Characterization of POC16/WDR90 Proteins in Centriole Integrity

Thesis

Reference

Characterization of POC16/WDR90 Proteins in Centriole Integrity

STEIB, Emmanuelle

Abstract

Les centrioles sont des organites essentiels aux processus de division cellulaire et de signalisation de nombreux organismes eucaryotes. Ils sont caractérisés par une organisation de neuf triplets de microtubules, polarisés longitudinalement en trois régions distinctes : proximale, centrale et distale. Précisément, la région centrale est définie par la présence d'une charpente interne hélicoïdale macromoléculaire, ancrée à la jonction interne des microtubules A et B du triplet, décrite comme « connexion en Y ». Sa composition moléculaire et sa fonction n'ont pas été décrites à ce jour. Nous avons identifié la protéine POC16 comme l'un des premiers composants de la partie centrale du centriole. Il a été suggéré que qu'elle compose la « connexion en Y ». J'ai entrepris une analyse fonctionnelle et structurelle de POC16 chez Chlamydomonas et Paramecium, ainsi que de son homologue humain WDR90.

J'ai mis en lumière son importance ainsi que celle de la charpente interne dans le maintien de l'intégrité de la paroi de microtubules qui préservent l'architecture et les fonctions du centriole.

STEIB, Emmanuelle. Characterization of POC16/WDR90 Proteins in Centriole Integrity. Thèse de doctorat : Univ. Genève, 2020, no. Sc. Vie 52

DOI : 10.13097/archive-ouverte/unige:137536 URN : urn:nbn:ch:unige-1375368

Available at:

http://archive-ouverte.unige.ch/unige:137536

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

1 / 1

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de Biologie

Département de Biologie Cellulaire Professeur P. Guichard Docteur V. Hamel

Characterization of POC16/WDR90 Proteins in Centriole Integrity

THÈSE

présentée aux Facultés de médecine et des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences en Sciences de la vie,

mention Bioscience Moléculaire

par

Emmanuelle STEIB

de

Mulhouse, France

Thèse N^o 52

GENÈVE 2020

Résumé

Les centrioles sont des organites conservés au cours de l’évolution, essentiels aux processus fondamentaux de division cellulaire et de signalisation de nombreux organismes eucaryotes.

Transversalement, les centrioles sont caractérisés par une organisation de neuf triplets de microtubules. Ils sont polarisés de façon longitudinale en trois région distinctes: proximale, centrale et distale. Plus précisément, la région centrale est définie par la présence d’une charpente interne hélicoïdale et macromoléculaire où les quatre protéines POC1B, POC5, FAM161A et Centrin-2 forment un complexe et sont localisées. Une partie de la structure ancrée à la jonction interne entre les microtubules A et B du triplet, connectant cette charpente interne à la paroi de microtubules, a été décrite en tant que « Y-shaped linker » ou « connexion en Y », néanmoins sa composition moléculaire et sa fonction n’ont pas été décrites à ce jour.

Nous avons identifié la protéine POC16 comme l’un des premiers composants de la partie centrale du centriole de Chlamydomonas. De part son homologie avec FAP20, la protéine ciliaire localisant à la jonction interne du flagelle, il a été suggéré que POC16 fasse partie de la « connexion en Y » du centriole.

Au cours de cette thèse, j’ai entrepris une analyse fonctionnelle et structurelle de la protéine POC16 chez Chlamydomonas reinhardtii et Paramecium tetraurelia, ainsi que de son homologue humain WDR90. J’ai combiné des méthodes biologie cellulaire et de biochimie et utilisé les techniques de pointe de microscopies électronique et à expansion pour révéler la localisation précise et la fonction de POC16/WDR90.

J’ai identifié des défauts de flagelles chez des mutants poc16 de Chlamydomonas reinhardtii. De même, la déplétion de WDR90 induit un défaut de ciliogénèse dans les cellules humaines. Grâce à la microscopie à expansion, j’ai révélé la localisation précise de POC16/WDR90 en tant que composant du triplet de microtubules de la paroi centriolaire. De plus, j’ai démontré in vitro que POC16 et WDR90 lient directement les microtubules de par leur domaine d’homologie avec FAP20. Enfin, j’ai mis en évidence que les cellules dépourvues de POC16/WDR90 sont caractérisées par des centrioles cassés dont le complexe de la charpente interne a été déstabilisé.

Cette étude met en lumière l’importance de POC16/WDR90 ainsi que de la charpente interne dans le maintien de l’intégrité de la paroi de microtubules afin de préserver l’architecture et les fonctions du centriole.

Abstract

Centrioles are evolutionary conserved organelles essential for fundamental processes ranging from cell division to cell signaling in many eukaryotes. Centrioles are characterized by a nine-fold radial arrangement of microtubules triplets and are polarized along their long axis with three distinct regions: proximal, central core and distal. In particular, the central core region is defined by the presence of a macromolecular helical inner scaffold where the four proteins POC1B, FAM161A, POC5 and Centrin-2 form a complex and localize.

