The Pennsylvania State University

The Graduate School

Eberly College of Science

THE ROLES OF C2CD3 IN CENTRIOLE MATURATION

AND PRIMARY CILIUM FORMATION IN MAMMALIAN CELLS

A Dissertation in

Biology

by

Xuan Ye

 2014 Xuan Ye

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

December 2014

The dissertation of Xuan Ye was reviewed and approved* by the following:

Aimin Liu Associate Professor of Biology Dissertation Advisor

Gong Chen Professor of Biology Chair of Committee

Graham H. Thomas Associate Professor of Biology, Biochemistry and Molecular Biology

Melissa M. Rolls Associate Professor of Biochemistry and Molecular Biology Chair of the Molecular, Cellular and Integrative Biosciences Graduate Program

Douglas R. Cavener Professor of Biology Head of the Department of Biology

*Signatures are on file in the Graduate School

iii

ABSTRACT

The cilium/flagellum is a microtubule-based cell surface organelle that exists broadly in many living organisms, from protozoa to metazoan. It is critical for mobility, sensory functions and signal transduction. The primary cilium plays critical roles in vertebrate development and physiology. Over the last decade, benefited from active biological and medical studies on this organelle, an increasing number of human genetic diseases have been found related to cilia defects, collectively known as “ciliopathy”.

Previously, our lab identified C2 calcium-dependent domain containing 3 (C2cd3) as an essential regulator for ciliogenesis. C2cd3 homozygous mutants exhibit defects in neural tube development, left-right asymmetry and limb patterning, consistent with the phenotypes of other cilia mutants. A great reduction in cilia number observed in C2cd3 mutants suggests it is essential for cilia formation. However, the molecular mechanism underlying C2cd3 function in ciliogenesis is still elusive. To understand the function of C2cd3, I examined its subcellular localization. A dynamic subcellular localization of C2cd3 is observed during the cell cycle.

C2cd3 forms punctae around centrosome during interphase, which are dispersed during mitosis but leaving two fine dots at each spindle pole. This implies that C2cd3 may be localized to both centriolar satellites and centrosomes.

To confirm its centriolar satellite localization, I found that C2cd3 punctae were dependent on dynein-mediated microtubule-dependent retrograde transport. Furthermore, C2cd3 colocalizes and physically interacts with known centriolar satellites components, Pcm1 and Bbs4.

I also discovered that C2cd3 was targeted to centriolar satellites through the interaction between its C-terminus and Pcm1. However, C2cd3 is dispensable for centriolar satellite integrity and many known centriolar satellite functions, including Rab8 ciliary localization, microtubule

iv arrangement and cell cycle progression. Surprisingly, I found that C2cd3 is essential for centriolar satellite recruitment of Centrin, suggesting its role in centriolar satellite-mediated transport.

To study the second localization of C2cd3, I pinpointed C2cd3 to the distal ends of centrioles. Interestingly, C2cd3 is essential for the recruitment of both distal and subdistal appendages to the mother centriole. Furthermore, loss of C2cd3 results in failure in the recruitment of Ttbk2 to the mother centriole as well as the removal of Ccp110 from the mother centriole, two critical steps in initiating ciliogenesis. C2cd3 is also required for recruiting the intraflagellar transport proteins Ift88 and Ift52 to the mother centriole during ciliogenesis.

Consistent with its role in distal appendage assembly, C2cd3 is essential for ciliary vesicle docking to the mother centriole. These results suggest that C2cd3 regulates cilium biogenesis by promoting the assembly of centriolar distal appendages, which are critical for docking ciliary vesicles and recruiting other essential ciliogenic proteins.

Finally, to elucidate the role of C2cd3 in distal appendage assembly, I found that C2cd3 contributes to the centriolar recruitment of Centrin, a centriolar distal end protein. Ofd1 and

Dzip1, two regulators of distal and subdistal appendages, fail to be recruited to centrioles in

C2cd3 mutant cells. I propose that C2cd3 may mediate distal appendage recruitment by regulating centriole elongation.

In summary, my results indicate that C2cd3 regulates cilia formation by facilitating the recruitment of centriolar distal appendages. In the absence of C2cd3, distal appendages fail to be installed onto the mother centriole and all the downstream events for initiating the ciliogenesis are affected. The distal appendage defect might be a secondary effect of disrupted centriole diatal structure when C2cd3 is absent. I have also described the road map for studying how C2cd3 regulates centriole biogenesis and interplays with other distal appendage regulators in the future.

v

TABLE OF CONTENTS

List of Figures ...... viii

List of Tables ...... x

List of Abbreviations ...... xi

Acknowledgements ...... xii

Chapter 1 Introduction ...... 1

1.1 Cell biology of the primary cilium ...... 1 1.1.1 Structure of the primary cilia...... 1 1.1.2 Centriole and centrosome assembly ...... 5 1.1.3 Centriolar satellites and cilia ...... 10 1.1.4 Intraflagellar transport ...... 16 1.1.5 Ciliary gate I: the transition fibers ...... 17 1.1.6 Ciliary gate II: the transition zone ...... 19 1.1.7 Other compartments in the cilia ...... 21 1.1.8 Cilia assembly ...... 22 1.2 The primary cilium and signal transductions ...... 26 1.2.1 The primary cilium and Hedgehog signaling ...... 26 1.2.2 The primary cilium and Wnt signaling ...... 30 1.2.3 The primary cilium and PDGFRα, Hippo and other pathways ...... 31 1.3 The primary cilium and human disease ...... 32 1.3.1 Polycystic kidney disease ...... 33 1.3.2 Hepatic and pancreatic cysts and situs inversus ...... 34 1.3.3 The primary cilium and sensory defects ...... 36 1.3.4 The primary cilium and obesity...... 37 1.3.5 The primary cilium and tumors ...... 38 1.4 References ...... 39

Chapter 2 Materials and methods ...... 59

2.1 Cell culture and transfection ...... 59 2.1.1 C2cd3GT mutant cell lines establishment ...... 59 2.1.2 Cell culture, transfection and nocodazole treatment ...... 59 2.1.3 DNA constructs ...... 60 2.1.4 RNA interference ...... 61 2.2 Imaging ...... 61 2.2.1 Immunocytochemistry (ICC) ...... 61 2.2.2 Immunohistochemistry (IHC) ...... 62 2.2.3 Antibodies for Immunofluorescence...... 62 2.2.4 Transmission electron microscopy (TEM) ...... 63 2.3 Biochemistry ...... 63 2.3.1 Western blot ...... 63 2.3.2 Co-immunoprecipitation (Co-IP) ...... 63 2.3.3 Antibodies for Western Blot and Coimmunoprecipitation...... 64

vi

2.4 References ...... 64

Chapter 3 C2cd3 is localized to centriolar satellites but dispensable for centriolar satellite integrity ...... 65

3.1 Introduction ...... 65 3.2 Results ...... 69 3.2.1 C2cd3GT homozygous mutants exhibit ciliogenesis defects in most tissues .... 69 3.2.2 Dynamic C2cd3 protein localizations throughout the cell cycle ...... 72 3.2.3 C2cd3 punctate staining relies on microtubule-dependent retrograde transport ...... 74 3.2.4 C2cd3 colocalizes and physically interacts with known centriolar satellites components...... 76 3.2.5 C2cd3 is targeted to centriolar satellites through the interaction between its C-terminus and Pcm1 ...... 78 3.2.6 C2cd3 is dispensable for centriolar satellite integrity, Rab8 ciliary localization, microtubule arrangement and cell cycle progression...... 81 3.2.7 C2cd3 is essential for Centrin centriolar satellite recruitment ...... 85 3.3 Discussion ...... 87 3.3.1 C2cd3 is required for ciliogenesis in mouse embryonic development in a tissue-specific manner...... 87 3.3.2 C2cd3 is a centriolar satellite component ...... 88 3.3.3 C2cd3 is dispensable for typical centriolar satellite functions ...... 89 3.4 References ...... 89

Chapter 4 C2cd3 is localized to distal end of centrioles and critical for the distal structure assembly ...... 92

4.1 Introduction ...... 92 4.2 Results ...... 94 4.2.1 C2cd3 is localized to the distal ends of centrioles ...... 94 4.2.2 C2cd3 is required for the recruitment of DAPs and SAPs ...... 97 4.2.3 C2cd3 is essential for Ttbk2 and Ift recruitment and Ccp110 removal during ciliogenesis ...... 100 4.2.4 C2cd3 is critical for ciliary vesicle docking ...... 103 4.2.5 C2cd3 contributes to the centriolar recruitment of Centrin ...... 105 4.2.6 Relationship among C2cd3, Ofd1 and Dzip1 ...... 109 4.3 Discussion ...... 112 4.3.1 C2cd3 is required for the initiation of ciliogenesis ...... 112 4.3.2 C2cd3 may mediate DAP assembly through Centrin recruitment ...... 112 4.3.3 Different penetrances of different phenotypes in C2cd3 mutant cells ...... 114 4.4 References ...... 115

Chapter 5 Conclusion, discussion and future directions ...... 119

5.1 The role of C2cd3 in the initiation of ciliogenesis ...... 119 5.2 The role of C2cd3 in centriole length control ...... 122 5.3 The role of C2cd3 in DAPs and SAPs recruitment ...... 124 5.4 Future directions ...... 125

vii

5.5 References ...... 127

viii

LIST OF FIGURES

Figure 1-1. Subcompartments of a mature cilium ...... 4

Figure 1-2. Centriole biogenesis during cell cycle...... 7

Figure 1-3. Ciliogenesis process...... 23

Figure 1-4. Sonic hedgehog signaling in mammals...... 28

Figure 3-1. C2cd3 gene information...... 67

Figure 3-2. C2cd3GT mutant embryos and MEFs show severe ciliogenesis defects...... 70

Figure 3-3. C2cd3GT mutant embryos exhibit normal ciliogenesis in the gut...... 71

Figure 3-4. Endogenous C2cd3 and GFPC2cd3 exhibit dynamic subcellular localization throughout cell cycle...... 73

Figure 3-5. C2cd3 punctae depends on dynein-mediated microtubule-dependent retrograde transport...... 75

Figure 3-6. C2cd3 colocalizes and physically interacts with Pcm1 and Bbs4...... 77

Figure 3-7. C2cd3 centriolar satellite localization depends on the interaction between its C-terminus and Pcm1...... 79

Figure 3-8. Overexpression of full-length C2cd3, but not C2cd3dC, misplaces endogenous Pcm1...... 80

Figure 3-9. C2cd3 is dispensable for centriolar satellite integrity...... 82

Figure 3-10. Loss of C2cd3 does not affect Rab8 ciliary localization...... 83

Figure 3-11. C2cd3 is not required for microtubule organization and cell cycle progression...... 84

Figure 3-12. C2cd3 is required for the centriolar satellite recruitment of Centrin...... 86

Figure 4-1. C2cd3 is localized to the distal ends of centrioles...... 96

Figure 4-2. C2cd3 is essential for the recruitment of distal appendages and subdistal appendages...... 98

Figure 4-3. Other distal appendage components fail to be recruited to the mother centriole in C2cd3 mutant cells...... 99

Figure 4-4. C2cd3 is required for Ttbk2 recruitment and Ccp110 removal during ciliogenesis...... 101

ix

Figure 4-5. C2cd3 is required for the centriolar recruitment of Ift88 and Ift52 during ciliogenesis...... 102

Figure 4-6. C2cd3 is important for ciliary vesicle docking during ciliogenesis...... 104

Figure 4-7. Overexpressed GFPCentrin1-3 and GFPPoc5 are recruited to centrosome in the absence of C2cd3...... 107

Figure 4-8. Centriolar localization of endogenous Centrin is affected in C2cd3 mutant cells...... 108

Figure 4-9. Centriolar localization of Ofd1 and Dzip1 is affected in C2cd3 mutant cells...... 110

Figure 4-10. Centriolar localization of Dzip1, but not C2cd3, is compromised in Ofd1 mutant cells...... 111

Figure 5-1. Current understanding of ciliogenesis integrated with my results...... 121

x

LIST OF TABLES

Table 1-1. List of centriolar satellite proteins, their subcellular localization, functions and the phenotypes of corresponding mutant mice...... 11

Table 4-1. Summary of various phenotypes in C2cd3GT mutant cells and wildtype control cells...... 115

xi

LIST OF ABBREVIATIONS

BBS Bardet-biedl syndrome

C2cd3 C2-domain containing protein 3

Cetn Centrin

Cep164 centrosomal protein of 164 kDa

DAP(s) distal appendages

EvC Ellis-van Creveld syndrome

GPCRs G-protein coupled receptors

HEK human embryonic kidney cells

IFT intraflagellar transport

ImmunoEM immunoelectron microscopy

INVS Inversin

PCM pericentriolar matrix

Pcm1 pericentriolar material 1

PKD polycystic kidney disease

SAP(s) subdistal appendages shRNA small hairpin RNA siRNA small interference RNA

TAP Tandem affinity purification

TEM transmission electron microscopy

xii

ACKNOWLEDGEMENTS

Ph.D study is never an easy thing, and I would not make it without many people’s help.

First of all, I would like to express my deepest gratitude to my advisor Dr. Aimin Liu. Thank him

for guiding me through my Ph.D study with tremendous support and patience. He taught me the

most important thing in scientific research and life, the perseverance. Whenever the research hit a

dead end or experiments went wrong, it is him who gave me the faith to try harder and not to give

up. I also would like to thank other members in Liu lab, Huiqing Zeng, Jinling Liu, Hongchen

Cai, for their helpful discussions and suggestions. I also appreciate the help from people on the first floor of Life Science Building, who offered me technical suggestions and experimental reagents.

I would like to thank my committee members, Drs Gong Chen, Graham Thomas and

Melissa Rolls, for their helpful suggestions and supports. I also really appreciate the help from

Penn State Microscopy Facility, including Dr. Gang Ning, Missy Hazen and John Cantolina. My

special thanks to Ms. Kathryn McClintock for helping me with all the paperwork and answering all my questions since the first day I came here.

Of course, Ph.D life would be unimaginable without all these dear friends, Xin Tang,

Zhen Ren, Ben Niu, Li Chen, Yang Wang and more. I still remember that we were laughing, sweating, eating, drinking, adventuring and being Ph.Ds together.

Last but not least, I would like to thank my parents and Yijing Zhou for their support and love. It is so good to have them in my life.

Thank these five years, which has been a very unique and unforgettable experience to me.

I will carry on and keep looking for new excitements and joys in my life.

Chapter 1

Introduction

The cilium/flagellum is a small projection on the cell surface with microtubule backbones. This tiny organelle plays important roles in sensory functions and signal transductions. It also becomes the focus to understand and treat many human diseases due to its pivotal roles in development and homeostasis. In this chapter, I reviewed the cell biology of the primary cilium, cilia-mediated signaling pathways and cilia-related human genetic diseases.

1.1 Cell biology of the primary cilium

The cilium is around 500nm in diameter and up to several micrometers in length and its volumn is less than a ten-thousandth of the entire cell. It is a separated compartment from the rest of the cell with unique compositions and functions. Despite its tiny size, multiple delicate subcompartmentalizations are revealed within the cilia. In this section, I summarized recent advances in the cell biology of the primary cilium, from its structure, composition to its assembly regulation.

1.1.1 Structure of the primary cilia

The cilium is extended from the basal body, a modified centriole. Microtubules extend from the basal body to form the axoneme of the cilium (Ye & Liu 2011). The axoneme is encapsulated by ciliary membrane, which is an extension of the plasma membrane with its unique

2 composition. The basal body comprises nine bundles of microtubule triplets arranged as a cartwheel. The axoneme comprises nine sets of microtubule doublets.

Based on their structures and functions, cilia are categorized into multiple motile cilia and solitary primary cilia in vertebrates (Bloodgood 2009). Multiple motile cilia grow on epithelial cells of respiratory and reproductive tracts and ependymal cells in the brain. These cilia move synchronously to clear the mucus, deliver gametes and circulate cerebrospinal fluid. The axoneme of the motile cilium is made of nine microtubule doublets arranged as a cartwheel and two microtubule singlets in the center (9+2). The solitary primary cilia grow on most vertebrate cells and are essential for many sensory functions, including photosensation, chemosensation, mechanosensation, thermosensation and signal transduction (Fliegauf et al. 2007). The primary cilium has a “9+0” structure lacking the central pair of microtubule singlets.

Usually the number of cilia on each cell correlates well with their motility and axonemal structure. However, there are a few exceptions. Multiple olfactory cilia projected from the dendrites of olfactory receptor neurons are essential for olfactory sensation and have “9+2” axoneme despite being non-motile (McEwen et al. 2008). The non-motile kinocilia on the apical surface of hair cells in vertebrate inner ears also have 9+2 axoneme (Duvall et al. 1966). Tail flagellum of the spermatozoa is required for spermatozoa motility and has a 9+2 axoneme

(Manton 1965). Finally, nodal cilia, solitary cilia at the ventral surface of the embryonic node, have 9+0 axoneme but are motile and generate a leftward nodal flow critical for establishing laterality of the embryo (Murcia et al. 2000; Takeda et al. 1999; Marszalek et al. 1999).

The cilium/flagellum exists from protozoa to metazoan. Experimental models for cilia study include the flagella of Chlamydomonas reinhardtii (a unicellular green algae flagellate) and

Tetrahymena pyriformis (a ciliated protozoan), the cilia on sensory neurons of Caenorhabditis elegans (a nematode), the motile cilia on Kupffer’s vesicles and embryonic kidney of Danio rerio

3 (Zebrafish),the motile cilia on the skin of Xenopus laevis, the nodal and the primary cilia of Mus musculus and the primary cilia on retinal pigmented epithelial (RPE) cells of Homo Sapiens.

Notwithstanding the divergences across species and differences across tissues, the basic structure of cilia is largely conserved. Studies on simpler models have greatly increased our understanding of the cilia in other species. Specifically, the pioneering discovery of intraflagellar transport (IFT) system in Chlamydomonas revealed the common ciliary transport machinery in all types of cilia in various species and laid the groundwork for connecting cilia and signal transduction (Rosenbaum & Witman 2002). Mammalian photoreceptor cells have highly specialized cilia, containing the outer segment (the enlarged distal part of the cilium filled with dense stacks of rhodopsin-containing disc membranes) and the connecting cilia (the proximal part of the cilia) (Roepman & Wolfrum 2007). Studies of these specialized cilia on mouse and human photoreceptor cells brought up the idea of different subcompartments with specific functions and protein compositions (Sedmak & Wolfrum 2010; Sedmak & Wolfrum 2011).

As illustrated in Fig. 1-1, the cilium comprises three major parts: the basal body, axoneme and ciliary membrane. Additional structures and specialized subcompartments are also important components of the cilium. At the base of the cilium, subdistal appendages (SAPs), pericentriolar material (PCM) and centriolar satellites surround the basal body. Moving upward, the transition fiber zone contains transition fibers, septin ring complex on the membrane and periciliary membrane. More distal is the transition zone containing Y-shaped linkers, ciliary necklace and basal plate. Both the transition fibers and the transition zone are proposed as ciliary gates regulating the import and export of soluble and membrane proteins. In addition, EvC zone, inversin (INVS) zone and tip structure are also discovered, but current knowledge about their components and functions is still limited.

4

Figure 1-1. Subcompartments of a mature cilium

The cilium is originated from the basal body, a modified mother centriole. The basal body is surrounded by PCM and centriolar satellites. The subdistal appendages are developed into basal feet to nucleate microtubules. At the distal end of basal body, there are pinwheel-shaped transition fibers, septin ring complex and ciliary pocket. Moving upward, there is the transition zone, including Y linkers, the ciliary necklace and the basal place. More distal is EvC zone and INVS zone. In the distal part of the cilium, a tip structure is proposed. Intraflagellar transport machinery facilitates the transport within cilia in two directions: anterograde transport comprises IFT-B complex and Kinesin2 motor; retrograde transport comprimses IFT-A complex and dynein motor.

5 1.1.2 Centriole and centrosome assembly

All cilia are extended from the basal body, a modified centriole. Moreover, the basal body stays as an anchor point for axonemal microtubules after ciliogenesis, with one exception in the sensory neurons of C. elegans, where the basal body degenerates after cilia formation leaving only the transition fibers at the proximal end of the cilia, suggesting that the basal body may not be essential for maintaining a cilium (Perkins et al. 1986).

Besides serving as the basal body of the cilia, centrioles also serve as the core of centrosomes, organelles important for mitotic spindle organization and cell polarity. The electron- dense matrix of proteins surrounding the centrioles is called pericentriolar material (PCM).

