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IFT accessory proteins
Besides the individual IFT proteins I introduced above, recent work also revealed that some other proteins including CrFAP22/CeDyf-3/Ceqilin/HsCLUAP1 and CeDyf-13/TbBIFTC3 participate in the IFT and are regarded as IFT accessory proteins (Murayama et al., 2005; Ou et al., 2005; Takahashi et al., 2004; Blacque et al., 2005; Franklin & Ullu. 2010).
The obvious function of the IFT in cilia is supposed to be the transport of cargos (precursors) essential for ciliary building and maintenance. First evidence about the composition of cargos came from a study in Chlamydomonas, which showed that the ciliary localization of inner dynein arm, a part of axonemal components, required the activity of kinesin (Piperno et al., 1996). Other studies also showed that the IFT controls the tubulin balance in flagella (Marshall & Rosenbaum, 2001) and that the transport of outer dynein arms into flagella also needs IFT (Hou et al., 2007). In Chlamydomonas, some flagellar components can be preassembled in the cell body before entering the flagella. They can form a 12S complex transported by anterograde IFT from basal body to tip and then reassemble a 20S complex that turns around from tip to base by retrograde IFT (Qin et al., 2004; Behal et al., 2012). In addition, in mammalian retina, the photopigments, which are important for animal light detection, are synthesized in the inner cell body and then transported to the distal outer segment by the IFT system through the connecting cilium (Insinna & Besharse, 2008).
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).
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).
I.1.4.2. 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).
Bardet‐Biedl Syndrome (BBS)
BBS represent one wide range of human multisystemic developmental disorders including obesity, retina defect, polydactyly and learning obstacle. Until now, 14 genes (BBS1-BBS14) have been identified, which encode proteins localized to the primary cilium, basal body or centrosome. Recent studies showed that some BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9) form a complex named BBSome, which associates to RAB8, and regulate vesicle travel from cytoplasm to cilium, an important step for ciliogenesis and ciliary maintenance. In addition, the other three BBS proteins, BBS6, BBS10 and BBS12 form a second group that regulate the assembly of the BBSome. In addition, recent studies revealed that the BBSome is involved in intraflagellar transport (IFT) in the cilium, in Chlamydomonas.
The BBSome has been found to control IFT assembly at cilia base and turnaround at flagella tip. A study in Caenorhabditis elegans also showed that two BBS proteins (BBS7 and BBS8) are required for the stabilization of IFT particles (Blacque et al., 2004).
Chlamydomonas reinhardtii is a unicellular green alga of ∼10μm in length, with a chloroplast, a pyrenoid, a senses light ‘eyespot’ and two anterior flagella of 10∼12μm in length. During vegetative growth, the two flagella beat, which provokes cell swimming. Cell can direct their movements in response to various light stimuli. In the early stage of mating process, flagella are also required for gamete adhesion. During mitosis, the flagella are not essential and are disassembled, allowing the two centrioles (basal bodies) to migrate and organize the spindle pole to contribute to mitosis. Several advantages of Chlamydomonas make it a good model for research on flagella. First, it is possible to grow them synchronously in a medium and large amounts of flagella can be easily purified for biochemical analyses. Secondly, Chlamydomonas has a clear genetic background, all three genomes (nucleus, chloroplast, mitochondrion) are sequenced and there are standard biological protocols that can be applied for the genetic and cellular research. Further more, a large number of mutants have been screened and identified. Over 500 proteins have been detected by proteomics to be components of the flagella. Many of them have homologs in mammals, which make Chlamydomonas an interesting model for some human cilium-related disease. In addition, a conserved mechanism essential for ciliogenesis, intraflagellar transport (IFT), was first discovered in Chlamydomonas. Most IFT proteins were first purified in Chlamydomonas, and then found in other organisms.
Trypanosoma brucei is a parasite with a flagellum. It causes sleeping sickness in human and nagana in animals. It is also an attractive model for flagellar research because of the unique biological flagellar feature, that it assembles a new flagellum while keeping the old one (Absalon, et al., 2008). Therefore, it is possible to study both flagellum construction and maintenance in one individual cell, using the gentical and molecular tools provided by this organism. In addition, the morphology of flagella is changed according to different life cycle and this differentiation can be reproduced in vitro, that provide us a wonderful model for researching the regulation mechanism of flagellar assembly. Recent works in Trypanosoma brucei provide some supplement evidence about IFT recycling process (Buisson, et al., 2013).
Paramecium tetraurelia and Tetrahymena thermophila
Ciliates such like Paramecium and Tetrahymena represent models harboring a huge number of cilia. In ∼50μm long Tetrahymena cell, there are ∼750 cilia and basal body while there are ∼4000 cilia and basal body in ∼120μm long Paramecium cell. Specific cilia arrangement over the cell surface allowed for long morphogenetic studies at the cell level by following basal body duplication pattern. Using GFP-fusion and gene depletion method, localization and function of basal body and ciliary proteins can be easily detected.
Ciliates are also excellent models for studying tubulin post-translational modifications, which are important processes for tubulin diversity and for ciliary function. Since almost all known tubulin members were found in Paramecium or Tetrahymena, it has been possible to approach the roles of the different post-translational modifications in cilia and other microtubule-based organelles. Elimination of β-tubulin polyglycylation in Paramecium or Tetrahymena induces various phenotypes including lethality, slow swimming, and division defects. Mutant clones produce non-motile cilia lacking the central pair or abnormally short cilia. Polyglycylation is also required for maintenance of length of already assembled cilia. Basal bodies in mutant cells show redundant number of microtubule (Thazhath et al., 2004). Another posttranslational modification, polyglutamylation, was also identified in Paramecium and found to be important in the interactions of tubulin with microtubule-associated proteins and calcium, essential for microtubule dynamics (Edde et al., 1990). Tetrahymena is also a tool often used for IFT research in ciliogenesis (Beals et al., 2007; Dave et al., 2009).
Table of contents :
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.
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.2.2. 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