Part of the structure rooted at the inner junction of the A- and B- microtubule triplets, connecting the helical scaffold to the microtubule wall, has been described as the Y- shaped linker but its molecular composition and its function have not been identified yet.

We found the conserved protein POC16 to be one of the first central core component of Chlamydomonas centrioles. Interestingly, through its homology with the flagellar inner microtubule doublet protein FAP20, POC16 has been proposed to be part of the Y-shaped linker.

In this thesis, we undertake a functional and structural analysis of POC16 in Chlamydomonas reinhardtii and Paramecium tetraurelia as well as the characterization of its human ortholog WDR90. We combine cell biological approaches to biochemical technics and use cutting-edge electron- and expansion-microscopy to reveal POC16/WDR90 precise localization and function. Importantly, we found that Chlamydomonas reinhardtii poc16 mutants exhibit flagellar defects and that depletion of WDR90 leads to defective ciliogenesis in human cells. Using expansion microscopy, we reveal POC16/WDR90 precise localization as a microtubule-triplet component of the centriolar wall. Moreover, we demonstrate in vitro that both POC16 and WDR90 directly bind to microtubules through their homology domain with FAP20. Eventually, we show that cells depleted for POC16/WDR90 display broken centrioles through the destabilization of the inner scaffold complex. This work reveals the importance of POC16/WDR90 as well as of the inner scaffold in maintaining the characteristic microtubule wall integrity that is critical for centriole architecture and functions.

Acknowledgements

I have thought about this part so many times throughout my PhD… I have imagined short or long versions. Direct or coded. Formal or personal. In the end, it will be long, emotional and a bit encrypted.

I would like to thank the members of my Jury: Dr. Charlotte Aumeier, Dr. Alice Meunier and Dr. Juliette Azimzadeh for having accepted to dedicate time and attention to my work.

I promise I did not consciously choose a panel exclusively made of women. But as a feminist, I am thrilled that it is the case.

I would already like to thank the people I collaborated with during this project.

Thanks to the Steinmetz’s team, especially to Natacha Olieric, I have purified recombinant proteins from 24L of bacteria at least once in my life. So thank you for the Moscow Mule that followed.

I also learned how to fish for paramecia in Dr. Tassin’s lab. Some people describe it as a

“princess” experience as I slept in a castle (in Gif-sur-Yvette…). Others would compare it to a jungle survival due to the 52 mosquito bites I received in one night. So thank you France Koll for the insecticide-plug. And thank you AMT for having injected the beasts with GFP-ptPOC16 DNA while I was heading to Ed Sheeran’s concert at the Stade de France.

I have learned a lot with you. I have seen Science from multiple perspectives and in different environments. Thank you for having welcomed me in your labs and for having pushed me out of my comfort zone.

I owe a huge “Thank you!” to Dora Godinho and to Anita for the PhD Booster. This experience has been incredibly empowering to me and it has opened doors I never even imagined existed. So thanks for Colorado, thanks for China. And thanks for the timing. I had forgotten the competence and you brought back the confidence.

Time to dive now …

I am grateful for having been co-supervised by Paul Guichard and Virginie Hamel. Thank you for having trusted me with the first steps of your lab. Transmitted me your scientific tools. Invested in my work. Tolerated my need for autonomy. Supported my scientific communication. And thank you for having mentored me into the scientist I have become.

I deeply know it was a chance to get the opportunity to work with you two. I keep in my mind, in my heart and on my arm the precious lessons you have helped me to learn.

I have been very lucky to be part of the Centriole Architecture Lab.

Thank you Maeva for having grounded me since Day1. Thanks Nik for the support when my emotions were overflowing. Thank you Susanne for having kept everything organized.

Thanks Davide for the peperoncini. Thank you Eloïse for your guidance regarding my postdoc choice.

Now I understand what it feels like to be part of a team.

Marine, I have kept you for the perfect transition between my professional and my personal life …

I still cherish the day in October 2016 when you made me feel like I belonged in Geneva for the first time. Thank you for having held my hand on a Sunday afternoon in July 2019.

Thanks for everything in between and for everything ever since.

I would like to thank Juliette, Sandra and Sofia for having accepted me into the PeachClub.

Fede, thank you for having helped me practice patience.

Merci Isa de m’avoir soutenue sans relâche, d’avoir été juste de l’autre côté du couloir et d’avoir sû receptionner même les émotions les plus rudes.

Mariel, merci d’avoir partagé tous tes bons plans et de m’avoir accueillie dans la région.

Marine et Max, avec vous j’ai grandi et gagné à comprendre pourquoi nous sommes qui nous sommes. Sur un terrain et dans la vie.