Centrioles and PCM together build the centrosome that serves as microtubule-organizing center

(MTOC) to nucleate and regulate the microtubule network. Although centrioles exist at spindle poles, they are dispensable for mitosis (Debec et al. 2010). The ablation of centrioles affects cell- cycle progression and cytokinesis but likely through indirect effects (Piel et al. 2001; Hinchcliffe et al. 2001; Mikule et al. 2007). Nevertheless, centrioles have been shown to be essential for spindle positioning and asymmetric cell division, which are important for planar cell polarity and morphogenesis (Montcouquiol & Kelley 2003; Park et al. 2006).

New centrioles are generated through two different mechanisms: the canonical pathway and de novo pathway (Nigg & Raff 2009). In the canonical pathway deployed by most animal cells, the number of centrioles in each cell is tightly controlled: new centrioles are assembled only once per cell cycle and only one new centriole is assembled perpendicularly to the parental centriole each time. These newly assembled yet immature centrioles are called procentrioles.

Although the assembly of procentrioles starts in late G1 or early S phase, the license for centriole duplication is granted in late mitosis of previous cell cycle, when the tight association between procentrioles and their parental centrioles is dissolved by Polo-like kinase 1 (Plk1) and protease

6 separase, a process termed centriole disengagement (Tsou & Stearns 2006; Tsou et al. 2009). The

procentrioles are only assembled adjacent to their parental centrioles possibly due to the

requirement for PCM environment (Loncarek et al. 2008). It is likely that the PCM around parental centriole creates an environment with all the essential factors for the initiation of centriole biogenesis. It was also shown that the size of the PCM of parental centriole correlates with the number of new centrioles formed around it (Loncarek et al. 2008).

Multiple new centrioles are assembled de novo in the absence of existing centrioles or far away from them, suggesting that existing centrioles do not serve as templates for the assembly of new centrioles (Loncarek & Khodjakov 2009). This idea is also supported by the fact that the cartwheel, the first structure assembled during centriole biogenesis, can be self-assembled in vitro

(Gavin 1984). As discussed above, initiation of centriole biogenesis may require the favorable

environment with essential factors surrounding the parental centrioles, instead of the parental centrioles per se.

As illustrated in Fig. 1-2, the key regulators for triggering the initiation of centriole biogenesis include polo-like kinease 4 (Plk4), Cep152 and Cep192. Plk4 is required for centriole duplication in both Drosophila and human cells and its overexpression results in supernumerary centrioles (Sillibourne & Bornens 2010). Cep152 is also essential for centriole assembly in flies and human cells, and it recruits Plk4 and bridges the interaction between Plk4 and Centrosomal

P4.1-associated protein (CPAP) (Cizmecioglu et al. 2010; Dzhindzhev et al. 2010). Cep192 recruits PCM around centrioles and is critical for centriole duplication in human cells, although the latter might be an indirect result of the former (Zhu et al. 2008).

7

Figure 1-2. Centriole biogenesis during cell cycle.

The biogenesis of new centrioles starts at late G1 to early S phase, triggered by Plk4, Cep152 and Cep192. Cartwheel structures are assembled close to the walls of parental centrioles. Cartwheel comprises SAS6 as the hub and Cep135 as the spokes. Later, microtubules are recruited around the cartwheel. During late S to G2 phase, microtubules continue to elongate under the regulations of CPAP, Ccp110, Poc1 and Ofd1. During mitosis, newly assembled centrioles, also called procentrioles, are disengaged from parental centrioles and centrosomes are separated from each other. In G1 phase, previous daughter centriole becomes the mother centriole when recruiting the distal and subdistal appendages. Procentrioles become the daughter centrioles by disassembling part of the cartwheel structure and accumulating Poc5 and Centrin.

8 Procentriole assembly starts with building the cartwheel close to the parental centriole walls in late G1 to early S phase. Sas6 is localized to the hub of the cartwheel and is required for

centriole formation (Azimzadeh & Marshall 2010). Cep135 is a component of cartwheel spokes

and is critical for generating centrioles with correct diameters (Hiraki et al. 2007; Matsuura et al.

2004). Sas5, also known as STIL1/Ana2, physically interacts with Sas6 and is essential for centriole formation (Stevens et al. 2010). Interestingly, it was recently reported that SAS6 cartwheel was assembled in the proximal lumens of the cartwheel-less parental centrioles before transported outside of the parental centriole walls (Fong et al. 2014).

Subsequently, the nine-fold microtubule triplets are added to the cartwheel. The triplets connect to cartwheel through the A-tubule, the only complete microtubule first assembled in a triplet. It was shown that A-tubules were nucleated from γ-tubulin ring complex (γ-TuRC) cap at the proximal ends, whereas B-tubule and C-tubule, two uncapped incomplete microtubules, were assembled bidirectionally later along A-tubule and B-tubule respectively (Guichard et al. 2010).

After the new centrioles reach their full length, A- and B-tubules are longer than C-tubule and form doublets at the distal ends (Paintrand et al. 1992; O’Toole et al. 2003). The nucleation of open B- and C-tubules is facilitated by δ- and ε-tubulins (Azimzadeh & Marshall 2010). CPAP and centrobin are believed to promote microtubule nucleation by sequestering tubulin dimers

(Kohlmaier et al. 2009; Hiraki et al. 2007).

The elongation of the procentrioles is regulated through multiple mechanisms. Centriolar

Coiled coil protein 110 (Ccp110) and Cep97 inhibit the over-growth of the centriole, as the depletion of Ccp110 or Cep97 results in abnormally long centrioles (Spektor et al. 2007; Schmidt et al. 2009; Tang et al. 2009). CPAP is a positive regulator for centriole elongation as its overexpression leads to abnormally long centrioles (Schmidt et al. 2009; Tang et al. 2009;

Kohlmaier et al. 2009). It is proposed that CPAP promotes the incorporation of new tubulins to the centrioles, and Ccp110 inhibits this process by forming caps at the plus ends of microtubules.

9 Ccp110 and CPAP antagonize each other to maintain the normal length of centrioles. Ofd1 is

another centriole length regulator, loss of which leads to abnormal elongation of the centriole

(Singla et al. 2010). Finally, the level of Poc1 is also critical for centriole duplication and length

control; knockdown of Poc1 results in centriole duplication defects, but its overexpression induces abnormally long centrioles (Keller et al. 2009). Calcium-binding protein Centrin and its interacting partner Poc5 are localized to the distal lumen of centrioles (Paoletti et al. 1996;

Azimzadeh et al. 2009). Centrin is required for centriole biogenesis in Tetrahymena and

Chlamydomonas (Koblenz et al. 2003; Stemm-Wolf et al. 2005), but its role in mammalian cells remains controversial (Salisbury et al. 2002; Kleylein-Sohn et al. 2007). Poc5 has been suggested to be required for the distal end assembly (Azimzadeh et al. 2009).

During mitosis, procentrioles are disengaged from the parental centrioles in a Plk1- and

Separase-dependent process that grants them the ability to assemble new centrioles in the next cell cycle (Tsou & Stearns 2006). Two centrosomes are separated from each other by dissolving proteinaceous cohesion through Nek2A-mediated C-Nap1 phosphorylation (Faragher & Fry

2003). After centrosome disjunction, two centrosomes are driven to opposite directions by microtubule-dependent motors to form spindle poles.

After mitosis, the procentrioles become daughter centrioles by disassembling part of the cartwheel structure and γ-TuRC cap of A-tubule (Vorobjev & Chentsov 1980; Vorobjev &

Chentsov Yu. 1982; Guichard et al. 2010). After the next mitosis, the daughter centriole becomes the mother centriole by incorporating distal appendages (DAPs) and subdistal appendages (SAPs)

(Kobayashi & Dynlacht 2011). DAPs are composed of five components, namely Cep164, Fbf1,

Sclt1, Cep83 and Cep89 (Tanos et al. 2013; Joo et al. 2013; Sillibourne et al. 2013). Odf2 is localized to both DAPs and SAPs and involved in their assembly (Ishikawa et al. 2005). SAPs comprise EB1, FOP, CAP350, Ninein, ε-tubulin and Cep170, which serve as a microtubule anchoring and nucleating complex (Kobayashi & Dynlacht 2011).

10 1.1.3 Centriolar satellites and cilia

Centriolar satellites are nonmembranous electron-dense granules around the centrosome.

Recent studies suggest centriolar satellites are involved in cilia formation. Centriolar satellites are recruited in the vicinity of the centrosome during the interphase and are disassembled when cells enter mitosis (Dammermann & Merdes 2002). The integrity of centriolar satellites is dependent on dynein-mediated retrograde transport along microtubule network (Dammermann &

Merdes 2002). Centriolar satellites were also observed around the basal body of ciliated cells, suggesting its potential involvement in ciliogenesis. PCM1 is the first centriolar satellites component discovered (Dammermann & Merdes 2002). By far, more than 30 proteins have been identified as centriolar satellite components, some of which are illustrated in Table 1-1.

Interestingly, all centriolar satellite proteins, except PCM1, were also found at the centrosomes, centrioles, the basal body or cilia (Tollenaere et al. 2014).

11

Table 1-1. List of centriolar satellite proteins, their subcellular localization, functions and the phenotypes of corresponding mutant mice.

12 Since centriolar satellites harbor many centrosomal proteins, centriolar satellites were

thought as a transit station for centrosomal proteins on the way to their destinations, or as a

reservoir to ensure the supply of these centrosomal proteins. Inhibition of PCM1 by various

approaches leads to compromised recruitments of centrin, pericentrin and ninein to the

centrosomes, which further causes disorganization of microtubules (Dammermann & Merdes

2002). Similar phenotypes were observed after the disruption of dynactin function or microtubule

network. Centrosomal enrichments of Nek2A and CaMKIIβ also require centriolar satellites

(Puram et al. 2011; Hames et al. 2005). So far some centriolar satellite components have been found to be critical for recruiting other components by physically linking them with molecular motors. Specifically, BBS4 and Par6α bind to p150Glued, a subunit of dynactin responsible for link

dynein motors to their cargos (Kim et al. 2004; Kodani et al. 2010). Knockdown of BBS4 and

Par6α lead to disrupted centriolar satellites. Cep290 physically interacts with both dynein and kinesin motors and attenuating Cep290 results in a tighter centriolar satellites structure (Kim et al.

2008; Mykytyn et al. 2004). In contrast, the centrosomal localizations of some centriolar satellite components, including Ofd1, Cep290, Cep90, Cep72, Cep131, Ccdc13 and SSX2IP, seem independent of centriolar satellites integrity and microtubule-mediated transport (Lopes et al.

2011; Kim et al. 2008; Kim & Rhee 2011; Oshimori et al. 2009; Staples et al. 2012; Staples et al.

2014; Klinger et al. 2014). This is consistent with the idea that PCM components are recruited through both microtubule-dependent and microtubule-independent mechamisms.

Many centriolar satellite components are crucial for maintaining the mitotic spindle pole function. Cep72 and Cep90 recruits Kizuna and γ-tubulin to mitotic spindle poles to facilitate spindle pole stability and chromosome alignments during metaphase (Kim & Rhee 2011;

Oshimori et al. 2009). Cep131 and Ccdc13 have been shown to be required for correct chromosome segregation and their knockdown leads to multipolar spindle poles, micronuclei and

13 chromatin bridges (Staples et al. 2012; Staples et al. 2014). SSX2IP is critical for γ-tubulin accumulation around centrosome, and further for microtubule nucleation and spindle assembly

(Bärenz et al. 2013). As mentioned above, these proteins are localized to the centrosome independent of centriolar satellites, implying that their centriolar satellite population and centrosomal population may have disctinct functions. This raises a question which population is responsible for their centrosomal function.

In addition, centriolar satellite components are also involved in centriole duplication and centrosome separation. Cep63 and Cep152 are critical for centriole duplication. Knockdown of either of them leads to great reduction of centrin foci and delayed incorporation of SAS6, suggesting that they are crucial for the initiation of centriole duplication (Brown et al. 2013).

Interestingly, Cep63 centrosomal localization is regulated by the balance between Ccdc14 and

KIAA0753, which are colocalized with Cep63 at centriolar satellites, stressing the role of centriolar satellites in centriole duplication (Firat-Karalar et al. 2014). In addition, the disruption of centriolar satellite integrity by knocking down PCM1 results in the fragmentation or loss of the centrosome, which further leads to ciliogenesis defects and cell cycle arrest through p38MAPK- mediated pathway (Dammermann & Merdes 2002; Srsen et al. 2006; Kimura et al. 2013; Mikule et al. 2007). Centriolar satellites are also involved in centrosome separation by recruiting Nek2A and its substrate C-Nap1 to the centrosomes (Hames et al. 2005; Faragher & Fry 2003).

Centriolar satellites are also implicated in ciliogenesis. The most prominent example is

BBSome, a protein complex consisting 8 Bardet-Biedl syndrome proteins: BBS1, BBS2, BBS4,

BBS5, BBS7, BBS8, BBS9 and BBIP10 (Nachury et al. 2007). BBSome acts as coat complexes to sort membrane proteins to the cilia. BBSome is recruited to membrane by Arl6/BBS3 and membrane-associated BBSome recognizes the ciliary localization signals on ciliary membrane proteins and then facilitates their ciliary transport (Jin et al. 2010). Although all the BBSome components are required for normal cilia function, only BBS1 and BBS5 are essential for

14 ciliogenesis in cultured cells (Loktev et al. 2008). Detailed studies of the intrinsic interactions among BBSome components suggest that BBS4 is the last component added to the complex

(Zhang et al. 2012). Interestingly, BBS4 is the only component stably localized at centriolar satellites and depletion of BBS1 or BBS5 causes ciliogenesis defect without affecting BBS4 satellite localization, suggesting that BBS4 is unique from the rest of BBSome component

(Nachury et al. 2007). Congruently, BBS4 interacts with p150Glued to recruits Pcm1 to centriolar satellites, meaning that BBS4 is a core component of centriolar satellites (Kim et al. 2004). Later study shows that Cep131/Azi1 is colocalized with BBS4 at centriolar satellites and regulates the incorporation of BBS4 to the BBSome (Chamling et al. 2014). Knockdown of Cep131 results in accumulation of BBSome in the cilia, suggesting that centriolar satellites may regulates ciliogenesis by controlling the amount of BBS4 incorporated into the BBSome.

Small GTPase Rab8 and its guanine exchange factor (GEF) Rabin8 are required for ciliogenesis (Nachury et al. 2007; Westlake et al. 2011). Rabin8 are recruited by TRAPPII and

Rab11 to the centrosome before cilia formation (Westlake et al. 2011). BBSome interacts with

Rabin8 through BBS1, but depletion of either BBS1 or Rab8 does not affect Rabin8 centrosomal localization. Knockdown of Rab8, Rabin8 or Rab11 in zebrafish cause abnormal Kupffer’s

Vesicles and a delay in melanosome retraction, phenocopying BBS-morpholino zebrafish, suggesting that Rab8 and Rab11-mediated transport is functionally associated with BBSome pathway (Westlake et al. 2011; Nachury et al. 2007; Yen et al. 2006). Some centriolar satellites proteins and their interacting partners, including PCM1, Cep290 and Talpid 3, are critical for

Rab8 ciliary recruitment and ciliary vesicle docking (Kobayashi et al. 2014). Interestingly,

Cep290 and Talpid3 are also required for the disassembly of centriolar satellites components, including PCM1, Cep290 and BBS4, upon ciliogenesis after serum starvation.

It was observed that centriolar satellites accumulated at the basal body when cilia were formed in the interphase and dispersed when cilia were disassembled in mitosis (Akiharu et al.

15 1999; Kubo & Tsukita 2003; Balczon et al. 1994; Dammermann & Merdes 2002). However, recent studies suggested that the relationship between centriolar satellites and ciliogenesis is not so simple. Ubiquitin E3 ligase MIB1 physically interacts and ubiquitinates PCM1 and Cep131 to inhibit ciliogenesis during mitosis. Cellular stress, such as UV irradiation, heat shock and transcription blocks, induces p38-mediated acute dispersals of PCM1, Cep290 and BBS4, but not

Ofd1 (Villumsen et al. 2013). In the meantime, cellular stress blocks MIB activity and promotes ciliogenesis. Centriolar satellites are required for PLK1 PCM recruitment, which promotes cilia resorption by activating HDAC6. Interestingly, CKD1-mediated phosphorylation of Thr-703 of

PCM1 facilitates its interaction with PLK1 (G. Wang et al. 2013; Pugacheva et al. 2007).

However, recent publications revealed an intricate relationship between centriolar satellites and ciliogenesis. Tang and colleagues found that Ofd1 at centriolar satellites, but not other centriolar satellite components, was degraded by autophagy upon ciliogenesis induced by serum starvation

(Tang et al. 2013). Abnormal accumulation of Ofd1 in Atg5 or Atg3 mutant cells results in fewer and shorter cilia and defective BBS4 ciliary localization, which can be rescued by partial knockdown of Ofd1. In addition, depletion of Ofd1 leads to abnormal cilia formation on cycling cells and non-ciliated cells. This suggests that Ofd1 is a unique centriolar satellite component inhibiting ciliogenesis, congruent with its different response to cellular stress (Villumsen et al.

2013). The other group observed autophagy machinery around the basal body and in the cilia

(Pampliega et al. 2013). The autophagic activity depends on IFT machinery and Shh signaling.

Interestingly, depletion of Atg5 slightly increases ciliation rates and cilia length. The relationship between autophagy and ciliogenesis requires further investigation.

16 1.1.4 Intraflagellar transport

Intraflagellar transport (IFT) is a transport mechanism within the cilium that comprises axonemal microtubules, molecular motors and protein complexes serving as cargo adaptors

(Rosenbaum & Witman 2002). Based on the direction of transport, IFT is categorized into

anterograde IFT that facilitates transport from the base to the tip of the cilium, and retrograde IFT

for the opposite direction. Anterograde IFT is driven by two kinesin-2 family members, the

heterotrimeric Kif3a/Kif3b/ KAP complex and the homodimeric Kif17 motor. Most of their

cargos are coupled to the motors through the IFT-B complex, a 710-760 kDa complex consisting of 10 proteins. Retrograde IFT is driven by cytoplasmic dynein motor, and their cargos are coupled through IFT-A complex, a 550 kDa complex consisting of 6 components. Interestingly, recent studies showed that IFT-A complex was required for the ciliary entry of a subset of proteins, like Tulp3 (Mukhopadhyay et al. 2010).

Despite being found in cilia, most Kinesin2 and IFT particles are localized around the basal body, indicating that the basal body might be a docking area for IFT machinery (Deane et al. 2001). It has been suggested that Kinesin-2, IFT-B and cargos are docked at basal body before entering the cilia. At the ciliary tip, the complex is reorganized and the cargos are loaded onto

IFT-A driven by cytoplasmic dynein for the transport from cilia tip to the base. Both IFT complexes and motors are recycled through this bidirectional transport.

Although IFT was first observed and studied in the biflagellate alga Chlamydomona, it is highly conserved in many other ciliated species including the nematode, fruit fly, mouse and human. In addition, IFT is required for cilia assembly and maintenance in different tissues.

However, two exceptions have been found in mouse and fly sperms. IFT is critical for the assembly of mouse sperm flagella, but no IFT proteins were found in the mature mouse sperm flagella, suggesting that IFT may not be required for their maintenance (Rosenbaum & Witman

17 2002). In fly, the only ciliated cells are sensory neurons and sperms. NomopB, the Drosophila

homolog of IFT88, is essential for sensory cilia assembly but dispensable for sperm flagella

formation or function (Han et al. 2003). These suggest the divergence of ciliogenic mechanisms among different tissues.

1.1.5 Ciliary gate I: the transition fibers

As a specialized compartment, the cilium is enriched with distinct soluble and membrane proteins, not only to maintain the structure but also to facilitate its function. These ciliary proteins need to be imported into the cilia as there appears to be no protein synthesis happened inside the cilia. It has long been believed that the transport between cilia and cytoplasm is strictly guarded by a “ciliary gate” localized at the base of the cilia. Recent studies indicated that this proposed ciliary gate contained two different functional regions: transition fibers and transition zone.

