Localization of IFT57A‐GFP and IFT57C‐GFP proteins in vegetative cells

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Light receptors in retina.

Vertebrate retina uses photoreceptors to detect light. This kind of cells is composed an inner and an outer segment linked by a structure derived from a primary cilium called connecting cilium. Photopigments are synthesized in the inner cell body and then transported to the distal outer segment through the connecting cilium. Defect of this cilium leads to retina degeneration and induces blindness (Boldt et al., 2011).

Olfactory sensory cilia.

Another kind of sensory cilia are found in olfactory receptor neurons. Each neuron cell has an apical dendritic knob containing several basal bodies and cilia. Depletion of some ciliaassociated proteins would reduce the ciliary layer, which leads to complete or partial anosmia (McEwen et al., 2007; Kulaga et al., 2004).

Biogenesis of cilia.

The biogenesis of cilia is performed in two steps, first the biogenesis and anchoring at the cell surface of the basal body, second the assembly of the cilium itself (Kim & Dynlacht, 2013).

Basal body biogenesis

Basal body generally maturates from a centriole that migrates towards the surface and anchor to the plasma membrane (Azimzadeh & Marshall, 2010). Several proteins have been demonstrated to be essential for their duplication and anchoring, SAS-4, SAS-6, BLD10 for early steps of duplication, with SAS-6 being a key element in the establishment of the ninefold symmetry (Nakazawa et al., 2007, van Breugel et al., 2011, Mizuno et al., 2012), γ- tubulin and other divergent tubulins for microtubule nucleation and elongation (Guichard et al., 2010), and centrins and FOP-like proteins for anchoring at the membrane (Aubusson- Fleury et al., 2012). As well dissected in the Paramecium model, basal body assembly can be divided in four successive steps: nucleation of microtubules, elongation, maturation of microtubular scaffold and docking at the cell surface (Aubusson-Fleury et al., 2012). (Fig. I- 6).

Microtubule elongation in basal body duplication

γ-tubulin is an essential actor in microtubule nucleation involved in basal body duplication. γ- tubulin has first been shown to recruit α-tubulin and β-tubulin for microtubule nucleation through the γTuRC complex (Moritz et al., 1998; Oegema et al., 1999; Detraves et al., 1997), but this role was also found for centriole/basal body microtubule assembly. Depletion of γ- tubulin in Paramecium caused the loss of new basal body assembly (Ruiz et al., 1999). Study in Tetrahymena revealed that γ-tubulin plays an important function in the initial process of basal body duplication by nucleotide binding domain (Shang et al., 2005). However, the role of γ-tubulin in basal body nucleation concerns only the A-tubule (Guichard et al., 2010).
Other divergent members of the tubulin family, more or less conserved in other eukaryotes, were also found to be involved in basal body biogenesis. δ-tubulin was first identified in Chlamydomonas as a mutation in the UNI3 gene, which causes a defect in flagella number. The observation by electron microscopy of Chlamydomonas uni3 mutants and of Paramecium depleted for δ-tubulin showed that this protein is important for the formation and maintenance of C-tubule in triplet (Dutcher et al., 1998; Garreau de Loubresse et al., 2001).
ε-tubulin was identified in human genome using a genetic approach and it localized at mammalian centrosomes (Shang et al., 2000). Studies in mammals and in Paramecium showed that complete loss of ε-tubulin was lethal and caused defects in centriole or basal body duplication (Dupuis-Williams et al., 2002; Shang et al., 2003). In addition, partial depletion of ε-tubulin was also found to causes the loss of B-tubule and C-tubule in Chlamydomonas and Paramecium, which indicate that B-tubule and C-tubule are needed for the formation and maintenance of new basal body (Dupuis-Williams et al., 2002; Dutcher et al., 2002).
η-tubulin has only been found in four organisms (Trypanosoma, Chlamydomonas, Ciona and Xenopus) (Dutcher, 2003). Mutations of η-tubulin in Paramecium inhibit basal body duplication and causes delocalization of γ-tubulin (Ruiz et al., 2000). Another study also identified an interaction between η-tubulin and β-tubulin (Ruiz et al., 2004). These results together suggest that η-tubulin is important for binding other tubulins to the basal body that is required for correct basal body duplication.

