Study the functional interaction between autophagy and primary cilium

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Ciliogenesis and Ciliary Subdomains

Ciliogenesis occurs in quiescent cells when the cells enter the G0 phase. When the cells decide to re-enter in a cell cycle, cilium resorption occurs followed by the S phase. Ciliogenesis and cell cycle is regulated by post-translational modifications (Avidor-Reiss and Gopalakrishnan). Sorokin proposed two models of primary ciliogenesis upon TEM observations from epithelial cells and fibroblasts (Sorokin 1962; Sorokin 1968); an ‘extracellular’ pathway and an ‘intracellular’ pathway. In the polarized cells, the ‘extracellular’ model the centrioles move directly to the plasma membrane and the mother centriole serve as a docking center for the growth of the axoneme thus forming the basal body of the cilium (Benmerah 2012). This model is observed in the ciliated cells of lung or kidney tissues. Alternatively, in non-polarized cells, as in the case for fibroblasts, an intracellular ciliary vesicle grows around the mother centriole together with the axoneme. This vesicle will later fuse with the plasma membrane, which will result in the protrusion of PC to the extracellular space. The BB is formed of a 9 triplet microtubules (Figure 3). The TEM images show that PC can remain invaginated to the plasma membrane which resembles to the flagellar pocket. The ciliary pocket is continuous with the plasma membrane, but functionally different. This small but significant domain of the PC is established after fusion of the plasma membrane with the intracellular ciliary vesicle that grows on top of the BB (Ghossoub, Molla-Herman et al. 2011). The origin and function of the ciliary pocket is not clearly known but it is currently a topic of intense research (Benmerah 2012). The ciliary pocket is characterized by the presence of clathrin coated vesicles resulting from the vesicular trafficking that originates and ends in this region. Acting as a specific endocytic membrane, the ciliary pocket has been described to regulate the transforming growth factor-β (TGF-β) pathway (Molla-Herman, Ghossoub et al. 2010; Clement, Ajbro et al. 2013). The presence or absence of the ciliary pocket depends on the cell type and can be explained by different kinetics during ciliogenesis; for example, ciliary pocket is common in fibroblasts but has rarely been observed in epithelial cells. This rare observation of the pocket in epithelial cells can be explained by a short-lived intracellular pathway of the ciliary pocket docking to the PM (Ghossoub, Molla-Herman et al. 2011). PC is not isolated from the cytoplasm but is highly compartmentalized; it is surrounded by a specialized region of the plasma membrane that holds a different concentration of channels and receptors and the axoneme is separated from the rest of the intracellular compartments by the “transition zone”. The transition zone consists of the “ciliary necklace” and transition fibers and is evolutionarily conserved. It is characterized by the ciliary necklace formed by Y-shaped fibers that connect the microtubule doublets to the ciliary membrane at the base of the cilium distal to the BB. Moreover, this zone is mostly characterized by TEM data (Reiter, Blacque et al. 2012). This ciliary subdomain is highly organized with a molecular structure resembling that of the nuclear pore and serves as a border control where different, precursors and intraflagellar transport (IFT) proteins, along with proteins of different signaling pathways are concentrated and regulated for entry to the axoneme (Kee, Dishinger et al. 2012). The composition of the transition zone and subdomains is not fully known, however several studies suggest that distal appendage proteins play an important role (Avidor-Reiss and Gopalakrishnan 2013). During ciliogenesis, the mother centriole anchors to the associated membrane; the ciliary vesicle membrane or the plasma membrane. The mother centriole can be distinguished from the daughter centriole by the presence of subdistal and distal appendages. Distal appendages are necessart for ciliogenesis. OFD1 (oral-facial-digital syndrome1) protein is associated with the distal ends of centrioler microtubules and is necessary for centriole docking via the distal appendages which forms the transition fibers at the base of the cilium (Reiter, Blacque et al. 2012; Singla, Romaguera-Ros et al. 2010).