Transition fibers were first described as pinwheel-shaped fibers or alar sheets, originating from DAPs of the mother centriole (Anderson 1972). DAPs are essential for early steps of ciliogenesis (see details in Chapter 4). In a mature cilium, transition fibers serve multiple functions. Transition fibers are believed to anchor the cilia onto cell membrane, which is supported by the fact that the DAPs, precursors of the transition fibers, are critical for the mother centriole to dock to the ciliary vesicle (Tanos et al. 2013). Furthermore, they serve as a diffusion barrier for the transport between cilia and cytoplasm. Electron micrographs of transition fibers suggest that the sizes of the openings are very small, limiting the passage of vesicles or large protein complexes (Anderson 1972). A recent study confirmed this speculation by showing that only soluble proteins with relative molecular weight under 30-40K are able to diffuse into the cilia (Kee et al. 2012). Finally, transition fibers are the places where IFT proteins dock, assemble into complexes and load cargos onto Kinesin. Immunoelectron microscopy showed that many

18 IFT proteins were localized at transition fibers (Deane et al. 2001; Sedmak & Wolfrum 2010;

Sedmak & Wolfrum 2011).

Recent discovery of nucleopore-like structure at the base of cilia confirmed the existence of a flagellar/ciliary pore complex (CPC) that was proposed 12 before (Rosenbaum & Witman

2002; Dishinger et al. 2010; Kee et al. 2012). It is demonstrated that like nuclear pore complex

(NPC), CPC deploys a size-exclusion mechanism which limits the passive diffusion of the molecules with relative molecular mass above certain threshold (around 30K-40K) (Kee et al.

2012). Molecules or complexes above the threshold are transported between compartments through an active transport mechanism similar to nuclear import, involving NLS signal, nuclear transport receptors, Ran GTPase and its regulatory proteins. A subset of nucleoporins (NUPs) are found at the base of the cilia, suggesting the existence of the CPC structure (Kee et al. 2012).

Interestingly, the absence of transmembrane ring NUPs and nuclear FG (phenylalanine-glycine repeat domains) NUPs at cilia base suggests the different anchoring mechanisms between CPC and NPC. Other components of the machinery, including importin β-1 and 2, RanGTP, RanBP1, are also accumulated in the cilia (Fan et al. 2011; Dishinger et al. 2010; Kee et al. 2012). Proteins using this mechanism to enter cilia include Kif17, a Crumbs3 isoform (CRB3-CLPI) and Retinitis

Pigmentosa 2 (RP2), all of which physically interact with importin proteins (Dishinger et al.

2010; Fan et al. 2007; Hurd et al. 2011). KKRK classic NLS on Kif17 is critical for its interaction with importin β-2 and M9-core-like sequence of RP2 binds to importin β-2. It was also shown that the level of RanBP1 (Ran binding protein 1) regulated ciliogenesis, by promoting RanGTP hydrolysis and dissociation between Ran and importin (Fan et al. 2011).

At the cilia base, a ring-shaped Septin diffusion barrier was found between the transition fibers and the axoneme (Hu et al. 2010). Septins are a group of GTPases that assembles into higher-order structures such as filaments and rings (Weirich et al. 2008). Depletion of Septin2 leads to increased exchange of ciliary membrane proteins between cilia and cell membrane,

19 shorter or the absence of cilia and defective sonic hedgehoge (Shh) response, indicating that

Septin2 is part of a diffusion barrier for the ciliary membrane proteins (Hu et al. 2010). Other

Septins are also associated with cilia. Septin-2, -4, -6 and -7 have been found at cilia base and

Septin-2, -7, -9 and -11 are found in the axoneme in airway epithelium cells (Fliegauf et al.

2014). Consistently, Septin-2, -7 and -9 colocalize with actin in cytoplasm and with microtubule in the axoneme of retinal pigmented epithelial cells (Ghossoub et al. 2013). Microtubule- associated protein 4 (MAP4) binds with Septin2 and negatively regulates the axoneme length by controlling the amount of Septin2 accessible to axonemal microtubules (Ghossoub et al. 2013).

1.1.6 Ciliary gate II: the transition zone

The other part of the ciliary gate is the transition zone, which is close to the base of cilia, but distal to the transition fibers. In the transition zone, Y-shaped linker structures bridge the axonemal microtubules and the ciliary membrane (Craige et al. 2010). On the ciliary membrane of the transition zone, several parallel strands of intramembrane particles found in electron micrographs are described as ciliary necklace (Gilula & Satir 1972). However, the compositions of Y linker and ciliary necklace have not been characterized.

Proteins associated with cilia-related human genetic diseases, including Nephronophthisis

(NPHP), Meckel syndrome (MKS) and Senior-Loken syndrome (SLSN), are localized to the transition zone (Szymanska & Johnson 2012). Many transition zone proteins interact with each other and form distinct functional modules. A recently identified transition zone protein complex contains many MKS-associated proteins, whose transition zone localization depends on each other (Chih et al. 2012; Garcia-Gonzalo et al. 2011; Williams et al. 2011). Disruption of this complex leads to the displacement of many ciliary membrane proteins including Somatostatin receptor 3 (SSTR3) and 5-hydroxytryptamine (HTR6) receptors, suggesting that this complex

20 acts as a diffusion barrier for maintaining proper ciliary membrane protein localization.

Complexes with similar composition have also been identified in C. elegans (Sang et al. 2011;

Huang et al. 2011). Interestingly, when Septin2 is knocked down, these transition zone proteins are either failed to be recruited or restricted to the transition zone, suggesting that this complex functions downstream or parallel to Septin (Chih et al. 2012).

In addition to this MKS complex, four complexes containing NPHP-associated proteins

were also identified (Sang et al. 2011). NPHP-1, -4 and -8 are critical for apical organization of

kidney epithelial cells, and are localized to both cell-cell boundaries and the transition zone.

NPHP-2, -3 and -9 are localized more distal to transition zone and are required for the ciliary localization of some G-protein coupled receptors (GPCRs). Two more complexes, one containing

NPHP-5, -6 and Ataxin 10, and the other containing AHI1, MKS1, TCTN2 and MKS6, are also important for ciliogenesis and cilia-dependent signaling respectively. The transition zone harboring multiple functional modules of ciliopathy-associated proteins suggests its multiple roles in ciliary function and ciliopathy pathogenesis.

How transition zone proteins function as a diffusion barrier for ciliary membrane proteins is still elusive. Many of the transition zone proteins, like B9D1 and TMEM 231, are membrane- bound proteins that may restrict the diffusion of ciliary membrane proteins by modifying the lipid composition in transition zone. Alternatively but not exclusively, transition zone protein complexes may regulate the ciliary membrane protein localization by interacting with IFT machinery. For example, several transition zone proteins, including B9D2 and LCA5, physically interact with IFT particles to facilitate opsin transport in photoreceptor cells (Zhao & Malicki

2011; Boldt et al. 2011).

Transition zone proteins are also shown to be required for cilia structure by maintaining the association between axonemal microtubules and ciliary membrane. Immunoelectron microscopy showed that Cep290 was localized to the Y-linkers in Chlamydomonas and a

21 mutation in Cep290 disrupts the association between microtubules and the membrane in the

transition zone and changes protein composition of cilia (Craige et al. 2010). Furthermore, two

transition zone proteins, MKS6 and NPHP4, were also required for the association between microtubules and the membrane in the transition zone (Sang et al. 2011; Huang et al. 2011).

Interestingly, besides its centriolar localization, Centrin is also enriched in the terminal plates of Tetrahymena flagella and the connecting cilia of photoreceptors, equivalent to transition zone in regular cilia (Kilburn et al. 2007; Wolfrum 1995; Wolfrum & Salisbury 1998). It was reported that Centrin colocalized and formed complex with visual G-protein transducin in the connecting cilia of photoreceptors, suggesting that Centrin might play a role in ciliary transport

(Pulvermüller et al. 2002; Giessl et al. 2004). Surprisingly, recent study suggests that Centrin2 was also localized to nuclear pores and was involved in mRNA and protein nuclear exports

(Resendes et al. 2008). The involvement of Centrin in both nuclear pore-mediated transport and ciliary transport suggests the shared machinery between these two transport systems.

Taken together, structures at transition fibers and transition zone constitute the gate guarding the transport between the cilium and the rest of the cell. Some models have been proposed to explain how they function, but details about their structures, compositions and mechanisms require further elucidation.

1.1.7 Other compartments in the cilia

Recent studies suggested that other compartments may exist in the cilium.

Inversin/NPHP2 (Invs) marks the Inversin compartment, which is more distal to the transition zone (Shiba et al. 2010). NPHP3 and Nek8/NPHP9 are also localized to the Inversin compartment, and their localization is dependent on Invs. These proteins are also critical for the ciliary localization of some ciliary membrane proteins (Sang et al. 2011).

22 The EvC (Ellis-van Creveld syndrome) zone, harboring the EvC and EvC2 proteins, lies

in between the transition zone and Invs compartment (Shiba et al. 2010). EFCAB7 and IQCE tether EvC/EvC2 to the EvC zone, and loss of these proteins results in the spread of EvC/EvC2 along the entire cilium (Pusapati et al. 2014).

The distal tips of the axonemal microtubules are attached to the ciliary membrane through microtubule-capping structure (Dentler 1980; Suprenant & Dentler 1988). It was believed that this cilia tip structure regulates the microtubule assembly and cilia length. The fact that some proteins specifically accumulate at the cilia tip raises the possibility that a specialized ciliary-tip compartment may exist. A recent study suggested that Kif7, a Kinesin-4 family protein, may play an essential role in maintaining such a ciliary-tip compartment (He et al. 2014).

1.1.8 Cilia assembly

In mammals, the primary cilium is a dynamic organelle, which is assembled during G0 or

G1 phase and disassembled upon mitotic entry (Kim & Dynlacht 2013). For the in vitro cultured cells, cilia formation is commonly induced by serum starvation and high cell density (Pugacheva et al. 2007). Why mammalian cells assemble cilia during interphase and disassemble cilia during the mitosis is still elusive. The prevalent opinion is that cilia formation requires the liberation of the mother centriole from its role as the spindle pole during mitosis (Seeley & Nachury 2010).

Interestingly, the centrioles were found to serve as spindle poles and basal bodies of cilia simultaneously in insect meiotic spermatocytes and similar events were found in flagellate protozoa as well (Bloodgood 2009). This suggests that the mother centrioles are capable of playing two roles at the same time at least in certain species or tissues.

23

Figure 1-3. Ciliogenesis process.

Ciliogenesis starts with the mother centriole docking to the ciliary vesicle through distal appendages. Then secondary vesicles are transported and fused with the primary ciliary vesicle. In the meanwhile, microtubules are grown from the distal end of the mother centriole toward the ciliary vesicle. The structure is transported to the cell surface and ciliary vesicle fused with plasma membrane. After the protrusion of the cilium, it keeps growing to the normal length and recruiting components for its normal function.

24 Ciliogenesis, the process of cilia formation, is a multi-step process first described by

Sorokin (1962; 1968) in mid-20th century through electron microscopy. As shown in Fig. 1-3, ciliogenesis starts from docking the mother centriole to a ciliary vesicle. Subsequently microtubules at the distal end of the mother centriole commence to extend towards the ciliary vesicle. As axoneme grows, secondary ciliary vesicles are recruited to and fused with the primary ciliary vesicle. The docked basal body moves toward cell surface and ciliary vesicle fuses with the plasma membrane, allowing the cilium protrusion. The machinery responsible for transporting between the cilium and cytoplasm ensures the cilium to maintain proper length, chemical composition and functions.

When cells exit cell cycle and start to grow cilia, a group of positive regulators of ciliogenesis is recruited to the mother centriole. DAPs, the later transition fibers, play critical roles in these early events of ciliogenesis. Cep164, a DAP component, promotes the ciliary vesicle docking by physically interact with a small GTPase, Rab8, and its guanine nucleotide exchange factor, Rabin8 (Graser et al. 2007; Schmidt et al. 2012). In addition, Cep164 is essential for recruiting Tau Tubulin Kinase 2 (Ttbk2) to the distal end of the mother centriole, which promotes the centriolar recruitment of Ift proteins and removal of Ccp110 (Tanos et al. 2013; Joo et al. 2013; Goetz et al. 2012). Ccp110, in conjunction with its binding partners Cep97 and Kif24, negatively regulates ciliogenesis (Spektor et al. 2007; Tsang et al. 2008; Schmidt et al. 2009;

Kobayashi et al. 2011). Ccp110 is proposed to inhibit the microtubule growth by forming a cap at the distal end. Both Ccp110 and Cep97 are localized to the distal ends of the centrioles and they are removed from the mother centriole upon ciliogenesis. Their interacting protein Kif24 also inhibits ciliogenesis by depolymerizing centriolar microtubules. Similar to the role of Ttbk2, another Ser/Thr kinase , Mark4, was found to control the Ccp110 removal upon ciliogenesis

(Kuhns et al. 2013). Another protein similar to Ccp110 function is trichoplein, which negatively regulates the ciliogenesis (Inoko et al. 2012).

25 After ciliogenesis is initiated, the cilium continues to grow to its normal length. The cilia

length is positively regulated by the abundance of IFT-B complex and the availability of soluble tubulin (Besschetnova et al. 2010; Sharma et al. 2011; L. Wang et al. 2013). The cilia length is also regulated by the retrograde transport. Two regulators are implicated in this regulation, including Tctex1, a putative component of dynein, and Nde1, a dynein light chain subunit (LC8) interacting protein (Palmer et al. 2011; Kim et al. 2011). In addition, actin filaments are also involved in ciliary length control. Ciliary length is affected by many actin filament modulators, including cell shape and density (Pitaval et al. 2010), actin filament regulators (GSN, AVIL and

ACTR3) (Kim et al. 2010), and drugs regulating actin polymerization (cytochalasin D and

Jasplakinolide) (Sharma et al. 2011; Kim et al. 2010).

Upon mitotic entry, cells start to disassemble the cilia. Many mechanisms discovered so far, related to cilia resorption, are through regulating stability of axonemal microtubules.

Axonemal tubulins bear many post-translational modifications, including acetylation, detyrosination, polyglutamylation and glycylation (Gaertig & Wloga 2008). Acetylation and detyrosination stabilize the axonemal microtubules, while polyglutamylation and polyglycylation regulate the recruitment of proteins to the axoneme micrtobutules (Konno et al. 2012). In response to growth factor stimulation, HEF1 is stabilized to activate Aurora A kinase, which in turn leads to phosphorylation and activation of Histone Deacetylase 6 (HDAC6), a tubulin deacetylase (Pugacheva et al. 2007). HDAC6 promotes cilia disassembly by destabilizing the axonemal microtubules. The centrosomal recruitment of Plk1 upon mitotic entry also promotes cilia resorption by directly phosphorylating HDAC6 (G. Wang et al. 2013). Pitchfork (Pifo) also facilitates cilia disassembly by activating HDAC6 (Kinzel et al. 2010). In addition, Kif19A and

Kif24, two microtubule depolymerizing kinesins, are involved in cilia resorption by depolymerizing axonemal microtubules after the post-translational modifications are removed

(Kobayashi et al. 2011; Niwa et al. 2012).

26 In summary, the cilium is a tiny but complicated structure, whose assembly and

disassembly are under strict regulation. In the past decade, our knowledge of ciliary structure and

regulation has grown tremendously. New components, structures and processes involved in ciliogenesis will keep adding to our understanding of this enigmatic organelle.

1.2 The primary cilium and signal transductions

The primary cilium has been known for their sensory functions, including photosensation, chemosensation, mechanosensation and thermosensation. However, the most surprising recent discovery is that the primary cilium is involved in signal transduction and vertebrate development. In this section, I summarized signaling pathways related to primary cilia.

1.2.1 The primary cilium and Hedgehog signaling

The Hedgehog (Hh) signaling pathway was initially found in Drosophila for its critical roles in regulating embryonic segmentation, as well as the patterns of the eyes and wings

(Nüsslein-Volhard & Wieschaus 1980; Heberlein et al. 1993; Ma et al. 1993; Tabata & Kornberg

1994). In Drosophila, Hh ligand binds to its receptor Patched (Ptc) and releases the repression on

Smoothened (Smo) by Ptc. In the absence of Hh, the downstream transcription factor Cubitus interruptus (Ci) is cleaved into a repressor for target gene expression. In the presence of Hh, the proteolytic processing of Ci is inhibited and the full-length Ci acts as an activator for target gene expression. In mammals, there are three homologues of Hh, namely Sonic hedgehog (Shh),

Indian hedgehog (Ihh) and Desert hedgehog (Dhh). Among them, Shh received the most attention because of its involvement in multiple key events in mammalian development, including spinal cord and limb patterning (Chiang et al. 1996; Chiang et al. 2001; Krauss et al. 1993; Kraus et al.

27 2001). There are also three mammalian homologues of Ci, including Gli1 (Glioma-Associated

Oncogene Homolog 1), Gli2 and Gli3 (Matise & Joyner 1999). Gli1 is an obligatory activator, whereas Gli2 and Gli3 have both activator and repressor domains. Genetic studies suggest that

Gli2 mainly acts as an activator and Gli3 as a repressor (Pan et al. 2006; Wang et al. 2000).

Cilia were first linked to Hh signaling by studying mouse mutants with defective intraflagellar transport (Huangfu et al. 2003). Mouse mutants of Ift88, Ift172 and Kif3a exhibit abnormal neural tube patterning due to abnormal Hh signaling. Ift88 and Ift172 are IFT-B complex components and Kif3a is a subunit of Kinesin2 (Pazour et al. 2000; Pedersen et al.

2006). The defective Hh signaling in these cilia mutants suggests that Hh signaling in mouse may require cilia (Huangfu et al. 2003). Subsequent analyses of many additional mouse mutants with ciliogenesis defects substantiated the essential roles of the primary cilia in Hh signaling.

Interestingly, genetic data suggested that the primary cilium is required for both the activator and repressor activities of Gli proteins (Huangfu et al. 2003; Liu et al. 2005; May et al.

2005). The loss of Shh-dependent ventral cell types in the neural tube suggests diminished Gli activator activity in the absence of cilia. On the other hand, the polydactyly and posteriorized limb patterning indicates impaired Gli repressor activity. Indeed, biochemical analysis indicated that the proteolytic processing of Gli3 was severely disrupted in many cilia mutants, leading to a drastic reduction of Gli3 repressors.

28

Figure 1-4. Sonic hedgehog signaling in mammals.

In the absence of Shh, Smo is excluded from cilia and inhibited by Ptc1. Gli protein ciliary recruitment is inhibited. Glis are phosphorylated and proteolytically processed into Gli repressors. Gli repressors inhibit the expression of downstream target genes. In the presence of Shh, Shh binds to Ptc1 to relieve its inhibition on Smo. Smo and Glis are accumulated in the cilium. Glis become transcriptional activators to activate downstream target gene expression. This schematic is adopted from Ye & Liu 2011.

29 In addition, many Hh signaling core components, including Ptch1, Smo, Glis and Sufu

(Suppressor of Fused), were found in the cilia and their ciliary localizations are regulated by Hh

signaling, implying that Hh signaling is mediated by cilia (Goetz & Anderson 2010). As

illustrated in Fig. 1-4, in the absence of Hh ligand, Ptch1 is accumulated in the primary cilium

and the ciliary recruitments of Smo, Gli2 and Gli3 are inhibited. Gli2 and Gli3 become repressors

after proteolytic processing to repress target gene expression. In the presence of Hh, Ptch1 is

removed from the cilia. Smo, Gli2 and Gli3 accumulate in the cilia. Gli2 and Gli3 are dissociated

from Sufu to become activators. It is believed that when Hh signaling recruits Gli2/3 into the cilia, Gli2/3 are post-translationally modified and freed from the inhibition of Sufu. However, the nature of this modification is not yet clear.

Unlike other cilia mutants, mutations in IFT-A genes lead to increased Hh signaling. IFT-

A is traditionally thought as exclusively required for retrograde transport (Pedersen &

Rosenbaum 2008). A recent study suggested that IFT-A is also involved in ciliary entry of Tubby like protein 3 (Tulp3), which serves as an adaptor to promote ciliary trafficking of some G

Protein Coupled Receptors (GPCRs), including melanin concentrating hormone receptor 1

(MCHR1) and SSTR3, but not Smo (Mukhopadhyay et al. 2010). Further studies discovered a novel negative regulator of Hh signaling, Gpr161, which is recruited into the cilia by IFT-A and

Tulp3 (Mukhopadhyay et al. 2013). Gpr161 promotes protein kinase A (PKA) activity by increasing the cAMP level. The upregulated PKA activity inhibits Hh signaling by facilitating

Gli3 processing. Hh signaling excludes Gpr161 from cilia to prevent its activity.

