secretion. The ratio of principal to

10 intercalated cells is 3:1 in the cortical collecting duct and 2:1 in the medullary collecting duct. This proportion can vary between species (Roy, Al-bataineh et al. 2015). The intercalated cells participate in urinary acid secretion, bicarbonate secretion/reabsorption and ammonium excretion. They accumulate large amount of round mitochondria at their apical side, lack primary cilium but display numerous irregular apical microvilli (Fig3B) (Roy, Al-bataineh et al. 2015).

Figure 3. A. rat kidney immunostaining reveals aquaporin 2 (AQP2) (green) and the V- ATPase (red) (Brown, Paunescu et al. 2009). Principal cells contain a tight apical band of AQP2 and also a weaker staining at their basolateral pole. Intercalated cells (IC) exhibit either a strong apical staining only [A-type intercalated cells (A-IC)] or a basolateral/bipolar staining for the V-ATPase [B-type intercalated cells (B-IC)]. Nuclei are stained in blue with DAPI. Scale bar=5 μm. B- B) Transmission electron micrograph of rat cortical collecting duct illustrates further the two configurations of intercalated cells. The type A-IC (right) and the type B-IC (left) present a cytoplasm with more abundant mitochondria and apical microvilli. Original magnification, ×5000) (Roy, Al-bataineh et al. 2015).

Intercalated cells are characterised according to the presence of the chloride- bicarbonate exchanger AE1 (Slc4a1) or Pendrin and localization of the H⁺-ATPase. There are three subpopulations of these cells: type A (α-intercalated cells) which secrete H⁺ and reabsorb HCO3- via an apical H⁺-ATPase and a basosolateral AE1, type B (β- intercalated cells) which secrete HCO3- and reabsorb Cl^- via an apical Pendrin and a

11 basolateral H⁺-ATPase, and non-A/non-B intercalated cells. which express both Pendrin and the H⁺-ATPase at the apical membrane.

1.3 Na⁺ and Cl^- transport by the nephron

The kidneys maintain the body extracellular fluid (ECF) volume by modulating the amount of Na⁺ excreted in the urine. The proportion of Na⁺ and water is maintained constant by osmoregulation therefore, any variation of body Na⁺ content is associated with a proportional variation of water content. Transport mechanism at the tissue level rely on functional organization of the plasma membranes into apical and basolateral domains in each cell, these domains differ one from another by lipid and protein compositions.

Figure 5. Na⁺ reabsorption along nephron. Na⁺ reabsorption (Jna) along the nephron is shown as percentage of filtered Na⁺. Major regulatory mechanisms such as glomerulo- tubular (GT) balance and tubulo-glomerular (TG) feedback as well as major hormonal control are indicated. CCD, cortical collecting duct; CNT, connecting tubule; DCT, distal convoluted tubule; J(Na-subscript), sodium flux; OMCD, outer medullary collecting duct;

IMCD, inner medullary collecting duct; PCT, proximal convoluted tubule; PST, proximal straight tubule; tAL, thin ascending limb; TAL, thick ascending limb of Henle's loop; tDL, thin descending limb (Palmer and Schnermann 2015).

The proximal tubule and the loop of Henle reabsorb about 70% and 25% of filtered Na⁺, respectively (Fig.5). The DCT and collecting system inclusive reabsorption is around 8%

12 of the filtered Na⁺ load in which the CD contribution is only 3%. The ASDN is responsible of the fine tuning of Na⁺ balance and is therefore tightly regulated by hormones such as aldosterone and arginine vasopressin (AVP).

Transcellular Na⁺ reabsorption in all nephron segments starts with passive Na⁺ entry across the apical membrane which follows its electrochemical gradient. Indeed, Na⁺ concentration inside of the cell is lower than outside and negative cell voltage with respect to interstitium and lumen is generated by basolateral Na,K-ATPase and K- channels. The PT, the TAL and the DCT use a combination of Na⁺-coupled transporters and exchangers to move Na⁺ across the apical membrane. In the collecting system, Na⁺ enters via apical ENaC channel (Fig.6). The next step is active extrusion of Na⁺ though the basolateralmembrane (extracellular Na⁺ reabsorption) in every cells type. This step is performed by the Na-K-ATPase, which functions in a way to keep low intracellular Na⁺ (about 15mM) and high intracellular K⁺ (about 120mM) and develops a -70mV potential difference (cell interior negative) between external and internal leaflets of the plasma membrane.

Ion transport along the kidney tubule is also dependent on the paracellular pathway.