Intraflagellar Transport Mechanism

A bi-directional movement in the Chlamydomonas flagella was fist observed by Rosenbaum lab and identified as the intraflagellar transport (IFT) (Kozminski, Johnson et al. 1993). IFT complexes are conserved in ciliated organisms and have different functions. The IFT mechanism plays an essential role in the assembly and maintenance of the cilia or flagella and during the transport and trafficking of ciliary components such as cargo along the axoneme (Rosenbaum and Witman 2002; Sung and Leroux 2013). The bi-directional trafficking along the polarized microtubules of the axoneme is maintained through the action of two motor protein families: kinesins and dyneins (Figure 4) (Pedersen and Rosenbaum 2008). Anterograde IFT (aIFT) trafficking with base-to-tip cargo direction, depends on the heterotrimeric kinesin-2 motor, also known as KIF3 motor complex. This complex consists of two kinesin-2 family proteins KIF3A, KIF3B and KIF-associated protein 3 (KAP3). Retrograde IFT (rIFT) with tip-to-base cargo direction depends on the dynein motor that consists of cytoplasmic dynein 2 heavy chain 1 (DYNC2H1) and cytoplasmic dynein 2 light intermediate chain 1 (DYNC2LI1)) (Pedersen, Veland et al. 2008). The kinesin and dynein motors are conserved in all ciliated organisms with the exception of Plasmodium falciparum (Sung and Leroux 2013). Two large complexes composed of more than 20 proteins called IFT A and IFT B are essential for the building and trafficking along the cilium, the first for retrograde and the latter for anterograde trafficking. Mutations in the two IFT subunits have different consequences; IFT B protein defects lead to absent or shortened cilia whereas defects in IFT A proteins lead to a bulged cilia (Goetz and Anderson 2010). The docking and assembly mechanism of IFT particles are not well characterized. Several IFT proteins are identified over the years in several organisms and a number of these proteins possess specialized roles in ciliogenesis, antero- or retrograde trafficking and transport of ciliary components to the cilia or flagella. Some subunits of IFT complexes, such as IFT20 and DYF-11/MIP-T3, which are part of the IFT B complex are found in other cellular compartments from where they facilitate transport of vesicles with specific cargo to the BB and axoneme (Pedersen, Veland et al. 2008). Continuous shuttling of IFT20 protein between the Golgi and the PC is required for ciliogenesis and cilia dependent signalizations (Follit, Tuft et al. 2006; Keady, Le et al. 2011). OFD1 protein plays an important role in association with IFT assembly and is required for the recruitment of IFT88 protein (Singla, Romaguera-Ros et al. 2010). Maintenance of the cilia also depends on the BBsome, a complex formed by proteins encoded by genes mutated in Bardet–Biedl syndrome (BBS). Ciliary targeting sequence (CTS) is recognized by the BBsome and serves target the proteins with CTS to the cilia in a BBSome dependent manner. Several proteins involved in the vesicular trafficking such as the small GTPase Rab8 or Rab11 are evolutionarily conserved in the vesicular trafficking and also in cilia assembly via IFT and BBsome (Sung and Leroux 2013). The mechanisms of the transport and trafficking of proteins involved in various cilia-dependent signaling pathways are not known in details (Goetz and Anderson 2010). However various proteins are observed to interact with IFT. For example the Chlamydomonas orthologue of the calcium channel PKD2 is IFT-dependent (Huang, Diener et al. 2007).

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Fluid flow and Fluid Shear Stress

The mechanosensory role of PC is evolutionarily important for various organs. PC can sense different fluid movements such as urine in the kidney tubules, blood in the vascular system, bile in the hepatic biliary system, lacunocanalicular fluid in the bone and cartilage, digestive fluid in the pancreatic duct, nodal flow in the Hensin’s node, and cerebral spinal movement in the nervous system. The transduction of mechanical stimuli by the PC depends on different receptors and channels such as calcium channels polycystin 1 (PC1) and polycystin 2 (PC2) on the axoneme depending on the tissue and thus can induce various intracellular signalizations explained above. See Table 1 for an overview of cilia-dependent mechanical stress in different tissues. There are other mechanosensory cellular compartments in different tissues including microvilli, actin filaments in the kidney, glycocalyx, a membrane-bound filament structure found or caveolae, an invagination of the plasma membrane rich in lipids, receptors and channels including K+ channels, Cl channels, and Ca2+ channels on the surface of endothelial cells. Tyrosine kinase receptor (TKR), G-protein-coupled receptors (GPCRs) and G-protein itself have been shown to be activated by fluid shear stress. Adhesion proteins (PECAM-1, and VE-cadherin) at sites of cell–cell and cell– matrix attachment are subjected to tension under shear stress. Cytoskeleton is responsible for cell shape regulation and can respond to mechanical forces that deform cells (Ando and Yamamoto 2013) (Figure 6).

Autophagosome Machinery and Biogenesis

Macroautophagy, referred as autophagy from now on, depends on a tight and complex molecular regulation that involves more than 30 Atg proteins that were firstly identified in yeast. Autophagy related proteins involved in the formation and maturation of autophagosomes are almost entirely conserved in all eukaryotes (Mizushima, Yoshimori et al. 2011). Autophagy is characterized by a dynamic membrane re-organization which starts with the formation of a cup-shaped double-membraned sac called the isolation membrane (also referred to as phagophore) in the cytoplasm which expands in size and later seals as the double-membraned autophagosome. Autophagosome size can differ from 0.3-0.9 µm in yeast (Baba, Osumi et al. 1997) and 0.5-1.5 µm in mammals (Mizushima, Ohsumi et al. 2002). Autophagosomes later fuse with lysosomes (with vacuoles in yeast), forming the autolysosomes in which the intracellular content and the inner membrane of the autophagosome are degraded by lysosomal hydrolases. Formation of autophagosome and autolysosomes are highly dynamic. The two processes together can take 7-9 minutes in yeast (Geng, Baba et al. 2008) and autophagosome formation can take 5-10 minutes in mammals (Fujita, Hayashi-Nishino et al. 2008).
There are three important stages in autophagic machinery: 1. Initiation and nucleation (formation of the phagophore); 2.Vesicle elongation (growth and closure of autophagosome); 3.Maturation into autolysosomes. These stages depend on different core Atg protein units: Atg1/ULK complex, the class III phosphatidylinositol 3-kinase (PI3K) (Beclin-1-PtdIns3KC3-Atg14L) complex, the Atg2-Atg18/ WIPI complex, the Atg12-Atg5-Atg16L1 conjugation system, the Atg8/LC3 conjugation system, and Atg9 vesicles. For the relevance of the manuscript, the main focus will be on mammalian autophagic machinery (Mizushima, Yoshimori et al. 2011) (Figure 8).