EvC and EvC2 at EvC zone have been found to be required for Hh signaling. Hh signaling promotes the physical interaction between EvC/EvC2 complex and Smo. It was reported that EvC/EvC2 complex was required for Gli3 recruitment to the ciliary tip and

Sufu/Gli3 dissociation (Caparrós-Martín et al. 2013). However, EvC and EvC2 are dispensable for Hh-mediated Smo phosphorylation and ciliary accumulation.

30 Kif7, a Kinesin-4 family protein, has been implicated in Hh-mediated accumulation of

Glis and Sufu at cilia tips (He et al. 2014). In the absence of Kif7, Gli proteins and Sufu are distributed along the entire cilia instead of accumulated at the tip. Kif7 is localized to the tips of axonemal microtubules and essential for controlling cilium length and maintaining the distal structure.

1.2.2 The primary cilium and Wnt signaling

Besides Hh signaling, the primary cilium is also involved in Wnt pathway. Wnt signaling has been implicated in embryonic development and adult homeostasis (Clevers 2006; Logan &

Nusse 2004). It is critical for the body axis establishment, cell fate specification, cell proliferation and migration in various tissues. Mutations affecting Wnt signaling were also found to underlie breast cancer, prostate cancer, glioblastoma and many other malignancies. Depending on different downstream events, Wnt signaling can be categorized as canonical (β-catenin dependent) and noncanonical (β-catenin independent) (Logan & Nusse 2004). In canonical Wnt signaling pathway, Wnt ligands bind to the heterodimer of Frizzled receptor and LRP5/6 coreceptor. β- catenin is released from a destruction complex and translocated to the nucleus to activate

TCF/LEF1. In noncanonical Wnt signaling pathway, when Wnt ligands bind to Frizzled receptor,

β-catenin is degraded and Dvl is targeted to the cell membrane to transduce the signal to RhoA,

JNK, G protein and calcium, which regulate Drosophila planar cell polarity (PCP) and vertebrate convergence and extension (Veeman et al. 2003).

Several studies have linked cilia to Wnt signaling. Invs has been found to target cytoplasmic Dvl to degradation (Simons et al. 2005). Loss of Dvl results in PCP defects in

Xenopus embryos. However, Invs has multiple subcellular localizations, including the cilia, adherens junction and nucleus. Therefore, it is possible that Invs has two independent functions in

31 ciliogenesis and PCP. In addition, ablations of BBS proteins, which are localized around the basal

body, in mouse and zebrafish lead to typical manifestations of defective PCP signaling and

ectopic activation of β-catenin transcriptional targets (Gerdes et al. 2007; Ross et al. 2005).

Finally, loss of Kif3a, Ift88 and Ofd1 leads to hyperactivation of β-catenin-dependent Wnt

signaling and hypersensitivity to Wnt3a through canonical Wnt pathway (Corbit et al. 2008).

However, some evidence suggests the opposite. Canonical and noncanonical Wnt signaling

appeared normal in both midgestation embryos with primary cilia defects and mouse embryonic

fibroblasts (MEFs) derived from these embryos, suggesting that the cilia are dispensable for Wnt

signaling at least in early embryonic development (Ocbina et al. 2009). Taken together, the link

between cilia and Wnt signaling is still controversial and waits to be further examined.

1.2.3 The primary cilium and PDGFRα, Hippo and other pathways

PDGFRα (platelet-derived growth factor receptor alpha) signaling is involved in cell migration, proliferation and apoptosis (Heldin and Westermark, 1999). It was found that

PDGFRα was localized to the primary cilia of NIH3T3 and MEFs upon serum starvation

(Schneider et al. 2005). Ligand-stimulated activation of PDGFRa induces activation of Akt and

Mek1/2-Erk1/2. Phosphorylated Mek1/2 is detected in the cilia and around the basal body. Ift88 mutant cells with cilia defects show impaired PDGFRa-mediated response, suggesting cilia are required for PDGFRα signaling.

The Hippo signaling pathway regulates organ size through controlling cell proliferation and apoptosis. Many components of the pathway have been known as tumor suppressors or protooncogenes (Harvey & Tapon 2007). NPHP4, a ciliopathy-associated protein, interacts with

LAST1 (large tumor suppressor 1) and suppresses LAST1-mediated phosphorylation of YAP

(Yes-associated protein ) and TAZ (transcriptional coactivator with PDZ-binding domain)

32 (Habbig et al. 2011). NPHP4, and its interacting protein NPHP1 and NPHP8, are localized to both the ciliary transition zone and cell-cell boundaries. They are not required for cilia formation but essential for tight junction formation, lumen formation and polarity establishment in MDCK cells (Sang et al. 2011).

Many other receptors also accumulate in the primary cilia, such as melanin concentrating hormone receptor 1 (MCHR1), HTR6 and SSTR3. In addition to Tulp3 and IFT-A mentioned above, other Tubby-related proteins, including Tubby and Tulp1, and BBSome proteins are also proven to mediate GPCRs ciliary localization in photoreceptors and neurons (Hagstrom et al.

2001; Sun et al. 2012; Berbari et al. 2008). These GPCRs ciliary localization defects are implicated in the obesity and retinal degeneration in these mouse models and human patients

(Mukhopadhyay & Jackson 2013).

In summary, the studies of cilia in signal transduction laid the foundation for understanding the etiology of the human genetic diseases related to cilia defects.

1.3 The primary cilium and human disease

The cilium/flagellum was first observed by Leeuwenhoek in 1677 and the primary cilium in mammals was first described by Zimmermann in 1898 (Bloodgood 2009). However, it failed to draw much attention until the discovery of its role in polycystic kidney disease in 2000 (Moyer et al. 1994; Pazour et al. 2000). Subsequent surprising discoveries of its integral role in signal transductions and development, especially in Hh pathway, send this tiny cell surface organelle into the spotlight. Since 2000, along with more vigorous scientific studies, an increasing number of human genetic diseases, collectively known as “ciliopathy”, have been shown to be related to defects in the primary cilia.

33 The advance of the knowledge about the cilia helped the understanding of the etiologies

of ciliopathies, which includes Bardet-Biedl syndrome (BBS), Alstrom syndrome (ALMS),

polycystic kidney disesase (PKD), nephronophthisis (NPHP), Merckel syndrome (MKS), Joubert

syndrome (JBTS), Jeune asphyxiating thoracic distrophy (JATD), Oral-Facial-Digital syndrome

(OFD), Senior-Loken syndrome (SLSN), Ellis-van Creveld syndrome (EVC), retinal pigmentosa,

Leber’s congenital amaurosis (LCA) and Sensenbrenner syndrome (Waters & Beales 2011).

Many of these diseases are pleiotropic syndromes highlighting the versatile functions of primary cilia in different tissues (Quinlan et al. 2008). Their shared manifestations include renal disease, hepato-biliary disease, retinal degeneration and cerebral abnormalities. Additional clinical features are laterality defect, polydactyly, obesity, skeletal defects, anosmia, hearing loss and male infertility. Some ciliopathy genes exhibit strong allelism, meaning that different mutant alleles of the same gene lead to different phenotypes. The best illustration is CEP290, which is involved in 6 different ciliopathies, including MKS type 4, JBTS type 5, LCA type 10, BBS type

14, NPHP type 6 and SLSN type 6 (Waters & Beales 2011).

1.3.1 Polycystic kidney disease

Polycystic kidney disease (PKD) patients develop multiple cysts in kidneys caused by an imbalance of proliferation and differentiation, and these cysts lead to kidney failure by crowding out normal nephrons (Grantham 2008). Two types of PKD have been characterized: autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease

(ARPKD). ADPKD is caused by mutations in genes PKD1 or PKD2, and ARPKD is caused by mutations in PKHD1 (Chapin & Caplan 2010). The connection between PKD and the primary cilia was first established by the study of the mouse PKD model with mutations in Ift88. The

ORPK (Oak Ridge Polycystic Kidney) mouse carries a hypomorphic mutation in Ift88 (also

34 known as Tg737 or Polaris), which partially affects its expression and function (Moyer et al.

1994). Ift88 was later found to be required for cilia and flagella assembly in mouse and

Chlamydomonas, suggesting a role of the cilia in PKD pathogenesis (Pazour et al. 2000).

Mechanical sensory function of the kidney primary cilia was revealed in a study showing

that bending of the primary cilia results in an increased cytoplasmic calcium level (Praetorius &

Spring 2001). Soon after, polycystins (proteins encoded by PKD1 and PKD2 genes),

mechanoreceptive ion channels, were found in cilia membrane (Pazour et al. 2002; Yoder et al.

2002). Polycystin 2 (PC2), the protein product of PKD2, belongs to the transient receptor

potential (TRP) ion channel family. Polycystin 1 (PC1), encoded by PKD1, binds to PC2 and

assists its mechanosensory function. Both PC2 and PC1 are transmembrane proteins, localized to

the cilia membrane. In a normal kidney, the urine flow deflects the primary cilia on kidney

epithelium, causing the influx of calcium and PC1 binding to transcriptional factor STAT6 and its

coactivator P100 through its C-terminus (Low et al. 2006). These intracellular signals inhibit the

proliferation of kidney epithelial cells. In the absence of urine flow or in PKD patients whose

PC1 or PC2 are defective, calcium influx is blocked and the C-terminus of PC1, together with

STAT6 and P100, are released through intramembrane proteolysis. STAT6 and P100 are

translocated to nucleus to activate their target genes, and trigger proliferation and cyst formation.

1.3.2 Hepatic and pancreatic cysts and situs inversus

PKD patients also manifest damages in liver and pancreas and even heart and brain.

Consistently, in addition to polycystic kidney disease, ORPK mice also exhibit hepatic and pancreatic ductal abnormalities and cysts, retinal degeneration, skeletal defects, cerebellar hypoplasia, hydrocephalus, situs inversus and skin and hair abnormalities (Lehman et al. 2008).

These data from ciliopathy patients and mouse models suggest the role of cilia in maintaining

35 homeostasis in these tissues. Mechanical sensory functions of the primary cilia are indeed found

in cardiovascular system, bone tissues and bile ducts. Vascular endothelial cells respond to fluid

shear stress through cilia-mediated cytosolic calcium and nitric oxide release, which are impaired

in Pkd1 null mutant and Ift88 mutant mice (Nauli et al. 2008). The cilia on cardiac endothelium

are required for shear stress-induced shear marker upregulation (Hierck et al. 2008). The primary

cilia on bone cells are essential for osteogenic and bone resorptive responses to dynamic fluid

flow, independent of calcium influx and stretch-activated ion channels (Malone et al. 2007).

Cholangiocyte cilia in bile duct epithelium are important to translate luminal fluid flow signal to

intracellular calcium and cAMP signal (Masyuk et al. 2006).

The cilia are also implicated in left-right determination during vertebrate development.

Nodal cilia, cilia growing on the ventral surface of the node, were first described in 1994 (Sulik et

al. 1994), and later a leftward fluid flow generated by clockwise rotations of nodal cilia was

discovered (Nonaka et al. 1998). Subsequent studies showed that this leftward fluid flow is

necessary for left-right determination (Okada et al. 1999; Supp et al. 1999). The current “two cilia” model, which is modified from the “morphogen flow” model, proposes that there are two populations of primary cilia in the node. The motile cilia generate leftward flow and the non- motile sensory cilia respond to the differential deflection by intracellular calcium signaling

(McGrath et al. 2003). The model is supported by the observation that mutant mice lacking PKD2 proteins, which are critical for the mechanosensory function of the primary cilia , develop situs inversus when motile nodal cilia are normal (Pennekamp et al. 2002). L-R asymmetry defects are also found in many cilia mutant mice, like ORPK mutants (Murcia et al. 2000) and Kif3a mutants

(Takeda et al. 1999; Marszalek et al. 1999).

36 1.3.3 The primary cilium and sensory defects

Besides the mechanosensory functions mentioned above, the primary cilium also harbors other sensory functions in different neurons, including chemosensory function in olfactory sensory neurons and photosensory function in photoreceptors. Not surprisingly, impaired olfactory function has been found in ciliopathy patients with BBS or LCA (Green & Mykytyn

2010). Retinal degeneration is manifested in most of the ciliopathies and has become a hallmark for ciliopathies (Waters & Beales 2011). In addition, impaired peripheral sensory innvervation and function is also found in ciliopathy mouse model, suggesting the involvement of cilia in peripheral thermo- and mechano-sensation (Tan et al. 2007).

On the dendrite of each olfactory sensory neuron, there are 10-30 olfactory cilia protrude into nasal cavity to sense the inhaled odorant molecules (Jenkins et al. 2009). Olfactory GPCRs accumulate on the ciliary membrane, which can bind to ordorant molecules and lead to an increased cyclic adenosine monophosphate (cAMP) level by activating adenylate cyclase III

(ACIII). The increased cAMP level causes calcium influx by opening the CNG channels, which subsequently leads to depolarization of neurons through opening calcium-gated choloride channels (Su et al. 2009). Disruption of cilia structure or displacement of olfactory GPCRs in ciliopathy patients deteriorates the olfactory sensory functions (Kulaga et al. 2004; McEwen et al.

2007).

Light sensation in mammals is mediated by the photoreceptor cells, including rod and cone cells, in the eyes. A photoreceptor cell comprises a specialized cilia (including the outer segment and connecting cilia) and cell body (inner segment) (Roepman & Wolfrum 2007). The outer segment is filled with dense stacks of rhodopsin-containing disc membranes, which can sense light and reduce cyclic guanosine monophosphate (cGMP) level by activating phosphodiesterase (PDE). Reduction of cGMP level further leads to depolarization through

37 closure of cGMP-gated channels (Leskov et al. 2000). The rhodopsin-containing membranes are synthesized in the inner segment and transported to the outer segment through the narrow connecting cilium at a very high turnover rate, estimated 2000 rhodopsin molecules and 0.1 µm2

of membrane per minute (Wolfrum & Schmitt 2000). Therefore, ciliary transport is critical for

photoreceptor maintenance and phototranduction and ciliary transport defects cause retinal

degeneration in many ciliopathies (Adams et al. 2007).

1.3.4 The primary cilium and obesity

Obesity is associated with some ciliopathies, including BBS, ALMS and JBTS (Waters &

Beales 2011). In addition, obesity is also observed in mouse models with mutations in Bbs,

Alms1, Ift88, ACIII and Tubby (Mukhopadhyay & Jackson 2013). Many efforts have been made to elucidate the etiology of the obesity phenotype. The first question to answer is in which tissue the cilia defects underlie the obesity. It was reported that transient cilia on differentiating preadipocytes played an important role in adipogenesis, suggesting peripheral adipogenesis dysfunction contribute to the obesity manifestation in BBS patients (Marion et al. 2009).

However, later study showed that conditional knockout of Ift88 in hypothalamus resulted in obesity phenotype similar to Bbs mice, suggesting the obesity is mainly ascribed to neuronal cilia defects instead of peripheral cilia malfunction (Davenport et al. 2007).

The second question is which pathway in hypothalamus neurons is responsible for the obesity phenotype. Hypothalamus is a critical brain area involved in leptin-mediated satiety regulation (Schwartz et al. 2000). Leptin is primarily secreted by adipocytes and circulates in blood (Sinha et al. 1996). Leptin binds to leptin receptors to induce the production of proopiomelanocortin (POMC) in hypothalamus neurons, which further inhibits appetite by activating melanocortin-3 and -4 receptors (Cowley et al. 2001). Interestingly, hyperphagia and

38 elevated leptin level were observed in both BBS patients and Bbs mutant mice (Feuillan et al.

2011; Rahmouni et al. 2008). In addition, it was reported that Bbs1 physically interacted with the

leptin receptor and might be involved in the trafficking of leptin receptor between Golgi and

plasma membrane, suggesting that Bbs proteins or cilia might regulate appetite control and

energy balance through leptin signaling (Seo et al. 2009).

However, previous studies suggested that almost all the obese animals and human have

elevated leptin levels and somewhat leptin resistance, suggesting that the leptin resistance

observed in Bbs mutant mice might be secondary to obesity (Considine et al. 1996 ; Maffei et al.

1995). Surprisingly, recent findings indicated that cilia were not directly involved leptin signaling

and leptin resistance might be a secondary effect of obesity, instead of the primary cause (Berbari

et al. 2013). The discrepancy between this study and previous study on leptin resistance of Bbs

mutant mice is explained by the so-called “food anticipatory behavior”, which alters the feeding

behavior in response to caloric restriction (Berbari et al. 2013; Seo et al. 2009). It was shown that

caloric restricted Bbs mice were sensitive to leptin when their weights were normal and food

anticipatory behavior disappeared (Berbari et al. 2013). The molecular mechanism underlying the

obesity in ciliopathies remains elusive, but many potential mechanisms are proposed, including

melanin-concentrating hormone pathway, mTOR pathway and Hh signaling (Berbari et al. 2013).

1.3.5 The primary cilium and tumors

The primary cilia also play roles in in medulloblastoma and basal cell carcinoma by regulating Hh signaling. However, the roles of cilia in these tumors depend on the mechanisms of tumorigenesis. Cilia ablation inhibits medulloblastoma formation in mouse with constitutively active Smo, which is consistent with the fact that cilia are genetically downstream of Smo in Hh signaling (Han et al. 2009). On the other hand, removal of cilia is required for medulloblastoma

39 formation in mouse with constitutively active Gli2, indicating that constitutively active Gli2

causes tumor only when Gli3 repressor function is blocked by cilia removal (Han et al. 2009).

Similar results were observed in a basal cell carcinoma mouse model (Wong et al. 2009). Taken together, cilia can play either a positive or a negative role in tumor formation dependent on the context.

In summary, this small but delicate organelle plays critical roles in development and homeostasis. Moreover, it is involved in human genetic diseases. Studying how cilia are formed will shed lights on the understanding of ciliopathies.

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59 Chapter 2

Materials and methods

This chapter was published in PNAS (Ye et al. 2014)

2.1 Cell culture and transfection

2.1.1 C2cd3GT mutant cell lines establishment

Mutant mouse embryos and their littermates are dissected at E10.5 (Embryonic day 10.5) in sterile DMEM. The embryos are then transferred to a gelatin pre-coated 24-well plate containing primary cell culture medium, which includes DMEM supplemented with 10% fetal

bovine serum (FBS), non-essential amino acid (NEAA), sodium pyruvate, Glutamax (Invitrogen),

antibiotics (penicillin and streptomycin). Embryos are broken into pieces by pushing them

through gauge-20 needles. Primary cells were then immortalized by transfecting with SV40 large

T antigen plasmid (a gift from B. Wang, Cornell University). The use of animals for this study

has been approved by the Institutional Animal Care and Use Committee at the Pennsylvania State

University.

2.1.2 Cell culture, transfection and nocodazole treatment

Primary cells are cultured in primary culture medium described above. Mouse embryonic

fibroblast (MEFs), including C2cd3Hty, C2cd3GT and their littermate controls, and HEK293T cells are cultured in DMEM supplemented with 10% fetal bovine serum (FBS), Glutamax (Invitrogen), antibiotics (penicillin and streptomycin). Ofd1GT and control ES cells (a gift from J. F. Reiter,

60 University of California, San Francisco) are cultured ES cell culture medium, containing GMEM,

FBS, Glutamax, NEAA, Pyruvate, β-mercapital ethanol (β-ME) and mouse leukemia inhibitory factor (mLIF). All cells are culture in the incubator at 37°C and 5% (vol/vol) CO2. Transfection

was done with Lipofectamine 2000 (Life Technologies) or JetPRIME (Polyplus Transfection) by

following the manufacturer’s instruction. For microtubule disassembly, cells were treated with 6

mg/mL nocodazole (a gift from M. M. Rolls, The Pennsylvania State University) for 1 h at 37 °C, followed by 0.5 h on ice before immunofluorescence analysis. Control cells were treated with

DMSO for 1.5 h at 37 °C.

2.1.3 DNA constructs

Mouse C2cd3, Bbs4, Ift88, Ift52, Sclt1, Cep89, Fbf1, Centrin1, Centrin2, Centrin3 and

Poc5 cDNAs were cloned through reverse transcriptase-polymerase chain reaction. Pcm1, Rab8a, p50/Dynamitin/Dctn2 and Ccdc41 cDNAs were purchased from Open Biosystems. hCep164 was amplified from a plasmid (a gift from J. F. Reiter, University of California, San Francisco).

FLAG- and GFP-tagged constructs were assembled by subcloning the above genes into pcDNA3-

FLAG-PRMT5 (a gift from Y. Wang, The Pennsylvania State University), pFLAG-CMV2

(Sigma) or pEGFP (Clontech).