Basal body anchoring

Centrin was first identified in Chlamydomonas and it is essential for calcium sensitive striate flagellar roots and for basal body assembly (Salisbury et al., 1984; Koblenz et al., 2003). A study of centrin homologs in mammal, Marsilea, Leishmania, Tetrahymena and Paramecium showed the conserved function of centrin in basal body or centriole formation (Middendorp et al., 2000; Salisbury et al., 2002; Klink et al., 2001; Selvapandiyan et al., 2004; Stemm-Wolf et al., 2005; Ruiz et al., 2005). Moreover, study in Paramecium found that centrin is responsible for basal body positioning at the surface rather than duplication (Ruiz et al., 2005), which is different from the observation in Tetrahymena (Stemm-Wolf et al., 2005). Another research on PtFor20 revealed that this protein is participates in the late step of basal body duplication, like PtCen2 and PtCen3, and is important for the transition zone maturation. The presence of PtCen2 is necessary to recruit PtFor20, itself necessary for PtCen3 recruitment.
These proteins are needed for transition zone formation and for new basal body docking (Aubusson-Fleury et al., 2012). In mammalian cells, the centrosomal protein CEP164 was found to mediate the vesicular docking to the mother centriole during early steps of ciliogenesis, which is important for the initial establishment of ciliary membrane at distal end of centriole (Schmidt et al., 2012). Similar conclusions on a role in basal body anchoring could be obtained by analysis of mutants for other genes such as talpid (Stephen et al., 2013) and OFD1 (Thauvin-Robinet et al., 2013).

Assembly of the cilium

Generally, the cilium is nucleated at the basal body and built from proteins synthesized in the cytoplasm that transit through the transition zone and are delivered to the distal part of the growing cilium by a so-called intraflagellar transport (IFT). This transport machinery was first observed in green alga Chlamydomonas, with two motile flagella, using differential interference contrast microscopy (Kozminski et al., 1993; Kozminski et al., 1995). The IFT ultrastructure was refined at the electronic microscopy level by identifying train-like particles moving along the axoneme microtubule in cilium (Pigino et al., 2013). Then, IFT was also identified in other organisms such as Caenorhabditis elegans (Cole et al., 1998), sea urchin (Morris, 2004), zebra fish (Tsujikawa M, et al., 2004) and mammals (Pazour et al., 2000), proving that the system is evolutionarily conserved in eukaryotes. Since then, the intraflagellar transport mechanism has been dissected at the molecular level. The IFT particle, carrying cargos along the axoneme, can be grouped in two sub-complexes with different motors during ciliogenesis, IFTA and IFTB. The IFTB sub-complex contains sixteen proteins connected to a kinesin-II motor responsible for the anterograde transport in ciliary building. The IFTA sub-complex contains six proteins connected to a dynein motor responsible for the retrograde transport form the ciliary tip to the base of the cilia during recycling processes (Table I-1; Fig. I-7).

Diverse functions of IFT proteins beyond ciliary transport.

As I presented above, the IFT plays a crucial role in cilium assembly through a conserved motile mechanism in various organisms. However, recent studies in ciliate and non-ciliate cells provide more and more evidence to reveal that some IFT proteins also perform functions in other cellular compartments than the cilium. This indicates that IFT proteins are more complicated than people firstly imagined. For example, IFT88 (IFTB) was shown to localize at centrosomes in actively proliferating cells and that it can regulate the G1-S transition in the cycle of non-ciliated cells (Robert et al., 2007). Research in human HeLa cells also showed that IFT88 is required for spindle orientation and chromosome segregation during mitosis (Delaval et al., 2011). As already mentioned, IFTB members such as IFT20, IFT57 and IFT88, were found to take part in immune synapse organization (Finetti et al., 2009).

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Ciliopathies: pathologies related to ciliary dysfunction.