Diverse Regulatory Pathways

Autophagy can be constitutively active in the cell however autophagic activity depends on integration of different inputs and stress stimuli such as various nutrient deprivations including amino acids or growth factor starvation along with stimuli like organelle damage, infection, hypoxia or mechanical stress. These signals are integrated and regulated by diverse cellular pathways. mTOR-dependent Regulation of Autophagy One of the best characterized regulatory pathways for autophagy is the mammalian target of TOR (mTOR) pathway. mTOR integrates multiple signaling pathways that are sensitive to the availability of amino acids, ATP, growth factors, and the levels of reactive oxygen species and negatively regulates autophagy (Singh and Cuervo 2011). Besides autophagy, mTOR pathway has diverse functions in the cell such as initiation of mRNA translation, cell growth and proliferation, ribosome biogenesis, transcription, cytoskeletal reorganization and long-term potentiation (Ravikumar, Sarkar et al. 2010). Downstream targets of the mTOR complex includes for example ribosomal protein S6 kinase-1 (S6K), translation initiation factor 4E binding protein-1 (4EBP-1), and eEF2 kinase which are involved in protein synthesis regulation. There are two mTOR complexes; a rapamacyn sensitive mTOR complex1 (mTORC1) and rapamycin insensitive mTORC2. mTORC1, the main regulator of autophagy, includes the catalytic subunit raptor, G protein β -subunit-like protein (GβL) and proline-rich Akt substrate of 40 kDa (PRAS40) (Meijer, Lorin et al. 2014). Rapamycin, a potent autophagy activator, inhibits the kinase activity of mTORC1. In the presence of nutrients, mTORC1 is responsible for the phosphorylation of ULK1 on Serine 757 and its subsequent sequestration therefore inhibiting autophagy. Activation of mTORC1 by amino acids occurs via the translocation of the complex to the lysosomal membranes where Rag GTPases reside. Insulin activates the complex via G protein Rheb (Meijer and Codogno 2011). Inhibition of mTORC1 by rapamycin is mediated by the reduced phosphorylation of S6K and 4EBP1 and releases ULK1 to phosphorylate FIP200 in the ULK1-Atg13-FIP200 complex to be recruited to the phagophore formation site on the ER (Jung, Jun et al. 2009).

Table of contents :

Table of Illustrations
I. Introduction
1. Cilium: Structure and Signaling
1.1. Structure
1.1.1. Motile Cilia and Flagella
1.1.2. Primary Cilia
1.1.3. Ciliogenesis and Ciliary Subdomains
1.1.4. Intraflagellar Transport Mechanism
1.2. Ciliary Signaling
1.2.1. Hedgehog Signaling
1.2.2. PDGF Pathway
1.2.3. Wnt Pathway
1.2.4. Calcium Signaling
1.2.5. Fluid flow and Fluid Shear Stress
2. Autophagy
2.1. Role of Autophagy
2.2. Different Types of Autophagy
2.2.1. Macroautophagy
2.2.2. Chaperone-mediated Autophagy
2.2.3. Microautophagy
2.3. Autophagosome Machinery and Biogenesis
2.3.1. Initiation
2.3.2. Elongation
2.3.3. Maturation
2.3.4. The origin of the Autophagosome
2.4. Regulation of Autophagy
2.4.1. Diverse Regulatory Pathways
2.4.2. Post-translational Modifications of Autophagic Machinery
2.4.3. Transcriptional and Epigenetic Regulations of Autophagy
2.5. Physiological Role of Autophagy
2.5.1. Selective Autophagy
2.5.2. Autophagy during pre- and post-natal state
2.5.3. Autophagy in the liver
2.5.4. Autophagy in the brain
2.5.5. Autophagy in the muscle
2.5.6. Autophagy in the intestine
2.5.7. Autophagy in the pancreas
2.5.8. Autophagy in the lung
2.5.9. Autophagy in innate and adaptive immunity
2.5.10. Pathophysiological Role of Autophagy
2.6. The Role of Autophagy in Kidney Physiology and Physiopathology
2.6.1. Physiology of the Kidney: An Introduction
2.6.2. Autophagy in the Kidney: Health and Disease
II. AIMS of the study
III. Materials and Methods
IV. Results
1. Objective1:Study the functional interaction between autophagy and primary cilium
1.2. Discussion 1
2. Objective 2: Study the role of autophagy in integration of mechanical stress via primary cilium
2.1. Results
2.2. Discussion 2
V. General Discussion and Perspectives


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