A GFP-Ttbk2 construct was a gift from K. V. Anderson (Sloan-Kettering Institute). A

GFPDzip1 construct was a gift from Y Jing (University of Illinois at Urbana-Champaign). A

GFPOfd1 construct was a gift from J F Reiter. GFP-Cep290 (#27379), deposited by J. Gleeson

(Valente et al. 2006); and GFP-Smo (#25395), deposited by P. Beachy (Chen et al. 2002) were obtained from Addgene (www.addgene.org).

61 2.1.4 RNA interference

A small interfering RNA (siRNA) specific for mouse Pcm1and a control scramble siRNA

were synthesized according to published sequences (Dammermann & Merdes 2002). siRNAs were introduced into MEFs using Lipofectamine RNAiMax (Life Technologies). Forty-eight hours after the first transfection, cells were replated and retransfected with the same siRNA.

Forty-eight hours after the second transfection, cells were processed for immunofluorescence.

Additional RNA interference experiments were performed by transfecting MEFs with plasmids expressing small hairpin RNAs (shRNAs; Sigma) against various regions of mouse Pcm1 using

JetPRIME (Polyplus Transfection). A plasmid expressing a shRNA against GFP was used as a control. Cells were processed for immunofluoresence 5 d after transfection.

2.2 Imaging

2.2.1 Immunocytochemistry (ICC)

For immunofluorescence analyses, cells grown on glass coverslips were fixed in 4%

(wt/vol) paraformaldehyde at room temperature or ice-cold methanol for 4 min, permeabilized in

PBS containing 0.1% Triton X-100, and incubated in primary antibodies for 2 h at room temperature. Subsequently, the cells were washed three times in PBS and incubated in fluorescently labeled secondary antibodies for 1 h at room temperature. After a final wash with

PBS, the coverslips were mounted on microscope slides with VectaShield mounting medium containing DAPI (Vector Laboratories). The cells were imaged with a Nikon E600 epifluorescence microscope or an Olympus FV1000 confocal microscope.

62 2.2.2 Immunohistochemistry (IHC)

Embryos were dissected and fixed in fresh 4% paraformaldehyde (PFA) at 4 °C for 1 h.

After rinsing with PBS, embryos were saturated in 30% sucrose over night. The embryo samples

were then embedded in optimum cutting temperature (OCT, Tissue-Tek) and cryo-sectioned at

microns using Leica CM1900 cryostat. The sections were incubated with primary antibodies at 4

°C overnight followed by 2-hour incubation with fluorescently labeled secondary antibodies, and

mounted with DABCO (Sigma). Pictures were taken using a Nikon E600 microscope.

2.2.3 Antibodies for Immunofluorescence.

Mouse α-acetylated α-tubulin (Sigma; T7451) at 1:1,000, mouse α-γ-tubulin (Sigma;

T5326) at 1:500, rabbit α-GFP (Life Technologies; A11122) at 1:500, mouse α-GFP (Life

Technologies; A11120) at 1:50, rabbit α-PCM1 (Santa Cruz Biotechnology; sc-67204) at

1:1,000, rabbit α-PCM1 (a gift from A. Merdes, Université de Toulouse, Toulouse, France) at

1:100,000, rabbit α-FLAG (Sigma; F7425) at 1:100, rabbit α-CEP164 (Sigma; SAB3500022) at

1:200, rabbit α -ninein (a gift from J. E. Sillibourne, Institut Curie, Paris) at 1:5,000, rabbit α-

Cp110 (Proteintech; 12780-1-AP) at 1:300, rabbit α-Ift88 (Jia et al. 2009) at 1:10,000, mouse α-β- tubulin (Sigma; T4026) at 1:200, rabbit α-Ofd1 at 1:100 (Singla et al. 2010), goat α-Cep164 (a gift from J. F. Reiter, University of California, San Francisco) at 1:500, mouse α-Centrin

(Millipore) at 1:50, mouse α-Arl13b at 1:1000 (Caspary et al. 2007) and rabbit α-pericentrin

(Abcam; ab4448) at 1:500. An antibody against mouse C2cd3 was generated by immunizing rabbits with a 400-aa peptide corresponding to the N terminus of mouse C2cd3 protein, and was used at 1:1,000.

63 2.2.4 Transmission electron microscopy (TEM)

Embryonic day 9.5 mouse embryos were dissected in fresh fixative [2mM CaCl2/2%

(wt/vol) paraformaldehyde/2.5% (wt/wt) glutaraldehyde/0.1Mcacodylate buffer, pH 7.4], prefixed in fixative at 4 °C overnight and rinsed with 0.1 M cacodylate buffer. The embryos were then postfixed in 1% OsO4/0.1 M cacodylate buffer for 1 h at 4 °C in the dark and rinsed with 0.1 M cacodylate buffer. Samples were en bloc stained with 2% (wt/vol) aqueous uranyl acetate for 1 h at room temperature in the dark, rinsed with water, dehydrated in ethanol and acetonitrile, infiltrated, and embedded in Eponite 12. Ultrathin sections (80 nm) were cut with a Leica UC6 ultramicrotome (Leica Microsystems), stained with uranyl acetate and lead citrate, and imaged with a JEOL 1200 transmission electron microscope.

2.3 Biochemistry

2.3.1 Western blot

Protein samples were extracted from cultured cells using 1% Triton lysis buffer. Proteins were separated by SDS-PAGE and immunobloted with standard procedure.

2.3.2 Co-immunoprecipitation (Co-IP)

Coimmunoprecipitation between overexpressed GFP- and FLAG-tagged proteins in

HEK293T cells was performed using a FLAG immunoprecipitation kit (Sigma) according to the manufacturer’s instructions.

64 2.3.3 Antibodies for Western Blot and Coimmunoprecipitation.

Mouse α-FLAG (Sigma; F1804) at 1:2,000, rabbit α-GFP (Life Technologies; A11122)

at 1:2,000, rabbit α-Ift88 (Jia et al. 2009) at 1:50,000, mouse α-Centrin (Millipore) at 1:50 and mouse α-β-tubulin (Sigma; T4026) at 1:5,000 were used.

2.4 References

Caspary, T., Larkins, C. E., & Anderson, K. V. (2007). The Graded Response to Sonic Hedgehog Depends on Cilia Architecture. Developmental Cell, 12, 767–778. doi:10.1016/j.devcel.2007.03.004

Chen, J. K., Taipale, J., Cooper, M. K., & Beachy, P. A. (2002). Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes and Development, 16, 2743–2748. doi:10.1101/gad.1025302

Dammermann, A., & Merdes, A. (2002). Assembly of centrosomal proteins and microtubule organization depends on PCM-1. The Journal of Cell Biology, 159(2), 255–66. doi:10.1083/jcb.200204023

Jia, J., Kolterud, A., Zeng, H., Hoover, A., Teglund, S., Toftgård, R., & Liu, A. (2009). Suppressor of Fused inhibits mammalian Hedgehog signaling in the absence of cilia. Developmental Biology, 330(2), 452–60. doi:10.1016/j.ydbio.2009.04.009

Singla, V., Romaguera-Ros, M., Garcia-Verdugo, J. M., & Reiter, J. F. (2010). Ofd1, a human disease gene, regulates the length and distal structure of centrioles. Developmental Cell, 18(3), 410–24. doi:10.1016/j.devcel.2009.12.022

Valente, E. M., Silhavy, J. L., Brancati, F., Barrano, G., Krishnaswami, S. R., Castori, M., … Gleeson, J. G. (2006). Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nature Genetics, 38, 623–625. doi:10.1038/ng1805

Ye, X., Zeng, H., Ning, G., Reiter, J. F., & Liu, A. (2014). C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals. Proceedings of the National Academy of Sciences of the United States of America, 111(6), 2164–9. doi:10.1073/pnas.1318737111

Chapter 3

C2cd3 is localized to centriolar satellites but dispensable for centriolar satellite integrity

Part of this work (3.2.2-3.2.6) has been published in PNAS (Ye et al. 2014).

3.1 Introduction

It has been more than a century since the primary cilium was first observed, but how cilia are initiated, assembled and maintained is still largely unknown. The revelation of its involvement in human diseases has attracted more and more scientists to study this tiny organelle.

ENU mutagenesis in mouse, aimed at screening genes involved in mouse embryonic patterning and morphogenesis, serendipitously identified many genes related to cilia (García-García et al.

2005; Zohn et al. 2005). This not only suggests that cilia are critical for embryonic development but also indicates that ENU mutagenesis screening is a powerful tool to find novel ciliogenic genes.

Previously our lab discovered an ENU-induced mutant mouse line called Hearty (Hty)

(Hoover et al. 2008). Hty homozygous mutant mice die between E11 and E13, and exhibit defects in neural tube development, left-right asymmetry, limb patterning and pericardial edema, consistent with the phenotypes of other cilia mutants. Hty was identified as a mutant allele of

C2cd3 with a G to A mutation that causes abnormal splicing (Fig. 3-1A). A second C2cd3 gene- trap (C2cd3GT) mutant allele was generated by the insertion of LacZ in the third intron (Fig. 3-

1A). C2cd3GT homozygous mice exhibit similar but more severe phenotype compared to Hty mutants. Electron micrographs showed severe ciliogenesis defects at the embryonic node of Hty

66 and C2cd3GT mutants, consistent with the immunofluorescence staining of C2cd3Hty primary cells.

GFP-tagged C2cd3 is localized around the centrosome/basal body of cilia suggesting its involvement in ciliogenesis. C2cd3 homologues were also found in other vertebrates including

human, chicken and Xenopus (Fig. 3-1 B). In addition, a remote homologue with low similarity

may also exist in Drosophila.

A recent study reported that C2cd3 mutations were involved in a new subtype of Oral-

Facial-Digital syndrome, a ciliopathy (Thauvin-Robinet et al. 2014). This suggests that studying

C2cd3 function in cilia formation is crucial for understanding and even treating its related human

genetic diseases. My goal is to fill the knowledge gap between C2cd3 and cilia formation. In this

chapter, I first confirmed the cilia defects in C2cd3GT homozygous mutant embryos. In order to study the molecular function of C2cd3, I started with examining the subcellualr localization of

C2cd3. I discovered that one group of C2cd3 was localized around the centrosome and the other group was localized to centrosome. In this chapter, I focused on C2cd3 population around the centrosome and I found that C2cd3 was associated with centriolar satellites. I also demonstrated that C2cd3 physically interacted with Pcm1 through its C-terminus and this interaction was essential for its satellite localization. However, C2cd3 is not required for the localization of other satellite compoenents and it is dispensable for many known functions of centriolar satellites, including Rab8 ciliary localization, microtubule arrangement and cell cycle progression

(Tollenaere et al. 2014). Surprisingly, satellite localization of Centrins depends on C2cd3, suggesting the possible role of C2cd3 in centriolar satellite-mediated trafficking.

67

Figure 3-1. C2cd3 gene information.

A. Schematics of wildtype C2cd3, C2cd3Hty and C2cd3GT alleles and their corresponding protein products. B. Gene tree of C2cd3 (from www.ensembl.org) shows the sequence similarity among different species.

68

69 3.2 Results

3.2.1 C2cd3GT homozygous mutants exhibit ciliogenesis defects in most tissues

Previous studies from our lab revealed severe ciliogenesis defects at the node in C2cd3GT

mutants (Hoover et al. 2008). The node is a transient cavity at the end of the notochord during embryonic development. Using Arl13b as a cilia marker, my immunofluorescent stainings of embryo sections and primary cells indicated that less than 10% of C2cd3GT mutant cells were able to generate cilia, compared to around 20% of C2cd3Hty cells (Fig 3-2B and C and Hoover et al.,

2008). According to transcript analysis and severity of developmental defects, C2cd3Hty was considered as a hypomorphic mutant allele and C2cd3GT was considered as a null allele. Most tissues in C2cd3GT mutants had severe ciliogenesis defects, such as the node (Hoover et al.,

2008), spinal cord and limb buds (Fig 3-2A). However, one notable exception was the gut of mutant embryos, which grow cilia normally (Fig 3-3). The tissue-specific ciliogenesis defects in

C2cd3GT mutants highlighted the divergence of ciliogenic machinery in different tissues.

70

Figure 3-2. C2cd3GT mutant embryos and MEFs show severe ciliogenesis defects.

A. In wildtype littermates, cilia (green, marked by Arl13b) are assembled near the centrosomes/basal bodies (red, marked by γ-tubulin) in the neural epithelium of neural tube and in the mesenchymal cells of limb buds. In contrast, mutant embryos exhibited near-complete loss of cilia in these tissues. Nucleus is visualized in blue by DAPI staining. B. Only 6.9% of cells bear cilia in mutant embryos, compared to near 100% in wildtype. Ciliation rate is calculated by number of cilia over number of centrosome in each picture of limb bud tissue. C. Only 2.9% of mutant cells assemble cilia, compared to 77.7% in wildtype. Ciliation rate is calculated by number of cilia over number of nucleus in MEFs. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

71

Figure 3-3. C2cd3GT mutant embryos exhibit normal ciliogenesis in the gut.

Cilia are formed normally in the guts of mutant embryos (A and dashline area with higher power shown in C) compared to wildtype littermates (B and dashline area with higher power shown in D), although cilia defects are prominent in other tissues, such as the neural tube and limb buds. Cilia are marked by Arl13b in green and centrosomes/basal bodies are marked by γ-tubulin in red. Nucleus is visualized in blue by DAPI staining.

72 3.2.2 Dynamic C2cd3 protein localizations throughout the cell cycle

In order to investigate the molecular mechanisms by which C2cd3 regulates ciliogenesis,

I examined the subcellular localization of this protein. I generated C2cd3 mutant and wildtype mouse embryonic fibroblast (MEF) cell lines from C2cd3GT mutant embryos and their littermates

for the cell biology studies. These MEF cells not only faithfully recapitulated the cilia defects in

mutant embryos, but also offered an easier way to culture, stain and image the samples and a

versatile platform for different treatments. A polyclonal antibody raised against a N-terminal peptide of C2cd3 revealed punctate staining around centrosomes marked by γ-tubulin staining in interphase mouse embryonic fibroblasts (MEFs) (Fig 3-4A). Interestingly, during mitosis, C2cd3 punctae were dispersed while leaving only two dots at each spindle pole marked by γ-tubulin (Fig

3-4A). No staining was detected in C2cd3GT mutant cells but similar staining pattern reappeared after transfecting the mutant cells with GFP-C2cd3, confirming the specificity of this antibody

(Fig 3-4A). Overexpressed GFP-C2cd3 in wildtype MEFs exhibits similar dynamic localization

(Fig 3-4B). However, GFP-C2cd3 occasionally accumulated in PCM or even more peripheral area at the spindle poles, which could be overexpression artifact (Fig 3-4B).

The punctate staining of C2cd3 around the centrosome is reminiscent of centriolar satellites, the nonmembranous electron-dense granule structures surrounding the centrosomes. In addition, the dispersal of C2cd3 punctae during mitosis is consistent with the cell cycle-dependent nature of centriolar satellites. The C2cd3 staining of two dots inside each spindle pole during mitosis suggests potential centriolar localization of C2cd3, which is discussed in Chapter 4.

73

Figure 3-4. Endogenous C2cd3 and GFPC2cd3 exhibit dynamic subcellular localization throughout cell cycle.

A. Endogenous C2cd3 (red) forms punctae around centrosome (green, marked by γ-tubulin) in wildtype MEFs during interphase. Two fine dots of C2cd3 at each spindle pole are revealed after the punctae are dispersed during mitosis. No red staining iss observed in C2cd3 mutant cells, but similar staining pattern reappeared after GFPC2cd3 overexpression, suggesting the specificity of C2cd3 antibody. B. GFPC2cd3 mimics the dynamic localization of endogenous C2cd3 in wildtype MEFs. However, sometimes two fine dots at each spindle pole were less prominent, possibly due to overexpression artifact.

74 3.2.3 C2cd3 punctate staining relies on microtubule-dependent retrograde transport

A previous study revealed that the recruitment of centriolar satellites to the centrosomes required dynein-mediated retrograde transport along the microtubules (Dammermann & Merdes

2002). In order to test whether C2cd3 is associated with centriolar satellites, I first inhibited microtubule polymerization by treating cells with nocodazole. The effect of nocodazole was confirmed by β-tubulin staining. Compared to intact microtubule network in DMSO-treated cells, no filament staining was observed in nocodazole-treated cells (Fig. 3-5B). C2cd3 punctae around the centrosome disappeared after nocodazole treatment, suggesting C2cd3 punctae are microtubule-dependent (Fig. 3-5A). Interestingly, the C2cd3 staining of two fine dots at the centrosome remained after the disappearance of punctae staining upon nocodazole treatment, supporting the idea that C2cd3 has a secondary localization on centrioles (Fig. 3-5A).

Dynactin (Dynein activator complex) is a multi-subunit protein complex required for bridging cargos and the molecular motor dynein. p50 (Dynactin2 or Dynamitin) is a 50-KD subunit of dynactin. Overexpression of p50 displaces other dynactin subunits and abolishes dynactin function, which in turn disrupt the retrograde transport (Schroer 2004). GFP-p50 overexpression disrupted C2cd3 punctae in a dose-dependent manner (Fig. 3-5C). High levels of

GFP-p50 overexpression greatly reduced C2cd3 satellite localization around the centrosome, but not its centriolar localization. Both nocodazole treatment and p50 overexpression experiments proved that C2cd3 punctae around the centrosome rely on microtubule-dependent retrograde transport.

75

Figure 3-5. C2cd3 punctae depends on dynein-mediated microtubule-dependent retrograde transport.

A. C2cd3 punctae are dispersed upon nocodazole treatment, revealing its fine dots at the centrosomes. DMSO-treated cells served as a control. B. Microtubule organization is disrupted in nocodazole-treated cells, compared to DMSO-treated control cells. C. C2cd3 punctae are also susceptible to the overexpression of p50/Dynamitin in a dose-dependent manner. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001). ANOVA p-value is calculated by F test and p<0.001 for p50 overexpression experiment.

76 3.2.4 C2cd3 colocalizes and physically interacts with known centriolar satellites components.

In order to pinpoint the localization of C2cd3 punctae around the centrosome, I co-

stained C2cd3 with Pcm1 and Bbs4, two known centriolar satellite components (Dammermann &

Merdes 2002; Kim et al. 2004). The immunoflourescence staining pattern of C2cd3 overlapped with Pcm1 and Bbs4, indicating that C2cd3 is localized to centriolar satellites (Fig. 3-6A). Pcm1 is the first known centriolar satellites component and it is required for the integrity of centriolar satellites. Depletion of Pcm1 causes misplacements of other centriolar satellites components

(Dammermann & Merdes 2002). To test whether C2cd3 centriolar satellite localization relies on

Pcm1, Pcm1 was knocked down by siRNAs (small interference RNAs) and shRNAs (small hairpin RNAs) targeting different regions of Pcm1 mRNA (Fig. 3-6B and C). When Pcm1 was knocked down, C2cd3 failed to be recruited around the centrosome, but the C2cd3 localization associated with the centrosome was unaffected. Coimmunoprecipitation analysis of overexpressed proteins in HEK293T cells indicated that C2cd3 physically interacts with Pcm1 and Bbs4. All together, C2cd3 localizes to centriolar satellites and associates with other centriolar satellites components, suggesting that C2cd3 is a bona fide centriolar satellite component.

77

Figure 3-6. C2cd3 colocalizes and physically interacts with Pcm1 and Bbs4.

A C2cd3 overlaps with Pcm1 and Bbs4 in immunefluorescence staining. B and C. C2cd3 centriolar satellite localization is disrupted in cells treated with Pcm1 siRNA or shRNA. The lack of Pcm1 staining indicates the effectiveness of the knockdown. Both Pcm1 and C2cd3 are localized to centriolar satellites in the control cells treated with a scramble siRNA. D. Overexpressed C2cd3 coimmunoprecipitates with Pcm1 and Bbs4. For quantitative analyses, SD is indicated. n = 3 independent experiments. IP, immunoprecipitation; WB, Western blot.