Paracellular Na⁺ reabsorption in PT is mostly driven by solvent drag through claudin-2 containing leaky tight-junctional complexes while in TAL it is driven by lumen positive transepithelial potential via specific tight junctional permeability conferred by claudin 10b (reference review by Alan Yu, JASN 2014). In the DCT, paracellular transport is very low. In the collecting system, the electrochemical gradient for Na⁺ reabsorption is negative by the combination of low luminal Na⁺ concentration and lumen negative electrical potential, leading to some degree of Na⁺ “backleak” from interstitium to lumen via tight junctional complexes and this despite a high transepithelial resistance.

1.4 The Renin-Angiotensin-Aldosterone System (RAAS)

The RAAS is a major physiological system that controls blood pressure and sodium homeostasis (Fig.7). Its functions are coordinated by renal metabolism, cardiovascular

13 system and the central nervous system (CNS). It is thought that the system appeared first in primitive chordates and tunicates. Approximately at the moment of the divergence of bony fish, all the necessarily components were present including the Mas receptor, which firstly appears after the bony-fish/tetrapod divergence. This evolutionary innovation appearance took place 400 million years ago. Angiotensinogen first appeared in cartilage fish. Originally RAAS genes had ancestral functions unrelated to their current role (Fournier, Luft et al. 2012).

Figure 6. General scheme and effects of high levels of the RAAS molecules: angiotensin II and aldosterone during pathological conditions (arterial hypertension and metabolic syndrome). K: Kidney; V: Vascular tissue; C: Cardiac tissue; EP: Endopeptidases; ACE:

Angiotensin-converting enzyme; ACE-2: Angiotensin-converting enzyme 2 (Munoz- Durango, Fuentes et al. 2016).

14 Angiotensinogen, the key precursor of the RAAS, is produced in liver and cleaved into a short 10 amino-acid peptide angiotensin-I (Ang-I) by the aspartyl protease renin. Renin is produced myoepithelial cells of the afferent glomerular arteriole that is part of the juxtaglomerular apparatus (JGA) (Fig.6). Then, the angiotensin-converting enzyme (ACE) cleaves Ang I into an 8-amino-acid peptide angiotensin-II (Ang-II). Ang II mainly acts on the adrenal cortex to induce aldosterone (Aldo) secretion and on blood vessels to induce vasoconstriction. These two effects are aimed at increasing blood pressure. Ang- II also stimulates thirst and salt appetite, sympathetic tone, vasopressin release and sodium reabsorption along the kidney tubule. Aldo acts in the ASDN as the main stimulator of sodium and chloride reabsorption (Sparks, Crowley et al. 2014).

Figure 7. A. Classical mechanism of aldosterone action. A. Schematic view of aldosterone signaling in principal cell. Aldo passes into the cell, binds to the MR receptor. MR translocates into the nuclei, forms a dimer and binds to the hormone response NDA elements (HRE). This binding induces the expression of the aldosterone-target genes including SGK-1. SGK1 positively regulates ENaC surface expression by preventing its degradation via UPS. B. Scheme of a kidney (K) with an adrenal gland (AG) where aldosterone is produced (shutterstock.com). C. Aldosterone targets principal cells in the collecting tubule and collecting duct (shown with an orange square).

15 Aldosterone acts on the principal cells of the collecting system by binding to the cytoplasmic mineralocorticoid receptor (MR; Fig.7). MR then translocates into the nucleus and enhances expression of its target genes. One of these is serum- and glucocorticoid-regulated kinase (SGK-1), a key player in early aldosterone action. SGK-1 inhibits the E3 ubiquitin ligase Nedd4-2 by phosphorylation. SGK1 and Nedd4-2, along with other regulatory proteins, interact with each other and with ENaC. This results in decreased ENaC degradation and significant increase of the ENaC expression in the apical membrane of the principal cells, leading to an increase in Na⁺ permeability and therefore intracellular Na⁺ entry (Gaeggeler, Gonzalez-Rodriguez et al. 2005, Soundararajan, Lu et al. 2012, Lang and Pearce 2016). The simultaneous activation of Na⁺ entry leads to more intensive Na⁺ extrusion through the basolateral membrane via increased Na-K-ATPase abundance and activity to ensure stable cell volume and intracellular Na⁺ concentration (Boron and Boulpaep 2009). Aldo also displays relatively rapid non-genomic effects such as ROS production via NADPH oxidase. It causes oxidative stress at very low Aldo concentrations in renal tubule cells (Queisser, Oteiza et al. 2014). Aldo exposure also leads to downregulation of miRs including mmu-miR-335–

3p, mmu-miR-290–5p and mmu-miR-1983 in mCCD cells (Edinger, Coronnello et al.