Due to the diverse roles of cilia during development and in various organs at the adult stage, ciliopathies can be very diverse in symptoms and very pleiotropic. They can be associated with a wide range of human disorders, including obesity, polydactyly, kidney and liver disease, brain and nervous system defect, retina degeneration, anosmia, etc. Each syndrome can be caused by a mutation in several genes, and each ciliopathy gene can cause different syndromes according to the nature of the mutation, which makes the study of ciliopathies very complex (Gerdes et al., 2009).

Polycystic kidney disease (PKD).

Kidney is the most commonly affected organ in ciliopathies. Pathological structure changes in kidney are displayed in several kinds of illness including PKD, Nephronophthisis (NPHP) and Bardet-Biedl syndrome (BBS). ADPKD (Autosomal Dominant Polycystic Kidney disease) and ARPKD (Autosomal Recessive Polycystic Kidney disease) represent the largest proportion of PKD. Mutation of PKD1 (encoding polycystin-1) and PKD2 (encoding polycystin-2) were identified in ADPKD patients and are believed to cause the disease. ARPKD are more common in childhood, while mutations of PKHD-1 (encoding fibrocystin/polyductin) are confirmed in ARPKD patients  (Harris & Torres, 2009).

Nephronophthisis (NPHP).

Similar to PKD, NPHP is a recessive inherited renal cystic disease that more often happens in young adults. Eleven mutated genes (NPHP1- NPHP11) have been identified during patient genome scanning and they were gathered in one group whose coding products are named nephrocystins and they are also confirmed to play roles in the pathology of other ciliopathies including Bardet-Biedl syndrome (BBS), Meckel Gruber syndrome (MKS) and Joubert syndrome (JBTS) (Shiba & Yokoyama, 2012; Hurd & Hildebrandt, 2011).

The Paramecium model: a powerful material for research on cilia.

The model that I used in my thesis project is Paramecium tetraurelia, a ciliate living freely in fresh water. This unicellular is covered with ~4000 cilia and possesses several specialized organelles making it a true differentiated organism. Paramecium has a full digestive track starting by the oral apparatus, a kind of funnel covered with hundreds of cilia whose coordinated beating drives food particle for phagocytosis in food vacuoles and ending at the cytoproct where undigested contents of the food vacuoles are rejected into the medium. It also possesses contractile vacuoles, which have a function close to the one of the kidney in regulation of osmotic pressure and elimination of liquid waste. Paramecium also possesses a regulated secretion pathway in the form of a thousand of trichocysts, which can be triggered by external stimulation. The change of conformation of trichocysts into thin needles when they are expelled into the medium make them easy to detect and make also easy to distinguish cells able from cells unable to release them, mutants or RNAi treated cells.

Tools available for Paramecium studies.

The genome sequencing of Paramecium tetraurelia was completed in 2006, which shows that this species contains nearly 40000 genes arising through at least three successive wholegenome duplications (Aury et al., 2006).
A database, ParameciumDB, was developed to access the genome sequence results, as well as all the other resources accumulating with time, such as genetic and RNAi data, transcriptome and proteome analysis, bibliography (Arnaiz et al., 2007; Arnaiz et al, 2010; Arnaiz and Sperling, 2011). The transformation of Paramecium to express exogenous DNA, for example GFP fusion genes, is easily performed by DNA microinjection into the macronucleus, in which the foreign DNA is replicated as a mini chromosome and is transcribed (Gilley et al., 1988). An efficient homology-dependent inactivation method by RNA interference (RNAi) was developed in Paramecium using a method in which cells are fed with bacteria expressing double-strand RNA homologous to the sequence to inactivate (Galvani & Sperling, 2002).

Thesis project: IFT57 in cilia and nuclei in Paramecium.

As I explained at the beginning, my thesis project rapidly focused on the protein IFT57, then on IFT in a more general context. My motivation was the dual localization of the protein, in basal bodies and cilia on the one hand and in the macronucleus in the other hand.
However, the diverse IFT57/HIPPI functions in the cilium and the nucleus are not clearly related in the literature. From my initial interest in relationships between cilia and nuclei, I therefore started my thesis about the localization and role of IFT57 in Paramecium.