78 3.2.5 C2cd3 is targeted to centriolar satellites through the interaction between its C- terminus and Pcm1

To investigate the functional significance of the interaction between C2cd3 and Pcm1, I

mapped the regions on C2cd3 required for this interaction. The protein domains on C2cd3 are

largely unknown except five C2 domains predicted by SMART in the central region

(http://smart.embl-heidelberg.de/). I divided this protein into three regions, namely the N- terminus, the C2 central region and the C-terminus. I found that the C-terminus of C2cd3 was required for both its centriolar satellite localization and interaction with Pcm1, suggesting C2cd3 might be recruited to centriolar satellites through its interaction with Pcm1 (Fig. 3-7A). In contrast, the deletion of N-terminus (C2cd3dN1 and C2cd3dN2) did not affect the satellite recruitment of C2cd3 (Fig. 3-7B and C). Removal of both the N-terminus and the C2 region

(C2cd3E1 and C2cd3CT) greatly reduced, but did not abolish, its satellite recruitment (Fig. 3-7B and C). All these truncations, including C2cd3dN1, C2cd3dN2, C2cd3E1 and C2cd3CT, were able to bind Pcm1 in co-immunoprecipitation experiments (Fig. 3-7D). Interestingly, deletion of the C-terminus completely abolished both C2cd3 satellite localization and its interaction with

Pcm1 (Fig. 3-7B, C and D). Taken together, both the C-terminus and C2 region facilitates the satellite recruitment of C2cd3, and the C-terminus is required for its interaction with Pcm1.

Consistent with the idea that C2cd3 interacts with Pcm1 through its C-terminus, I found that high levels of C2cd3, but not C2cd3dC, was able to misplace endogenous Pcm1 (Fig. 3-8).

This suggests that the overexpression C2cd3 leads to misplacement of Pcm1 possibly through their interaction; without the ability to bind Pcm1, overexpression of C2cd3dC does not affect

Pcm1 localization.

79

Figure 3-7. C2cd3 centriolar satellite localization depends on the interaction between its C- terminus and Pcm1.

A. The schematic represents wildtype full-length C2cd3 protein and its truncation variants. On the right summarized their centriolar satellite localization and physical interaction with PCM1. “- ” indicates negative, “+” indicates weak, “++” indicates medium, “+++” indicates strong. B. Localization of GFP-tagged full-length C2cd3 and its truncation variants are illustrated in green. C. The bar chart shows the quantitative summary on percentage of transfected cells with GFP centriolar satellite staining. D. Physical interactions between FlagPcm1 and GFP-tagged C2cd3 variants are analyzed by coimmunoprecipitation experiments in HEK293T cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01, ***p<0.001 and n.s. means not significant). In C, all C2cd3 variants were compared to C2cd3 wildtype.

80

Figure 3-8. Overexpression of full-length C2cd3, but not C2cd3dC, misplaces endogenous Pcm1.

A. Overexpression of C2cd3 misplaces endogenous Pcm1 in a dose-dependent manner. B. The bar chart shows the quantitative summary of Pcm1 centriolar satellite localizations in cells with different C2cd3 or C2cd3dC expression levels.

81 3.2.6 C2cd3 is dispensable for centriolar satellite integrity, Rab8 ciliary localization, microtubule arrangement and cell cycle progression.

To probe the function of C2cd3 at centriolar satellite population, I tested various aspects of known functions of centriolar satellites. We used C2cd3GT cells to study the function of C2cd3 because mutant cells have consistent and complete depletion of C2cd3 comparing to cells treated by RNA interference. First, I investigated the requirement of C2cd3 for centriolar satellite integrity. It was previously found that many centriolar satellites components, such as Pcm1, Bbs4

and Cep290, were important for centriolar satellite integrity and subcellular localizations of other

components (Dammermann & Merdes 2002; Kim et al. 2004; Kim et al. 2008). However,

C2cd3GT mutant cells exhibited normal centriolar satellites marked by Pcm1, Bbs4, Cep290 and

Ofd1, suggesting that C2cd3 is dispensable for centriolar satellites integrity (Fig. 3-9). Second, some centriolar satellite components, such as Pcm1 and Cep290, were involved in the ciliary recruitment of Rab8 (Kobayashi et al. 2014; Kim et al. 2008). By using the hypomorphic

C2cd3Hty mutant cells that exhibit a slightly higher ciliation rate comparing to C2cd3GT, I found

no significant difference in Rab8 ciliary recruitment rate between control and C2cd3Hty mutant cells (Fig. 3-10). Third, centriolar satellites are involved in microtubule disorganization and

recruitment of some centrosomal proteins, including Centrin, Ninein and Pericentrin

(Dammermann & Merdes 2002). Disruption of centriolar satellites by Pcm1 knockdown also causes fragmentation or loss of the centrosome, which further leads to ciliogenesis defects and cell cycle arrest (Srsen et al. 2006; Mikule et al. 2007). Therefore, I examined the localization of some centrosomal proteins, microtubule organization and spindle pole integrity. Pericentrin and

γ-tubulin staining appeared normal in C2cd3 mutant cells (Fig. 3-9 and Fig. 3-11A). Microtubules were organized and emanated from centrosomes in the absence of C2cd3 (Fig. 3-11A). Moreover, cell cycle progression and spindle apparatus appeared to be normal in the mutant cells (Fig. 3-

11A). Ki-67 is a nuclear protein associated with cellular proliferation. Using Ki-67 as a marker

82 for cells in active cell cycle, I did not identify any difference between control and mutant cells

(Fig. 3-11B). All together, it seems that C2cd3 does not function as a core centriolar satellite component and is dispensable for centriolar satellites integrity, Rab8 ciliary recruitment, microtubule arrangement or some centrosomal protein recruitments.

Figure 3-9. C2cd3 is dispensable for centriolar satellite integrity.

Centriolar satellite components, including Pcm1 (A), Bbs4 (B), Cep290 (C) and Ofd1 (D and E) are normally recruited in C2cd3 mutant cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (n.s. means not significant).

83

Figure 3-10. Loss of C2cd3 does not affect Rab8 ciliary localization.

A and B. No difference on Rab8 ciliary localization is found between C2cd3 mutant cells and wildtype cells. p-value is calculated by chi-square (n.s. means not significant). For GFPRab8 ciliary localization, p-value calculated by chi-sqaure test is 0.88>0.05, not significant.

84

Figure 3-11. C2cd3 is not required for microtubule organization and cell cycle progression.

A. Microtubules and spindle pole apparatus are normally assembled in C2cd3 mutant cells. Microtubules are marked by β-tubulin in red and centrosomes are marked by pericentrin in green. B. The percentage of cells in active cell cycle, marked by Ki-67, in C2cd3 mutant cells is similar to in control cells. However, C2cd3 mutant cells exhibit dramatic ciliogenesis defects. p-value is calculated by chi-square ((*p<0.05, **p<0.01, ***p<0.001 and n.s. means not significant). For Ki67, p-value calculated by chi-sqaure test is >0.05, not significant. For cilia, p-value calculated by chi-sqaure test is <0.001, significant.

85 3.2.7 C2cd3 is essential for Centrin centriolar satellite recruitment

Besides at the centrioles, Centrins are also found at pericentriolar matrix and centriolar

satellites (Salisbury 2007; Dammermann & Merdes 2002). Centrin2 is colocalized with Pcm1 at

centriolar satellites and physically interacts with Pcm1 (Dammermann & Merdes 2002). Studies

showed that when centrosome overduplication was triggered by S-phase arresting or DNA

damage, Centrin granules were exported from the nucleus to the cytoplasm and colocalized with

Pcm1 at centriolar satellites to facilitate centrosome amplification (Prosser et al. 2009; Löffler et

al. 2012). This suggests that Centrins at centriolar satellites and PCM might serve as a part of the

potent environment for centrosome duplication.

Consistent with previous studies, I found that overexpressed GFP-Centrin1-3 were

localized not only to the centrioles but also around the centrosome marked by γ-tubulin (Fig. 3-

12A). These punctae were further identified as centriolar satellites because of the overlapping

between GFPCentrin2 and Pcm1 staining (Fig. 3-12C). The satellite localizations of

GFPCentrin1-3 were greatly enhanced in wildtype cells after serum starvation, consistent with the

fact that serum starvation induces centriolar satellites accumulation. (Fig. 3-12E). The

overexpressed GFPCentrin1 granules aligned in straight lines toward satellites, supporting the

idea that GFPCentrin1 is recruited to centriolar satellites through microtubule-mediated transport

(Fig. 3-12B).

Interestingly, I found that centriolar satellite localizations of Centrin were greatly reduced

in C2cd3 mutant cells. Compared to wildtype cells, overexpressed GFPCentrin1-3 failed to be

recruited to centriolar satellites in C2cd3 mutant cells (Fig. 3-12A and C). The difference between

wildtype and mutant cells became more significant after serum starvation (Fig. 3-12E). Similar

phenotype was observed when visualizing endogenous Centrin (Fig. 3-12D). This suggests that centriolar satellite recruitment of Centrin depends on C2cd3.

86

Figure 3-12. C2cd3 is required for the centriolar satellite recruitment of Centrin.

The satellite localization of overexpressed GFPCentrin1-3 (A) and endogenous Centrin (D) is disrupted in C2cd3 mutant cells. B. GFPCentrin1 dots are aligned in straight lines towards centrosome, implying that it is recruited along microtubules. Dash line area on the left is shown in a higher power on the right. C. GFPCentrin2 colocalizes with Pcm1 in wildtype cells, but not in C2cd3 mutant cells. E. Quantitative summary of satellite localization of GFPCentrin1-3 and endogenous Centrin in wildtype cells and C2cd3 mutant cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

87 3.3 Discussion

3.3.1 C2cd3 is required for ciliogenesis in mouse embryonic development in a tissue-specific manner.

Our lab previously found that C2cd3 was essential for ciliogenesis and Shh signaling

during mouse embryonic development. Ciliogenesis defects were observed in the nodes of both

C2cd3Hty and C2cd3GT mutant embryos by scanning electron microscopy and in primary cells carrying C2cd3Hty mutations by immunofluorescence. Here I characterized the global requirement

of C2cd3for ciliogenesis in various tissues during mouse development, including the spinal cord,

limb buds, somites and mesenchyme cells around the neural tube. Interestingly, close-to-normal ciliogenesis was found in early gut tissues of mutant embryos, highlighting the divergence of ciliogenic mechanisms in different tissues (Fig. 3-2 and Fig. 3-3). The tissue-specific cilia phenotype is not unique in C2cd3GT mutants and has been noted in many other cilia mutants.

Centrin 1, Azi1/Cep131, BBS2 and BBS4 knockout mice specifically exhibit spermatozoa flagella defects leading to male infertility (Mykytyn et al. 2004; Hall et al. 2013; Avasthi et al.

2013; Nishimura et al. 2004). It was also reported that depletion of Tctn1, a transition zone protein, led to the absence of cilia in the node, neural tube, mesenchymal cells surrounding neural tube and those in limb buds, but leaving cilia unaffected in nodal cord and early gut epithelium

(Garcia-Gonzalo et al. 2011). These results suggested that it is critical to study ciliogenesis in multiple cell types and mouse models in order to obtain a thorough understanding of the molecular mechanisms of ciliogenesis in different tissues.

88 3.3.2 C2cd3 is a centriolar satellite component

The punctate staining of C2cd3 around centrosome during interphase disappeared upon mitosis entry, consistent with the dynamic localization of centriolar satellites through the cell cycle (Fig. 3-4). I further demonstrated that C2cd3 punctae were dependent on dynein-driven retrograde transport along the microtubules and Pcm1 (Fig. 3-5 and Fig. 3-6). In addition, C2cd3 colocalizes and physically interacts with Pcm1 and Bbs4, two centriolar satellite markers (Fig. 3-

6). In summary, C2cd3 is a centriolar satellite component.

By mapping the Pcm1-interacting domain on C2cd3, I found that N-terminus of C2cd3 is

dispensable for neither its satellite localization nor its interaction with Pcm1. Deletion of the C- terminus of C2cd3 abolished its interaction with Pcm1 and its satellite localizations. Additionally, high-levels of full-length C2cd3, but not C2cd3dC, led to misplacement of Pcm1, supporting that

C2cd3 interacts with Pcm1 through its C-terminus. The C-terminus of C2cd3 alone bound to

Pcm1 efficiently but was not efficiently targeted to centriolar satellites. Adding back the entire or part of the C2 region greatly improved its centriolar satellite targeting. This suggests that binding to Pcm1 through the C-terminus of C2cd3 is required but not sufficient for C2cd3 satellite recruitment. C2cd3 satellite targeting may require not only the interaction with Pcm1 through its

C-terminus, but also other mechanisms through its C2 region. It will be interesting to investigate whether C2 region also binds to Pcm1 since it also contributes to C2cd3 satellite localization.

Identifying additional proteins interacting with C2 region will be critical for investigating the unknown mechanisms.

89 3.3.3 C2cd3 is dispensable for typical centriolar satellite functions

Although centriolar satellites were first described a long time ago, their functions remain

elusive. Currently centriolar satellites are known to play roles in Rab8 ciliary localization,

microtubule arrangement and cell cycle progression (Tollenaere et al. 2014). Unfortunately

C2cd3 is not required for any of them. Unaffected centriolar satellite structures in the absence of

C2cd3 imply that C2cd3 may be a peripheral component (Fig. 3-9). Surprisingly, satellite localizations of Centrins are abolished by loss of C2cd3, implying the possible role of C2cd3 in centriolar satellite-mediated trafficking.

3.4 References

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Dammermann, A., & Merdes, A. (2002). Assembly of centrosomal proteins and microtubule organization depends on PCM-1. The Journal of Cell Biology, 159(2), 255–66. doi:10.1083/jcb.200204023

García-García, M. J., Eggenschwiler, J. T., Caspary, T., Alcorn, H. L., Wyler, M. R., Huangfu, D., … Anderson, K. V. (2005). Analysis of mouse embryonic patterning and morphogenesis by forward genetics. Proceedings of the National Academy of Sciences of the United States of America, 102, 5913–5919. doi:10.1073/pnas.0501071102

Garcia-Gonzalo, F. R., Corbit, K. C., Sirerol-Piquer, M. S., Ramaswami, G., Otto, E. A., Noriega, T. R., … Reiter, J. F. (2011). A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nature Genetics, 43, 776–784. doi:10.1038/ng.891

Hall, E. A., Keighren, M., Ford, M. J., Davey, T., Jarman, A. P., Smith, L. B., … Mill, P. (2013). Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes. PLoS Genetics, 9. doi:10.1371/journal.pgen.1003928

Hoover, A. N., Wynkoop, A., Zeng, H., Jia, J., Niswander, L. a, & Liu, A. (2008). C2cd3 is required for cilia formation and Hedgehog signaling in mouse. Development (Cambridge, England), 135(24), 4049–58. doi:10.1242/dev.029835

90 Kim, J. C., Badano, J. L., Sibold, S., Esmail, M. a, Hill, J., Hoskins, B. E., … Beales, P. L. (2004). The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression. Nature Genetics, 36(5), 462–70. doi:10.1038/ng1352

Kim, J., Krishnaswami, S. R., & Gleeson, J. G. (2008). CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium. Human Molecular Genetics, 17(23), 3796–805. doi:10.1093/hmg/ddn277

Kobayashi, T., Kim, S., Lin, Y.-C., Inoue, T., & Dynlacht, B. D. (2014). The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly. The Journal of Cell Biology, 204(2), 215–29. doi:10.1083/jcb.201304153

Löffler, H., Fechter, A., Liu, F. Y., Poppelreuther, S., & Krämer, A. (2012). DNA damage- induced centrosome amplification occurs via excessive formation of centriolar satellites. Oncogene. doi:10.1038/onc.2012.310

Mikule, K., Delaval, B., Kaldis, P., Jurcyzk, A., Hergert, P., & Doxsey, S. (2007). Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest. Nature Cell Biology, 9, 160–170. doi:10.1038/ncb1529

Mykytyn, K., Mullins, R. F., Andrews, M., Chiang, A. P., Swiderski, R. E., Yang, B., … Sheffield, V. C. (2004). Bardet-Biedl syndrome type (BBS4)-null mice implicate Bbs4 in flagella formation but not global cilia assembly. PNAS, 101(23).

Nishimura, D. Y., Fath, M., Mullins, R. F., Searby, C., Andrews, M., Davis, R., … Sheffield, V. C. (2004). Bbs2-null mice have neurosensory deficits, a defect in social dominance, and retinopathy associated with mislocalization of rhodopsin. Proceedings of the National Academy of Sciences of the United States of America, 101, 16588–16593. doi:10.1073/pnas.0405496101

Prosser, S. L., Straatman, K. R., & Fry, A. M. (2009). Molecular dissection of the centrosome overduplication pathway in S-phase-arrested cells. Molecular and Cellular Biology, 29, 1760–1773. doi:10.1128/MCB.01124-08

Salisbury, J. L. (2007). A mechanistic view on the evolutionary origin for centrin-based control of centriole duplication. Journal of Cellular Physiology. doi:10.1002/jcp.21226

Schroer, T. A. (2004). Dynactin. Annual Review of Cell and Developmental Biology, 20, 759–779. doi:10.1146/annurev.cellbio.20.012103.094623

Srsen, V., Gnadt, N., Dammermann, A., & Merdes, A. (2006). Inhibition of centrosome protein assembly leads to p53-dependent exit from the cell cycle. Journal of Cell Biology, 174, 625–630. doi:10.1083/jcb.200606051

Thauvin-Robinet, C., Lee, J. S., Lopez, E., Herranz-Pérez, V., Shida, T., Franco, B., … Nachury, M. V. (2014). The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation. Nature Genetics, 46, 905–911. doi:10.1038/ng.3031

91 Tollenaere, M. a X., Mailand, N., & Bekker-Jensen, S. (2014). Centriolar satellites: key mediators of centrosome functions. Cellular and Molecular Life Sciences : CMLS. doi:10.1007/s00018-014-1711-3

Ye, X., Zeng, H., Ning, G., Reiter, J. F., & Liu, A. (2014). C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals. Proceedings of the National Academy of Sciences of the United States of America, 111(6), 2164–9. doi:10.1073/pnas.1318737111

Zohn, I. E., Anderson, K. V., & Niswander, L. (2005). Using genomewide mutagenesis screens to identify the genes required for neural tube closure in the mouse. Birth Defects Research Part A - Clinical and Molecular Teratology. doi:10.1002/bdra.20164

Chapter 4

C2cd3 is localized to distal end of centrioles and critical for the distal structure assembly

Part of this work (4.2.1-4.2.4) has been published in PNAS (Ye et al. 2014).

4.1 Introduction

As the root of the primary cilium, the basal body/mother centriole plays a critical role in ciliogenesis. Distinct from the daughter centriole, the mother centriole bears DAPs and subdistal

SAPs, both of which are installed on the outside of the distal microtubule barrel. As the mother centriole becomes the basal body, the DAPs and SAPs develop into the transition fibers and basal feet, respectively.

As mentioned in chapter 1, transition fibers are considered as part of the ciliary gate in the mature cilium. In addition, previous studies indicated that DAPs, the precursors of the transition fibers, were also critical for the initiation of ciliogenesis by facilitating the mother centriole docking to the membrane (Schmidt et al. 2012; Tanos et al. 2013; Joo et al. 2013;

Sillibourne et al. 2013). DAPs contain at least five components, namely Cep164, Fbf1, Sclt1,

Cep83/Ccdc41 and Cep89/Ccdc123/Cep123(Tanos et al. 2013). These components are recruited in a hierarchical manner: Cep83 is required to recruit Cep89 and Sclt1; Sclt1 is essential for

Cep164 and Fbf1 localization. Cep164 has multiple independent functions in ciliogenesis. First,

Cep164 helps the mother centriole to dock to the ciliary vesicle by physically interacting with

Rabin8 and Rab8 through its C-terminus (Schmidt et al. 2012). Other components, including

93 Cep83, Ccdc41 and Cep123, are also required for ciliary vesicle docking, possibly through their

effects on Cep164 recruitment (Tanos et al. 2013; Joo et al. 2013; Sillibourne et al. 2013).

In addition, Cep164 is required for recruiting Tau Tubulin Kinase 2 (Ttbk2) and IFT

proteins to the mother centriole and removing Centriolar Coiled coil protein 110 (Ccp110) from

the mother centriole. Ccp110, in conjunction with its binding partner Cep97 and Kif24,

negatively regulates ciliogenesis (Spektor et al. 2007; Tsang et al. 2008; Schmidt et al. 2009;

Kobayashi et al. 2011). Both Ccp110 and Cep97 are localized to the distal ends of the centrioles.