2014). These miRs were recently found to be important intermediate steps of the Aldo regulation of Na⁺ transport. Artificial overexpression of some miRs leads to blunted aldosterone stimulation of ENaC transport. This data indicates that the hormone signals through pathways that includes both gene expression activation via MR binding and modulation of miRs abundance.

1.5 Role of ENaC in aldosterone escape

When exogenous Aldo is administered together with salt loading, excretion of Na⁺ occurs despite increased levels of mineralocorticoids. This is commonly known as

“aldosterone escape” (Stockand, Mironova et al. 2010). The mechanism of this phenomenon is poorly investigated. It has been suggested that ENaC regulation by purinergic signalling is important for appearance of aldosterone escape. High dietary

16 Na⁺ intake increases urinary ATP concentration in mice which activates ionotropic P2X and metabotropic P2Y receptors. The apical ATP/UTP/P2Y2 receptor tandem expressed in the CNT and CD mediates the inhibitory effect of dietary salt on ENaC open probability and inhibits its activity during aldosterone escape in a dose-dependent manner (Stockand, Mironova et al. 2010, Vallon and Rieg 2011).

1.6 Ciliary structure and ciliogenesis

The primary cilium is a solitary, non-motile organelle that extends from the cellular surface of most differentiated cells (Satir, Pedersen et al. 2010, Venkatesh 2017). It is suggested that cilia function as a sensory antenna that coordinates signal transduction (Jin, Mohieldin et al. 2014, Atkinson, Kathem et al. 2015, Malicki and Johnson 2016, Pala, Alomari et al. 2017). Defective primary cilium is a cause of several human diseases and a whole spectra of developmental disorders, commonly known as ciliopathies (Satir, Pedersen et al. 2010).

Figure 8. A. Primary cilium ultrastructure (Satir, Pedersen et al. 2010). B. Schematic representation of the intraflagellar transport (IFT) in primary cilium. IFT proteins associated with cystic kidney disease are listed on the left side of the picture (Davenport and Yoder 2005).

17 Primary cilium consists of an axonema of 9+0 doublets of microtubules that extends from a basal body (Davenport and Yoder 2005) and are unsheathed by a membrane (Fig.8A). Immotile primary cilia lack a central pair of microtubules and other elements involved in ciliary motility (Teves, Nagarkatti-Gude et al. 2016). The axonemal microtubules polymerize at the growing tip to which cargo, including ciliary proteins is delivered by intraflagellar transport (IFT). IFT is an evolutionary conserved process that provides growth and maintenance of both motile and primary cilium (Pazour, Dickert et al. 2000, Avasthi and Marshall 2012, Bhogaraju, Engel et al. 2013, Scholey 2013). It based on association of ciliary building blocks with the scaffold IFT machinery particles that are transported along axonemal microtubules by kinesin 2 family members in the anterograde (base-to-top) direction (Fig.8B). Retrograde (top-to-base) transport is provided by dynein 2 motors (Prevo, Scholey et al. 2017).

Primary cilium derives from the mother centriole of the centrosome. Process of building of the antenna is called ciliogenesis (Fig.9) and it starts in G0 phase when differentiated cells exit the cell cycle (Avasthi and Marshall 2012, Nikonova and Golemis 2015).

However, G1 phase cells are also competent to form cilia. When G1 cells progress towards S1 phase primary cilium disassembles. In S1 phase centrioles are not competent to form cilia because they start duplication (Sanchez and Dynlacht 2016).

18 Figure 9. Ciliogenesis in an epithelial cell. A mother centriole contacts a ciliary vesicle.

Axoneme elongates at the tip and its most proximal region gives rise to the transition zone. The ciliary vesicle expands around the axoneme and transforms into the ciliary sheath, which fuses with the plasma membrane externalizes the cilium and modifies the outer sheath into the periciliary membrane. Factors that regulate ciliogenesis in kidney are shown with arrows. Figure adapted from Garcia-Gonzalo and Reiter 2012.

1.7 Ciliary membrane biogenesis and organization

Ciliary membrane is about 1/100 to 1/5000 of the total cellular surface and it`s volume is about 1/30000 of the total cell volume (Nachury, Seeley et al. 2010). It is thought that ciliary membrane has similar biochemical origins than plasma membrane, but it has a distinct lipid and protein composition which enables the cilium to function as a cellular antenna and creates cell type specific morphology/curvature (Garcia, Raleigh et al.

2018).

19 Figure 11. Phosphoinositides localisation at distinct cellular membranes. PI4P and PI(4,5)P2 are distributed throughout the plasma membrane, PI(4,5)P2 is concentrated in a ciliary base (Nakatsu 2015)

The lipid composition of primary cilia is poorly investigated. It is known that ciliary membrane contains PI4P along the entire organelle while PI(4,5)P2 is localized to the ciliary pocket region (Fig.11). Phosphoinositide metabolism, mediated by INPP5E, regulates intraflagellar transport of ciliary proteins and Hh signalling (Nakatsu 2015).