Table of contents :

INTRODUCTION
I.1. Eukaryotic cilia and flagella
I.1.1 Structure of cilia
I.1.2. Sensory functions of cilia
I.1.3. Biogenesis of cilia.
I.1.4. Ciliopathies: pathologies related to ciliary dysfunction.
I.1.5. Important models for ciliary studies.
I.2. The Paramecium model: a powerful material for research on cilia.
I.2.1. Basal bodies and cilia of Paramecium
I.2.2. Nuclear duality in Paramecium
I.2.3. Tools available for Paramecium studies.
I.3. Thesis project: IFT57 in cilia and nuclei in Paramecium.
CHAPTER 1 IFT GENES USED IN THIS WORK
1.1. Paramecium IFT proteins used in this study.
1.2. IFT57 (synonyms: HIPPI; CHE‐13)
1.3. IFT46 (synonyms: DYF‐6; FAP32)
1.4. IFT139 (synonym: FAP60)
1.5. IFT172 (synonym: OSM‐1)
1.6. Qilin (synonyms: CLUAP1; DYF‐3; FAP22)
CHAPTER 2 IFT57 IN CILIOGENESIS
PART 2.1. LOCALIZATION STUDY OF IFT57
2.1.1. Localization of IFT57A‐GFP and IFT57C‐GFP proteins in vegetative cells
2.1.2. Localization of IFT57A‐GFP in growing cilia
2.1.3. Conclusion of Part 2.1
PART 2.2. EFFECTS OF INACTIVATION OF IFT57 GENES
2.2.1. Possibilities of co‐inactivation within the Paramecium IFT57 gene family
2.2.2. Effect on IFT57 RNAi on Paramecium growth rate
2.2.3. Effect on IFT57 RNAi on Paramecium cilia
2.2.4. Effect on IFT57 RNAi on Paramecium expressing IFT57A‐GFP
2.2.5. Conclusion of Part 2.2
PART 2.3. IFT57 WITHIN THE INTRAFLAGELLAR TRANSPORT
2.3.1. Localization of IFT46 and qilin GFP fusions.
2.3.2. Effect of the depletion of different IFT proteins.
2.3.4. Conclusion of Part 2.3.
CHAPTER 3 POTENTIAL NUCLEAR ROLE OF IFT57
PART 3.1. NUCLEAR TARGETING OF IFT57A
3.1.1. Localization of IFT57A‐GFP and IFT57C‐GFP proteins in vegetative cells
3.1.2. Localization of IFT57A‐GFP during autogamy
3.1.3. Looking for the signal that targets IFT57A to the macronucleus, compared to IFT57C.
3.1.3. Conclusion of Part 3.1.
PART 3.2. LOOKING FOR THE ROLE OF IFT57A IN THE MACRONUCLEUS
3.2.1. Attempts to induce RNAi during autogamy by expression of a hairpin RNA under the NOWA1 promoter.
3.2.2. “Regular” IFT57 RNAi during sexual events
3.2.3. Conclusion of Part 3.2.
DISCUSSION
D.1. IFT57 in the IFT system for ciliogenesis.
D.1.1. Ciliary growth and maintenance in relation to IFT recycling.
D.1.2. Cytoplasmic complexes of IFT proteins
D.2. The presence of IFT57A in the macronucleus, a still unsolved mystery
D.2.1. Nuclear targeting of IFT57A‐GFP
D.2.2. Possible nuclear roles of IFT57A deduced from its localization.
D.3. IFT57A as a possible revelator of a cross talk between cilia/basal bodies and nuclei at autogamy
MATERIALS AND METHODS
M.1. Strains and culture conditions
M.2. Physiological manipulations of Paramecium
M.2.1. Deciliation
M.2.2. Trichocyst discharge
M.2.3. India ink labeling of food vacuoles
M.3. Molecular biology methods
M.4. Vectors used
M.4.1. GFP‐fusion expression vectors
M.4.2. RNAi vectors.
M.5. Transformation of Paramecium.
M.6. Immunofluorescence microscopy.
M.7. Electron microscopy.
M.8. RNAi by the “feeding” method.
M.8.1. RNAi by feeding during vegetative growth.
M.8.2. RNAi by feeding during autogamy
M.8.3. RNAi by feeding during conjugation
BIBLIOGRAPHY 

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