They disappear from the mother centriole during ciliogenesis. Their interacting protein Kif24 also

inhibits ciliogenesis by depolymerizing centriolar microtubules. Ttbk2 is specifically recruited to

the distal end of the the mother centriole during ciliogenesis and is essential for Ccp110 removal

and Ift protein centriolar recruitment (Goetz et al. 2012). Cep164 is a substrate of Ttbk2 and the

N-terminus of Cep164 is essential for Ttbk2 recruitment through physical interaction (Cajánek &

Nigg 2014). Interestingly, overexpression of Ttbk2, but not its kinase dead form, promotes aberrant DAP assembly on the daughter centriole, highlighting the role of Ttbk2 in DAPs assembly. Other DAPs components, such as Cep83, have also been shown to be required for

Ttbk2 and Ift recruitment and Ccp110 removal, possibly through affecting Cep164 localization

(Tanos et al. 2013).

SAPs are essential for microtubule anchoring. Known subdistal appendage components include Ninein and Cep170. Knockdown of Ninein leads to loss of microtubule anchoring onto the centrosome (Dammermann & Merdes 2002). Overexpression or knockdown of Cep170 affects microtubule organization and cell morphology (Guarguaglini et al. 2005). However, no evidence shows that SAPs are directly involved in ciliogenesis.

Outer dense fiber of sperm tails 2 (Odf2, also known as Cenexin) is considered as a component for both DAPs and SAPs, and it is essential for both DAP and SAP assembly in F9 cells (a teratocarcinoma stem cell line) (Ishikawa et al. 2005). Later studies showed that different

94 domains of Odf2 were specifically responsible for DAP and SAP recruitment (Tateishi et al.

2013; Chang et al. 2013). Transgenic mice with exon 6 and exon 7 deletions on Odf2 grow cilia but exhibit disrupted alignment of basal bodies in ciliated epithelia due to the loss of basal feet, supporting the idea that SAPs are not essential for ciliogenesis (Kunimoto et al. 2012).

Currently, little is known about how DAPs and SAPs are recruited. Ofd1 is localized to the distal ends of the centrioles and is essential for ciliogenesis. Ofd1 is required for DAP assembly, Ift88 centriolar recruitment, and centriole length control (Singla et al. 2010). The centriolar protein Dzip1 is essential for both DAP and SAP assembly and Ift centriolar recruitment (C. Wang et al. 2013). Kif3a and p150Glued are required for SAP assembly and centriole cohesion, and Kif3a is essential for p150Glued centriolar recruitment independently of its motor function (Kodani et al. 2013).

In this Chapter, I focused on the centriolar localization of C2cd3. I found that C2cd3 was localized to distal ends of centrioles, and it was required for DAP and SAP assembly. The ciliogenic downstream events were affected in C2cd3 mutant cells, including Ttbk2 and Ift recruitment, Ccp110 removal and ciliary vesicle docking. In addition, I demonstrated that Centrin failed to be recruited to centrioles in the absence of C2cd3, suggesting disrupted distal structures of centrioles. Finally, I discovered that C2cd3 was required for the centriolar recruitment of Ofd1 and Dzip1, two other regulators for DAP and/or SAP assembly.

4.2 Results

4.2.1 C2cd3 is localized to the distal ends of centrioles

As mentioned in Chapter 3, besides centriolar satellites, C2cd3 has a second localization at the centrosome. This localization became prominent when the satellite population of C2cd3

95 was abolished. During mitosis, two fine dots were revealed at each spindle pole (Fig. 3-4). Fine dots of C2cd3 staining appeared at each centrosome marked by γ-tubulin when dynein-mediated microtubule-dependent transport was compromised by nocodazole treatment or overexpression of p50 (Fig. 3-5A and C), and when centriolar satellites were disrupted by Pcm1 knockdown (Fig. 3-

6C).

In order to pinpoint the exact localization of C2cd3 on the centrioles, I co-stained C2cd3 with other centriole markers. Cep164 is a DAP component decorating the distal rim of the centriolar cylinder. Depending on the observation angle, Cep164 staining can be ring-shaped or bar-shaped. C2cd3 appeared in the center of the ring or in the middle of the bar, suggesting that

C2cd3 is localized at the distal tips of centrioles (Fig. 4-1A). Ninein is localized to both SAPs and the proximal ends of both the mother and daughter centrioles. C2cd3 appeared on both mother centriole and daughter centriole, consistent with the observation of two dots at each spindle pole

(Fig. 4-2B). C2cd3 was more distal to Ninein, confirming that it was at the level of the DAPs.

96

Figure 4-1. C2cd3 is localized to the distal ends of centrioles.

A. C2cd3 is localized at the same level of Cep164, a distal appendage marker. C2cd3 staining is observed in the center of the ring-shaped Cep164 staining or in the middle of the bar-shaped Cep164 staining. B. C2cd3 is localized more distal to Ninein, a subdistal appendage marker. Schematics are shown on the right.

97 4.2.2 C2cd3 is required for the recruitment of DAPs and SAPs

As C2cd3 was localized to the distal ends of centrioles, I sought to investigate its role in

DAP and SAP assembly. The recruitment of endogenous Cep164 was abolished in the absence of

C2cd3 (Fig. 4-2A). The centriolar localization of overexpressed GFPCep164 was also disrupted

by C2cd3 depletion, suggesting that this misplacement was not due to the loss of Cep164 protein

(Fig. 4-2A). Overexpressed C2cd3 also physically interacted with Cep164 in HEK293T cells

(Fig. 4-2B). Other components of DAPs, including Sclt1, Ccdc41, Cep89 and Fbf1, were also lost

from centrioles in C2cd3 mutant cells, suggesting that C2cd3 is required for DAP assembly

(fig.4-3).

Ninein centriolar recruitment was also compromised but to a lesser extent. I found that only 40% of mutant cells exhibited subdistal localization of Ninein, compared to nearly 90% of wildtype cells (Fig. 4-2C). Notably, the intensity of Ninein staining in mutant cells was also weaker than in wildtype cells. Therefore, SAP recruitment is compromised in the absence of

C2cd3.

98

Figure 4-2. C2cd3 is essential for the recruitment of distal appendages and subdistal appendages.

A. The centriolar recruitment of both endogenous Cep164 and GFPCep164 is abolished in C2cd3 mutant cells. B. Overexpressed C2cd3 coimmunoprecipitates with Cep164 in HEK293 cells. C. The centriolar recruitment of Ninein is affected in C2cd3 mutant cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test or chi-square (*p<0.05, **p<0.01 and ***p<0.001).

99

Figure 4-3. Other distal appendage components fail to be recruited to the mother centriole in C2cd3 mutant cells.

The recruitment of other distal appendage components, including Sclt1 (A), Ccdc41 (B), Cep89 (C) and Fbf1 (D), relies on C2cd3. Wildtype and mutant MEFs were transfected with GFP-Sclt1, Ccdc41, Cep89, and Fbf1, which were stained in green. Centrosome marker, γ-tub, was stained in red. Nucleus marker DAPI is in blue. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

100 4.2.3 C2cd3 is essential for Ttbk2 and Ift recruitment and Ccp110 removal during ciliogenesis

Distal appendages have been implicated in Ttbk2 and Ift recruitment and Ccp110 removal in the initiation of ciliogenesis. Therefore, I examined these events in wildtype and

C2cd3 mutant cells treated with serum starvation to induce ciliogenesis. GFPTtbk2 failed to be recruited to the mother centriole in the absence of C2cd3 (Fig.4-4A). Ccp110 was located at the distal ends of mother centriole, daughter centriole and procentrioles. Ciliogenesis triggers the removal of Ccp110 specifically from the mother centriole (Spektor et al. 2007). Depletion of

C2cd3 led to Ccp110 detainment at the distal end of the mother centriole (Fig4-4B). Ift proteins reside in the axoneme and at the basal body of a mature cilium (Pedersen et al. 2008).

At the beginning of ciliogenesis, Ift proteins are recruited to the basal body before the cilium is formed (Goetz et al. 2012). Consistent with previous studies, two IFT-B complex components, Ift88 and Ift52, were localized in the cilia and at the basal body in ciliated wildtype cells, at the centrosome in the non-ciliated cells (Fig. 4-5A and D). However, endogenous Ift88 was not detected at the centrosome in C2cd3 mutant cells (Fig. 4-5A). And this is not due to a decreased Ift88 protein level (Fig. 4-5B). Similarly, overexpressed GFPIft52 was absent from the basal body in C2cd3 mutant cells (Fig. 4-5D). Therefore, Ift88 and Ift52 failed to be recruited to the centrosome in C2cd3 mutant cells. In addition, the physical interaction between C2cd3 and

Ift88 was also detected (Fig. 4-5C). Together, the recruitment of Ttbk2 and Ift and the removal of

Ccp110 are blocked when C2cd3 is depleted.

101

Figure 4-4. C2cd3 is required for Ttbk2 recruitment and Ccp110 removal during ciliogenesis.

A. Ttbk2 fails to be recruited to the mother centriole in C2cd3 mutant cells after serum starvation. B. Ccp110 is removed from the mother centriole in wild-type cells but not in C2cd3 mutant cells after serum starvation. Note that Ccp110 staining is dynamic through the cell cycle. Before centrosome duplication, Ccp110 are observed as one dot in wildtype cells, representing the daughter centriole (“1”). In contrast, two dots are observed in C2cd3 mutant cells, representing both the mother and daughter centrioles (“2”). After centrosome duplication, only one centrosome is positive for Cp110 (“1+0”) in wildtype cells, but both centrosomes are positive for Cp110 (“1+1”) in C2cd3 mutant cells. In the G2 phase, the procentrioles are capped with Cp110 such that Cp110 appears as two dots in the centrosome containing the daughter centriole, and one dot representing the procentriole in the centrosome containing the mother centriole (“2+1”) in wild- type cells. In contrast, C2cd3 mutant cells exhibit two Cp110 dots in each centrosome in the G2 phase (“2+2”). For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

102

Figure 4-5. C2cd3 is required for the centriolar recruitment of Ift88 and Ift52 during ciliogenesis.

A. Ift88 is localized to the basal body and along the cilium in ciliated wild-type cells and to the mother centriole in unciliated wildtype cells. The centriolar staining of Ift88 is absent in C2cd3 mutant cells after serum starvation. B. Western blot analysis reveals comparable levels of Ift88 protein in C2cd3 mutant and wildtype cells. C. Coimmunoprecipitation analysis reveals the physical interaction between overexpressed C2cd3 and Ift88. D. GFPIft52 is localized to the basal body and along the cilium in ciliated wildtype cells and to the mother centriole in unciliated wildtype cells. The centriolar staining of GFPIft52 is absent in C2cd3 mutant cells after serum starvation. The centrosomes are labeled with γ-tubulin, and the nuclei are visualized with DAPI. The quantitative analyses of Ift88 and Ift52 centriolar recruitment only include unciliated cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

103 4.2.4 C2cd3 is critical for ciliary vesicle docking

The mother centriole docking to the ciliary vesicle is an important early event of ciliogenesis. A previous report suggested that the transmembrane protein Smo was a faithful marker of the ciliary vesicle (Joo et al. 2013). In wildtype cells, overexpressed GFPSmo was detected on the ciliary membrane in ciliated cells, or at the mother centriole in non-ciliated cells

(Fig. 4-6A). GFPSmo failed to be recruited to the mother centriole in the absence of C2cd3 (Fig.

4-6A). I also examined this docking process using Transmission Electron Microscopy (TEM). 5 out of 16 centrioles in wildtype cells were associated with ciliary vesicle. However, among 16 centrioles in C2cd3 mutant cells I examined, no vesicle-associated centriole was found (Fig. 4-

6B). In summary, C2cd3 is required for the mother centriole docking to the ciliary vesicle.

104

Figure 4-6. C2cd3 is important for ciliary vesicle docking during ciliogenesis.

A. GFPSmo is present in the cilia and ciliary vesicles docked to the mother centriole in wildtype cells, but is absent at the mother centriole in C2cd3 mutant cells, suggesting a defect in ciliary vesicle docking. The quantitative analysis only includes nonciliated cells. B. TEMs of centrioles in E10.5 wildtype and C2cd3 mutant mouse embryos. Longitudinal sections (Left) and cross- sections (Right) are presented. Asterisks indicate docked ciliary vesicles. C. A model summarizes the role of C2cd3 in ciliogenesis: C2cd3 is critical for the assembly of centriolar distal appendages, which in turn underlies the recruitment of Ttbk2, Ift88, and Ift52 as well as the removal of Cp110 and the docking of ciliary vesicles. C2cd3 may play a more direct role in recruiting Ift88 to the distal appendages. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

105 4.2.5 C2cd3 contributes to the centriolar recruitment of Centrin

Previous study suggests that knocking down C2cd3 leads to mislocalization of centriolar

Centrin and Poc5 (Balestra et al. 2013). Centrin and Poc5 are localized at the distal part of the centrioles and implicated in centriole replication and distal structure assembly (Salisbury et al.

2002; Azimzadeh et al. 2009). There are four Centrin genes in rodents, namely Centrin1-4.

Centrin2 and Centrin3 are ubiquitously expressed in all somatic cells (Salisbury 2007). However,

Centrin2 is not expressed in male germ cells (Tanaka et al. 2010). Centrin1 is expressed in male

germ cells, photoreceptors and other ciliated cells. Centrin4 was only detected in brain, kidney,

lung and ovary (Gavet et al. 2003). In terms of the subcellular localization, Centrin1 was found at

the mother centriole but not at the daughter centriole in mouse retina, whereas Centrin2 and

Centrn3 were found at both the mother and daughter centrioles (Giessl et al. 2004). Centrin4 is

only localized to the daughter centriole in the mouse retina, but overexpressed GFPCentrin4 is

localized to both the mother and daughter centrioles in Hela cells (Giessl et al. 2004; Gavet et al.

2003).

Surprisingly, I found that overexpressed GFPCentrin1-3 and GFPPoc5 were efficiently

recruited to the centrosome in C2cd3 mutant cells (Fig. 4-7). In addition, unlike their satellite

localization, their centriolar localization was independent of serum starvation (Fig. 4-7B). Since

expression level of Centrin might have an effect on its centriolar recruitment, I also examined the

endogenous Centrin by using Centrin antibody. Interestingly, endogenous Centrin were absent or

greatly reduced at the centrosomes in C2cd3 mutant cells, suggesting that C2cd3 contributes to

the centriolar recruitment of Centrin . Since the four mouse Centrin proteins are highly similar in

their protein sequences, I tested the specificity of this Centrin antibody by immunobloting

overexpressed GFPCentrin1-3. This Centrin antibody strongly recognized GFPCentrin1 and

106 GFPCentrin2 but not GFPCentrin3 (Fig. 4-8B). This suggests that the attenuated endogenous

Centrin staining may be due to defective centriolar recruitment of Centrin1 and/or Centrin2.

Since overexpressed GFPCentrin2 was efficiently localized to the centrioles in both wildtype and C2cd3 mutant cells, I sought to determine whether it could rescue DAP assembly in

C2cd3 mutant cells. Surprisingly, Cep164 centriolar recruitment is partially rescued in the transfected mutant cells (Fig. 4-8C). Notably, the intensity of Cep164 staining in rescued mutant cells is weaker than that in wildtype cells. Therefore, C2cd3 may regulate DAP assembly partially by facilitating Centrin localization to the centrioles.

107

Figure 4-7. Overexpressed GFPCentrin1-3 and GFPPoc5 are recruited to centrosome in the absence of C2cd3.

A. Overexpressed GFPCentrin1-3 are localized to centrosomes in C2cd3 mutant cells. B. Quantitative summary of the localization of overexpressed GFPCentrin1-3 in wildtype cells and C2cd3 mutant cells. C. Overexpressed GFPPoc5 is recruited to centrosomes in C2cd3 mutant cells. D. Quantitative summary of the localization of overexpressed GFPPoc5 in wildtype cells and C2cd3 mutant cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01, ***p<0.001 and n.s. means not significant).

108

Figure 4-8. Centriolar localization of endogenous Centrin is affected in C2cd3 mutant cells.

A. Endogenous Centrin is absent or weakly recruited at the centrosomes in C2cd3 mutant cells compared to wildtype cells. B. Centrin antibody strong recognizes GFPCentrin1 and GFPCentrin2, but not GFPCentrin3 in western blot. C. Overexpress GFPCentrin2 partially rescues the Cep164 recruitment defect in C2cd3 mutant cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

109 4.2.6 Relationship among C2cd3, Ofd1 and Dzip1

Two other proteins, Ofd1 and Dzip1, have also been implicated in DAP assembly. To study the relationship between C2cd3 and the other two regulators, I investigated whether C2cd3 was required for their recruitment. Centriolar recruitment of both endogenous Ofd1 and overexpressed GFPOfd1 were reduced by the loss of C2cd3 (Fig. 4-9A). Dzip1 has been shown to be localized to the centrioles but its exact localization has not reported. By co-staining Dzip1

and Cep164, I found that Dzip1 is in the center of the Cep164 ring in the top view, and in the

middle of the Cep164 bar in the side view, suggesting that Dzip1 is localized at the distal end of

the centrioles (Fig. 4-9B). I found that Dzip1 centriolar localization was abolished in the absence

of C2cd3 (Fig. 4-9C). Together, both Ofd1 and Dzip1 centriolar localization is affected by loss of

C2cd3.

I also investigated C2cd3 and Dzip1 localization in Ofd1 mutant cells. C2cd3 was normally recruited to the centrioles in the absence of Ofd1 (Fig. 4-10A). However, Dzip1 centriolar localization was significantly affected by loss of Ofd1 (Fig. 4-10B). In summary, Ofd1 is required for centriolar recruitment of Dzip1 but not C2cd3.

110

Figure 4-9. Centriolar localization of Ofd1 and Dzip1 is affected in C2cd3 mutant cells.

A. Centriolar recruitment of both endogenous Ofd1 and overexpressed GFPOfd1 is perturbed in C2cd3 mutant cells. B. Dzip1 is localized at the distal end of the mother centriole. Dzip1 staining is found in the center of Cep164 ring or in the middle of Cep164 bar. C. Dzip1 centriolar recruitment is disrupted in C2cd3 mutant cells. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01 and ***p<0.001).

111

Figure 4-10. Centriolar localization of Dzip1, but not C2cd3, is compromised in Ofd1 mutant cells.

A. C2cd3 is recruited to centrioles in Ofd1 mutant cells. B. Dzip1 centriolar recruitment is compromised in the absence of Ofd1. For quantitative analyses, error bar stands for standard deviation. n = 3 independent experiments. p-value is calculated by t test (*p<0.05, **p<0.01, ***p<0.001 and n.s. means not significant).

112 4.3 Discussion

4.3.1 C2cd3 is required for the initiation of ciliogenesis

My results suggest that C2cd3 plays a critical role in early events during ciliogenesis

(Fig. 4-6C). C2cd3 is essential for the assembly of the distal appendages, which are important for recruiting Ttbk2 and removing Ccp110. I confirmed that C2cd3 is indeed required for these downstream events. The centriolar recruitment of Ift proteins during ciliogenesis depends on

Ttbk2, and this process is also disrupted in C2cd3 mutant cells. Interestingly, C2cd3 also physically interacts with Ift88, suggesting that C2cd3 might be directly involved in Ift recruitment too. A recent study suggested that Ift recruitment may be important for strengthening the centriole-membrane association after membrane docking (Joo et al. 2013). The ciliary vesicle

docking to the mother centriole requires the interaction between DAPs and Rabin8/Rab8

(Schmidt et al. 2012). Both immunofluorescence and TEM results suggest that centriole- membrane association is abolished in the absence of C2cd3.

4.3.2 C2cd3 may mediate DAP assembly through Centrin recruitment

Our results suggest that the ciliogenic defects in C2cd3 mutant cells result from the failure in DAP assembly, but the mechanism by which C2cd3 regulates DAP assembly is still a mystery. Intriguingly, C2cd3 is required for the recruitment of Centrin to both the centrioles and centriolar satellites. Centrin2 has been implicated in centriole duplication in Tetrahymena and

Chlamydomonas (Koblenz et al. 2003; Stemm-Wolf et al. 2005), but its role in mammalian cells remains controversial (Salisbury et al. 2002; Kleylein-Sohn et al. 2007). In addition, its binding partner, Poc5, is required for assembling the distal ends of the centrioles (Azimzadeh et al. 2009).

Since both C2cd3 and Centrin are localized to the distal ends of the centrioles and loss of C2cd3

113 leads to Centrin centriolar recruitment defect, it is possible that the distal structure of centrioles

are disrupted in C2cd3 mutant cells. Consequently, DAPs and SAPs fail to be assembled.