Endoplasmic reticulum, Golgi apparatus, and recycling endosomes vesicles are being intercalated into the ciliary membrane or the ciliary membrane emerges from the plasma membrane through multiple membrane remodelling during ciliogenesis (Nachury, Seeley et al. 2010, Garcia, Raleigh et al. 2018). Vesicular trafficking into the primary cilium is mostly dependent on a Rab cascade comprising Rab8 and Rab11 (Nachury, Loktev et al. 2007, Westlake, Baye et al. 2011, Madugula and Lu 2016). The following model of ciliary membrane composition control is currently proposed : (1) the transition zone components via specific binding capacity select membrane-associated proteins to entry and exit the cilium (2); the BBSome function as an exporter/importer of

20 the ciliary membrane proteins; (3) a membrane diffusion barrier excludes non-ciliary membrane proteins only; and (4) intraflagellar transport complexes enrich or deplete ciliary proteins in coordination with BBSome (Garcia, Raleigh et al. 2018).

1.8 Transport of the ciliary components into the cilioplasm

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 growth factor β (TGF- β) receptor. It regulates bone development and metabolism. TGF- β stimulation is transduced into cellular response via SMAD2-3/ERK1-2 signalling (Pala, Alomari et al. 2017).

1.15 Ciliopathies: classification, general symptoms and kidney phenotype

The dysfunction of both motile and immotile cilia causes a spectrum of human disorders commonly known as “ciliopathies” (Benmerah, Durand et al. 2015, Reiter and Leroux 2017, Kempeneers and Chilvers 2018). This term was firstly used in 1984 and became popular in 21^st century.The ciliopathies are symptomatically heterogeneous and mostly inherited autosomal recessive disease (Fig16). The ciliopathies can be classified into two major categories: motor ciliopathies (associated with motile cilia and mutations that

35 lead to motility loss) and sensory ciliopathies (associated mostly with primary cilia and defects in sensory/signalling functions). Additionally, first order ciliopathies (caused by disruption of ciliary proteins) are distinguished from the secondary order ciliopathies (associated with non-ciliary proteins which are required for cilia function) ^104-105.

Figure 16. Different organ systems affected in ciliopathies and corresponding phenotypic manifestations of the disease are shown. Ciliopathies that are caused by defects in motile cilia are shown in orange, those that result from defects in primary cilia are shown in blue, those that are caused by defects in both cilia types are shown in green (Reiter and Leroux 2017).

Ciliopathies are distinguished from “extraciliary disorders”, a situation generated by a mutation in a protein which has both ciliary and non-ciliary functions; this causes a phenotype unrelated to the cilium. For example, IFT20 plays also a role in collagen trafficking, the ift20 mouse phenotype characterized by craniofacial skeletal abnormalities (Noda, Kitami et al. 2016).

36 The abnormalities found in ciliopathies includes dysfunction in the kidney, liver, respiratory organs, brain, eye, ear, skeleton and reproductive system (Fig.17).

Specifically, sensory ciliopathies are often characterized by kidney and liver diseases, organ laterality defects, polydactyly, retinal degeneration, skeletal and central nervous system malformations, obesity and diabetes (Fliegauf, Benzing et al. 2007, Kempeneers and Chilvers 2018).

1.16 Cystic kidney disorders

Primary cilia defects are often characterized by renal and liver cysts. The kidney disease spectrum in this case varies between the polycystic kidney disease (PKD) and syndromes where the renal cyst formation is accompanied by a broad range of abnormalities (Fig.8B, Fig.17). The mechanisms associated with renal cyst formation include increased cell proliferation, epithelial fluid and chloride secretion, extracellular matrix abnormalities, somatic and/or germ line mutations, planar cell polarity defects, apoptosis and inflammation (Paul and Vanden Heuvel 2014).

37 Figure 17. Major categories of ciliopathies (blue boxes) that are associated with the named proteins (right side) and a scheme of ciliary regions where these proteins localise are shown. The asterisks indicate proteins that are localized to other ciliary regions during ciliogenesis or participate in ciliary trafficking (Reiter and Leroux 2017).