Consistent with this idea, it was recently reported that centrioles in C2cd3 mutant cells were one third shorter compared to in wildtype cells (Thauvin-Robinet et al. 2014). Interestingly, overexpression of GFPCentrin2 in C2cd3 mutant cells partially rescued DAP assembly, suggesting that increasing Centrin2 level might partially restore the distal structure of centrioles.

It will be helpful to test whether other events or even ciliogenesis can be rescued in C2cd3 mutant cells by overexpressing Centrin2.

How Centrin is recruited to the centrioles is still unclear. Knockdown of Pcm1 results in decreased centriolar recruitment of Centrin, suggesting that centriolar satellites contribute to this process (Dammermann & Merdes 2002). Since C2cd3 and Centrin are both localized to satellites and distal ends of the centrioles, I hypothesize that the satellite population of C2cd3 is responsible for recruiting Centrin to centrioles, whereas centriolar C2cd3 installs Centrin onto the distal end of the centrioles. It will be intriguing to see whether C2cd3 physically interacts with Centrin.

Interestingly, overexpressed GFPCentrin have normal centriolar localization in C2cd3 mutant cells. This suggests that the centriolar recruitment of Centrin not completely dependent on C2cd3, and loss of C2cd3 can be partially compensated by increasing the Centrin protein level. It will be helpful to compare the endogenous levels of Centrin proteins in C2cd3 mutant cells to those in wildtype cells. Previous study showed that both Centrin and Poc5 centriolar localization was affected when C2cd3 was knocked down (Balestra et al. 2013). Although overexpressed

GFPPoc5 is correctly recruited to centrioles in C2cd3 mutant cells, it will be interesting to test whether the recruitment of endogenous Poc5 to the centrioles, as well as its protein level, is compromised.

In addition, as centriolar satellites have been implicated in SAP assembly, it is possible that the satellite C2cd3 is responsible for SAPs assembly defects. It will be interesting to test

114 whether C2cd3 physically interact with Ninein in centriolar satellites. It was reported that Kif3a

and p150Glued (a dynactin subunit) were localized to the subdistal level of the mother centriole and

responsible for the recruitment of some SAP components, like Ninein and Cep170, but not Odf2.

It will be helpful to see whether Kif3a or p150Glued localization is dependent on C2cd3 and whether Cep170 and Odf2 recruitment depends on C2cd3.

4.3.3 Different penetrances of different phenotypes in C2cd3 mutant cells

As shown in the Table 4-1, the molecular events regulated by C2cd3 can be categorized into three groups. The ones highlighted in green, including ciliogenesis, Centrin satellite recruitment, DAP assembly, the recruitment of Ttbk2, Ift88, Ift52 and GFPSmo, and Ccp110 removal, are highly dependent on C2cd3,. In contrast, centriolar recruitment of endogenous

Centrins, Ninein recruitment and Cep164 centriolar localization when GFPCentrin2 is overexpressed, are partially affected by loss of C2cd3. Notably, the intensities and/or patterns of the staining are also somehow compromised in these 40~50% of mutant cells with positive staining. Endogenous Centrin staining and Ninein staining is weaker in C2cd3 mutant cells compared to wildtype cells. Cep164 staining is weaker and looks like a small dot instead of a ring or bar in GFPCentrin2 overexpressed C2cd3 mutant cells. Intriguingly, overexpressed

GFPCentrins and GFPPoc5 are efficiently recruited to the centrioles in C2cd3 mutant cells. The partial loss of Centrin and Ninein at the centriole is reminiscent of that in Pcm1 knockdown cells, suggesting that it may result from the loss C2cd3 function at centriolar satellites. The partial restoration of the centriolar recruitment of overexpressed GFPCentrin2 in C2cd3 mutant cells suggests that increasing the levels of Centrin proteins may compensate for the compromised transport capacity of centriolar satellites.

115

Table 4-1. Summary of various phenotypes in C2cd3GT mutant cells and wildtype control cells.

Events highlighted in green are greatly abolished in C2cd3 mutant cells. Events highlighted in blue are not affected in C2cd3 mutant cells. Events highlighted in red are partially compromised in C2cd3 mutant cells.

4.4 References

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Koblenz, B., Schoppmeier, J., Grunow, A., & Lechtreck, K.-F. (2003). Centrin deficiency in Chlamydomonas causes defects in basal body replication, segregation and maturation. Journal of Cell Science, 116, 2635–2646. doi:10.1242/jcs.00497

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117 Kunimoto, K., Yamazaki, Y., Nishida, T., Shinohara, K., Ishikawa, H., Hasegawa, T., … Tsukita, S. (2012). Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet. Cell, 148(1-2), 189–200. doi:10.1016/j.cell.2011.10.052

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Schmidt, K. N., Kuhns, S., Neuner, A., Hub, B., Zentgraf, H., & Pereira, G. (2012). Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis. The Journal of Cell Biology, 199(7), 1083–101. doi:10.1083/jcb.201202126

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118 Thauvin-Robinet, C., Lee, J. S., Lopez, E., Herranz-Pérez, V., Shida, T., Franco, B., … Nachury, M. V. (2014). The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation. Nature Genetics, 46, 905–911. doi:10.1038/ng.3031

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Chapter 5

Conclusion, discussion and future directions

5.1 The role of C2cd3 in the initiation of ciliogenesis

It has been more than 50 years since the ciliogenesis process was first described by

Sorokin (1962), but we are still far from the complete understanding of its underlying mechanism.

Several studies published in the last year and even early this year greatly helped us to understand this process. There are four key events required for ciliogenesis: the mother centriole docking to the ciliary vesicle, recruitment of the Ift proteins, transition zone protein recruitment and axonemal microtubule growth. However, the relationship between these events is still puzzling.

Published studies suggested that at least some of them were independent from each other.

Depletion of Ttbk2 abolished Ift recruitment and Ccp110 removal, but did not affect ciliary vesicle docking or transition zone protein recruitment (Goetz et al. 2012). Loss of Tctn1, a transition zone protein, did not affect ciliary vesicle docking, but disrupted the axonemal microtubule growth (Williams et al. 2011). One study suggested that Ift recruitment was not required for the initial docking process but might strengthen the centriole-membrane association after docking (Joo et al. 2013). In addition, IFT mutants of Chlamydomonas and C. elegans are still able to grow normal transition zone (Williams et al. 2011; Brazelton et al. 2001; Perkins et al.

1986). Inhibiting Rab8 activity did not affect IFT centriolar recruitment or Ccp110 removal

(Cajánek & Nigg 2014).

However, the current view is far from complete. New events/processes wait to be discovered and the chronological order of these events needs to be unraveled. It was reported that loss of Cep89, a DAP component, blocked the big ciliary vesicle formation while leaving many

120 tiny vesicles at the distal end of the mother centriole (Sillibourne et al. 2013). This implies that

the ciliary vesicle docking step might not be as simple as that one single big ciliary vesicle

directly docks to the mother centriole, instead multiple tiny vesicles dock to the mother centriole before fusing into a big ciliary vesicle.

My study on C2cd3 uncovered the molecular mechanism of how a novel regulator contributes to ciliogenesis (See how my results are integrated into current understanding of ciliogenesis in Fig. 5-1). My results suggest that C2cd3 is upstream of these events and critical for

DAP assembly. All the disrupted downstream events in C2cd3 mutant cells might be the secondary effect of loss of DAPs. We are the first group to reveal the role of C2cd3 in ciliogenesis and C2cd3 is the third-reported factor essential for DAPs and/or SAPs assembly (Ye et al. 2014; Hoover et al. 2008).

Latest studies on C2cd3 in human and chicken revealed the conserved role of C2cd3 in vertebrates (Thauvin-Robinet et al. 2014; Chang et al. 2014). Intriguingly, C2cd3 mutations have been identified in human patients with a new subtype of oral-facial-digital (OFD) syndrome, a ciliopathy (Thauvin-Robinet et al. 2014). The mutant alleles identified in human and chicken are valuable assets for studying the molecular function of C2cd3. In these two human cases, one has a homozygous nonsense mutation leading to a severely truncated protein with only the first 62 amino acids. The other patient has compound heterozygous mutations, including a missense mutation (causing substitution of Cysteine1029 by Glycine) and a frameshift mutation (disrupting the amino acid 1304 and after). Talpid2, a mutant allele in chicken, has a nonsense mutation resulting in the deletion of amino acids 2210-2330. These studies highlighted the importance of

Cys1029 and the C-terminal120 amino acids of the C2cd3 protein. Studying the subcellular localizations of these mutant alleles and their functions will help to dissect the molecular functions of different domains on C2cd3.

121

Figure 5-1. Current understanding of ciliogenesis integrated with my results.

Events in blue boxes are the ones affected by loss of C2cd3. Black solid line indicates the relationship demonstrated in my work. Gray solid lines represent the relationship found in others’ work. Gray dash lines represent the relationship speculated based on my results and current understanding of ciliogenesis. Lighter gray dash lines with question marks represent possible pathways.

122 5.2 The role of C2cd3 in centriole length control

A recent paper found that C2cd3 was also required for centriole elongation (Thauvin-

Robinet et al. 2014). They found that centrioles in C2cd3 mutant cells were one third shorter

compared to wildtype cells. In addition, overexpressing C2cd3 resulted in hyperelongated

centrioles in 16% of U2OS cells. My finding that Centrins failed to be recruited to the centrioles

in C2cd3 mutant cells supports this idea. The mechanism of how C2cd3 contributes to centriole

elongation is worth further study. The current view of centriole length regulation is through

controlling the centriole microtubule polymerization and depolymerization. It was proposed that

CPAP and Cep120 facilitated centriole elongation by promoting the incorporation of new tubulins

to the centriolar microtubules (Kohlmaier et al. 2009; Schmidt et al. 2009; Tang et al. 2009;

Comartin et al. 2013; Lin et al. 2013). Ccp110 was proposed to inhibit this process by forming

caps at the plus ends of microtubules (Spektor et al. 2007; Tsang et al. 2008). Whether C2cd3 has

similar effects on microtubules waits to be uncovered. Intriguingly, since overexpressing

GFPCentrin2 partially rescued Cep164 recruitment defects in C2cd3 mutant cells, Centrin might play a role in centriole elongation in vertebrates. Although Centrin2 is required for centriole

biogenesis in Tetrahymena and Chlamydomonas (Koblenz et al. 2003; Stemm-Wolf et al. 2005), its role in mammalian cells has been controversial (Salisbury et al. 2002; Kleylein-Sohn et al.

2007).

In addition, how C2cd3 interplay with existing centriole elongation factors is also an interesting question. As mentioned in Chapter 1, centriole elongation regulators include CPAP

(Kohlmaier et al. 2009; Schmidt et al. 2009; Tang et al. 2009), Cep120 (Comartin et al. 2013; Lin et al. 2013), Poc1 (Keller et al. 2009), Ofd1 (Singla et al. 2010) and Ccp110 (Spektor et al. 2007;

Tsang et al. 2008). CPAP, Cep120 and Poc1 are positive regulators, overexpression of which lead to overly long centrioles, whereas Ofd1 and Ccp110 are negative regulators, knockdown of which

123 causes hyperelongated centrioles. It was reported that overexpression of Ofd1 inhibited the

formation of hyperelongated centriole in C2cd3 overexpressing cells, suggesting C2cd3 and Ofd1

may antagonize each to regulate the length of centriole (Thauvin-Robinet et al. 2014). It will be

interesting to study how C2cd3 interplays with other regulators to control the centriole length.

As C2cd3 is essential for both DAP and SAP assembly and centriole elongation, it will be

interesting to investigate whether these two functions are independent. Although how DAPs are

assembled is still unclear, it is possible that DAP defect is secondary to centriole elongation

defect in C2cd3 mutants. The fact that DAPs and SAPs are restricted in the doublet region of the

distal end of the mother centriole suggests that DAPs and SAPs may be specifically targeted to

the distal ends by recognizing adaptors in this region. Centriole elongation defects may lead to

loss of these adaptors at the disrupted distal structure. However, the nature of this disrupted distal

structure is still elusive. My results, along with some recent reports, suggested that some proteins, such as Ofd1, Dzip1 and Centrin, were lost from the distal ends of centrioles in C2cd3 mutant cells (Thauvin-Robinet et al. 2014). Interestingly, Ccp110 is still recruited to the distal ends of centrioles, suggesting that these distal proteins are targeted through different mechanism. It will be interesting to examine the localization of additional distal proteins in C2cd3 mutant cells.

Alternatively, these two functions may be independent. Ofd1, for example, was found to be required for both centriole length control and DAP assembly (Singla et al. 2010). Two Ofd1 missense mutant alleles were found to cause hyperelongated centrioles without affecting Cep164 localization, suggesting that these two functions of Ofd1 might be independent. Remarkably,

Cep164 is normally recruited in the hyperelongated centrioles induced by C2cd3 overexpression, supporting the idea that DAP recruitment and centriolar elongation could be independent

(Thauvin-Robinet et al. 2014). Furthermore, C2cd3 physically interacts with Cep164, suggesting

C2cd3 may directly recruit Cep164. Studying the mutations in human and chicken may identify a

124 mutant allele that can help to uncouple these two functions of C2cd3, i.e. a mutant allele that

causes DAP assemble defect without affecting centriole length.

5.3 The role of C2cd3 in DAPs and SAPs recruitment

C2cd3 is the third-reported regulator for DAP recruitments, following Ofd1 and Dzip1.

Ofd1 is specifically required for DAP recruitment and Dzip1 is responsible for both DAP and

SAP recruitments. We and other colleagues found that all three proteins are localized to the distal ends of the centrioles (Ye et al. 2014; Singla et al. 2010; C. Wang et al. 2013). I found that C2cd3 was required for the recruitment of both Ofd1 and Dzip1, whereas Ofd1 was only essential for

Dzip1 localization. It is possible that C2cd3 directly facilitates the recruitments of Ofd1 and

Dzip1. Alternatively, loss of Ofd1 and Dzip1 could be the secondary effect of defective centriole elongation. Therefore, investigating whether Ofd1 and Dzip1 are recruited to the hyperelongated centrioles induced by overexpressing C2cd3 will further clarify the relationship between the Ofd1 and Dzip1 recruitment and centriole elongation. In addition, assuming overexpressing

GFPCentrin2 will rescue centriole elongation defect in C2cd3 mutant cells, it will be interesting to test whether Ofd1 and Dzip1 recruitments will also be restored by overexpression of

GFPCentrin2.

To obtain a complete picture about the relationship among C2cd3, Ofd1 and Dzip1, I am generating Dzip1 mutant cell lines using CRISPR (clustered regularly interspaced short palindromic repeats) technique. First, I would like to confirm the previously described DAP and

SAP defects in Dzip1 mutant cells. Second, the role of Dzip1 in Ofd1 and C2cd3 recruitment can also be studied. Third, the question whether Dzip1 plays a role in centriole length control will be answered.

125 5.4 Future directions

Centriole is a tiny, yet delicate organelle. The centriolar microtubule cylinder is around

200nm in diameter and around 500nm in length. Immunofluorescence confocal microscopy is a

common method to pinpoint the localization of a centriolar protein. Immunoelectron microscopy

(ImmunoEM) is a more accurate method with higher resolution, but it requires high-quality

antibodes and sophisticated EM techniques. In the future, new microscope technologies,

including super-resolution microscopy, total internal reflection fluorescence (TIRF) microscopy

on live cells and correlative light electron microscopy (CLEM) will help us to decipher more

complicated questions in more delicate dimensions, such as spatiotemporal processes of

ciliogenesis, subcompartmentalization and signal transduction.

I and other colleagues found that C2cd3 was recruited to the distal ends of mother

centriole, daughter centriole and procentrioles (Thauvin-Robinet et al. 2014). By co-staining with other markers, C2cd3 is found colocalized with Centrin at the DAP level, and more distal to SAPs

(Thauvin-Robinet et al. 2014). Interestingly, the other distal end protein Ccp110 was found more distal to C2cd3, suggesting that the distal end can be further divided into more specific localizations (Thauvin-Robinet et al. 2014). It is proposed that Ccp110 forms a cap at the distal end of centriolar microtubules to inhibit microtubule growth. Since C2cd3 was found colocalized with Centrin in immunofluorescence staining and Centrin is localized in the distal lumen of the centrioles in immunoEM, I hypothesize that C2cd3 is also localized to the distal lumen (Paoletti et al. 1996; Thauvin-Robinet et al. 2014). To test this hypothesis, ImmunoEM should be used to further pinpoint the ultrastructural localization of C2cd3. In addition, the temporal aspect of

C2cd3 localization is also an intriguing topic. To answer the question when C2cd3 is incorporated to the procentrioles, one can use ImmunoEM to examine cells at different stages in the cell cycle, or use live cell imaging with TIRF. C2cd3 is colocalized with Centrin at procentrioles and

126 depletion of C2cd3 only leads to shorter contriole, suggesting that C2cd3 is incorporated after the

proximal end is assembled (Thauvin-Robinet et al. 2014).

The other intriguing question is how to distinguish the functions of C2cd3 at different localizations, including centriolar satellites and centriole. Currently, there are not well-defined boundaries for centriole, PCM and centriolar satellites. Many proteins are localized to both centriole, centriolar satellites and potentially PCM as well. Originally, centriolar satellites were considered as a transit station for centrosomal proteins on their way to the destinations, or as a reservoir to ensure the supply of these centrosomal proteins. However, recent studies showed that it has its own specific role in ciliogenesis. In particular, satellite accumulation of Ofd1 actually inhibits ciliogenesis (Tang et al. 2013). Since the centriolar localization of C2cd3 is independent of centriolar satellites, these two populations may be targeted to the centrioles and satellites through different mechanisms. It will be important to study the functions of different domains of

C2cd3 and try to uncouple its localizations/functions at the centrioles and satellites by creating deletions or truncations. Studying the mutations found in human and chicken will also shed light on this. My results about the domain requirement for its satellite localization can be used as important references for the future study.

One commonly neglected question is whether a factor is required for cilia maintenance.

Many studies only focus on the functions in cilia formation. However, studies showed that cilia formation and maintenance might require different machineries. Degradation of the basal body after cilia formation in C. elegans suggests that the basal body might not be essential for maintaining a cilium (Williams et al. 2011; Perkins et al. 1986). Cep123, a DAP component, is only required for cilia formation but not cilia maintenance (Sillibourne et al. 2013). Its role in cilia maintenance can be investigated by siRNA knocking down C2cd3 after serum starvation treatment.

127 Finally, screening for interacting partners is frequently used in the cilia field to find novel

candidates. The interacting partners of C2cd3 can be identified using affinity purification (TAP)

or yeast two-hybrid. In addition, a new method called BioID (biotin identification) has been

developed to identify not only binding partners but also proteins physically close to the protein of

interest (Roux et al. 2012; Morriswood et al. 2013; Comartin et al. 2013; Firat-Karalar et al.

2014). By tagging the protein of interest with a promiscuous biotin ligase and incubating the cells with biotin, biotin is added to the proteins physically close to it. The biotinylated proteins are pulled down by streptavidin beads and sent for mass spectrometry. To assess the interactions among centrosomal proteins, the centrosome must be disrupted and centrosomal proteins must be solubilized by harsh treatments. Some weak interactions might be affected during this process, which causes false negative in TAP. However, BioID overcame this difficulty by adding Biotin tag directly to the partners before harsh treatments. Combined with centrosome enrichment protocol, BioID makes a powerful tool to identify the proximal and interacting partners of centrosomal proteins.

5.5 References

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VITA

Xuan Ye

Education Ph.D., Biology 2009-2014 The Pennsylvania State University, University Park, PA B.S., Life Science and Technology 2005-2009 Wuhan University, China

Publications Ye, X., Zeng, H., Ning, G., Reiter, J. F., and Liu, A. (2014). C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals. Proceedings of the National Academy of Sciences of the United States of America, 111(6), 2164–9. doi:10.1073/pnas.1318737111 Ye, X., and Liu, A. (2011). Hedgehog signaling: mechanisms and evolution. Frontiers in Biology, 6(6), 504–521. doi:10.1007/s11515-011-1146-2

Presentations C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking, Poster Presentation at Keystone Symposia: Cilia, Development and Human Disease, Mar 2-7, 2014 C2cd3 is Critical for the Maturation of centrioles and Cilia Formation, platform presentation at Mid-Atlantic Regional Meeting of the Society for Developmental Biology, May 11-12, 2012.

Awards 1st place team mentor in SEECoS, Eberly College of Science (PSU) 2014 Travel grants, Biology department (PSU) 2014 Braddock Award, Biology department (PSU) 2009