Autosomal dominant (AD) PKD and autosomal recessive (AR) PKD are common potentially lethal hereditary disease in humans. ADPKD causes renal failure in approximately 50% of cases and its prevalence 1/400-1/1000 people. ADPKD is caused by mutation in PKD1 (85% affected individuals) on chromosome 16 and in PKD2 (15% of cases) on chromosome 4 (Ghata and Cowley 2017). The symptoms of the disease generally appear after 40 years and are hypertension, haematuria, abdominal pain, urinary tract infections, intestinal diverticulosis and cerebral aneurisms. PKD2 encodes

38 polycystin 2, a calcium channel of the TRP family that associates at the level of the primary cilium with polycytin 1, the PKD1 product. Cysts can originate from glomeruli or from any part of the kidney tubule including the collecting duct. Cysts begin as diverticula and slowly grow leading to compression of neighbouring tubules and vessels as well as interstitial inflammation and fibrosis. Cyst growth is driven by a combination of cell proliferation and fluid secretion. Suppression of autophagy may also play a pathogenical role in cyst formation and growth in ADPKD (Huber, Walz et al. 2011, Ravichandran and Edelstein 2014, Wang, Livingston et al. 2015). ARPKD is caused by mutation in PKDH1 gene encoding polyductin, a regulator of renal collecting duct and bile duct differentiation. The disease prevalence is 1/6000 live births and up to 75% of these patients die within a few days (Bergmann 2015). Nephronostisis is the most common cause of ESRD in children (end-stage renal disorders), it can be caused by mutations in the genes coding nephrocystins. Renal cysts are accompanied by congenital hepatic fibrosis, laterality defects, cerebellar vermis, hypoplasia and retinal degeneration. Mutations in HNF1ß or in other ciliopathy associated genes cause nephronophthisis, Bardet-Biedl or Joubert syndrome; these diseases are known to be PKD-mimetics (Kempeneers and Chilvers 2018).

1.17 Caenorhabditis elegans as a model organism for kidney research.

The Caenorhabditis elegans reference strain N2 is a laboratory animal, it has been modified by domestication due to mutation appearance and stabilization. It was isolated in Bristol, (United Kingdom) and cultured on agar plates seeded with E.coli as a food source. The natural milieu for C. elegans is a rich soil or compost where they are often found in a non-feeding “dauer” stage, it is found worldwide, mostly in areas with humid warm climate (Frézal and Félix 2015). Two alternative life cycles have been described for C. elegans. If well fed, they pass through four larval stages and reach the adult stage after 3 days. Under stress conditions they may shift during L1 stage to predauer stage (L2d) followed by non-feeding diapause called dauer. This stage is very resistant to a variety of stress conditions and able to survive several months without food. C. elegans

39 have boom-and-bust lifestyle. A cycle of colonization starts when dauer larvae find a food source; they exit a dauer stage and seed a population of up to 10⁴ feeding nematodes. As they run out of the food source, dauers may start explore the neighbouring environment for new resources. Dauer larva display a specific type of behaviour called nictation, it thought to be help in finding invertebrate host. A nematode stay on the tale and wave one`s body in the air. C. elegans can form a column and synchronize the group nictation. The population size of nematode varies: the densest population is detected in autumn followed by its decrease to the bottle necks in winter. The reproduction mode of these nematodes is called androdioecy. It happened ether through self-fertilizing (selfing) hermaphrodites or by breeding of XX (hermaphrodites) with X0 (males occur by non-disjunction of X chromosomes during meiosis at 0.1% frequency).

The introduction of the nematode to molecular biology and genetics led to tree Nobel prize awards: S. Brenner, R. Horvitz, J. Sulston in 2002 for apoptotic cell death description; A. Fire and C. Mello in 2006 for the discovery of gene silencing by dsRNA;

M. Chalfie in 2008 for the discovery and development of the green fluorescent protein, GFP. Starting in 1999, kidney researchers took advantage of this simple and time-saving fashion model organism which allows using modern molecular biology and genetics techniques combined with phenotype screening (Barr and Sternberg 1999). It is known that most human kidney disease genes associated with cyst formation, proteinuria or renal carcinoma development are highly conserved and C. elegans displays homologous genes with similar functions (Muller, Zank et al. 2011, Hsu 2012). Ciliary abortion leads to several defined phenotypes characterised by dysregulated mechanosensation, synaptogenesis, motility and life span (wormbook.org). Several landmark research on LOV-1, PKD-2 (homologues of human polycitin-1 and polycistin-2) describing functions and role in ciliogenesis and cyst formation were done in C. elegans (Barr and Sternberg 1999, Muller, Zank et al. 2011, Hsu 2012, Frézal and Félix 2015, Chen, Bharill et al. 2016, Bezares-Calderon, Berger et al. 2018). Many genes involved in juvenile cystic kidney disease including juvenile nephronophthisis (NHPH), Bardet-Biedl syndrome (BBS),