Charlotte, j’ai du mal à admettre que Genève me manquera parfois. À cause des Rose Lychee Martini, mais aussi à cause de toi.

Louise, merci d’être devenue et restée mon meilleur souvenir d’études. Entre une thèse sur la thérapeutique de l’orgasme féminin et l’autre sur les conséquences sociales du VIH.

Entre une interne en medicine et une biologiste cellulaire … J’ai l’impression qu’on est

devenues les deux pharmaciennes les plus improbables qui soient et ça ravit mon petit esprit rebelle.

Et puis merci Julien. Même si j’ai perdu 50 euros. Précieuses minutes.

J’aimerais remercier Lavinia pour la bulle de sécurité qu’elle m’a aidée à créer tout au long des derniers mois de mon PhD. Je mesure très humblement la force de son soutien.

Je n’aurais pas l’opportunité de m’épancher en remerciements si je n’avais pas été propulsée ici par l’aide de ma famille.

Maman, merci de m’avoir éduquée à devenir cette personne indépendante qui bouscule accidentellement toutes tes peurs. Jean, merci de tout garder sous contrôle. Laurent, je n’aurais pas atteint ce niveau de satisfaction si tu ne m’avais pas enseigné la discipline et la détermination. Marion, merci de fluidifier toutes les situations. Merci Uncle d’avoir pris soin de la voiture qui me procure cette fantastique sensation de liberté. Merci Béa et Michel pour votre bienveillance à toute épreuve. Rose, merci d’avoir éclos et emergé comme la lumière au bout du tunnel. Enfin Gabin, merci de me faire croire aux miracles et de me donner toute la force nécessaire pour ne jamais rien lâcher.

Eventually, I will thank my C.R.E.W. Because you have kept me sane (and that’s no small thing!). Because no one else couples Bourgogne to laughs and tears as well as you two.

Because you are my family too. At the end of the day, no matter the city we live in, you are Home to me. I love you to a point that no guy will ever be able to understand. Three old ladies on a rocking chair, having killed them all.

There is no life journey that does not correlate to a proper soundtrack. Especially if it is very loud. And you sing on it. Poorly. In a car. ≠Cliché

I will spare you the endless playlist I have been listening to every time I was splitting my cells. But I will still share the most meaningful song: Orelsan - Epilogue.

Abbreviations

3D 3 Dimension IJ Inner Junction

AED Amino-ethyldextran MAP Microtuble Associated Protein AKAP A-Kinase Anchor Protein MCC MultiCiliated Cell

BUG Basal body Upregulated Gene MIP Microtubule Inner Protein BSA Bovine Serum Albumin MTD MicroTubule Doublet

CDK Cyclin Dependent Kinase MTOC MicroTubule Organizing Center CEP CEntrosomal Protein PFA ParaFormAldehyde

CHO Chinese Hamster Ovary PLK Polo-Like Kinase CMV CytoMegaloVirus POC Proteome Of Centriole

CP Central Pair PTM Post-Translational Modification Cryo-EM Cryo-Electron Microscopy RNA RiboNucleic Acid

Cryo-ET Cryo-Electron Tomography SAS Spindle ASsembly

DNA DesoxyriboNuclein Acid SIM Structured Illumination Microscopy DUF Domain of Unknown Function STED STimulated Emission Depletion

FAP Flagella Associated Protein STORM STOchastic Reconstruction Microscopy γ-TuRc Gamma-tubulin ring complex TIRF Total Internal Reflexion Fluorescence GDP Guanosine DiPhosphate TEM Tranmission Electron Microscopy GFP Green Fluorescent Protein TZ Transition Zone

GTP Guanosine TriPhosphate U-ExM Ultrastructure EXpansion Microscopy

KO Knock-Out WDR WD-40 Repeat

IDA Inner Dynein Arm

1 Introduction ... 2

1.1 Centriole, an evolutionary conserved organelle ... 2

1.2 Centriole Architecture ... 5

1.2.1 Microtubule-based structure ... 5

1.2.2 Centriole and advances in microscopy ... 6

1.2.2.1 Electron microscopy ... 6

1.2.2.1.1 Resin-embedded electron microscopy ... 6

1.2.2.1.2 Cryo-electron microscopy ... 10

1.2.2.2 Fluorescence light microscopy ... 14

1.2.2.2.1 Conventionnal light microscopy ... 14

1.2.2.2.2 Super resolution fluorescence microscopy ... 14

1.2.2.2.3 Physical expansion of biological samples and U-ExM ... 17

1.3 Centriole Functions ... 19

1.3.1 Centriole and the mammalian centrosome ... 19

1.3.1.1 Mammalian centrosome functions in cycling cells ... 19

1.3.1.2 Centrioles biogenesis and cell cycle ... 23

1.3.1.2.1 Cycling cells ... 23

1.3.1.2.2 Differentiated multiciliated cells ... 26

1.3.2 Centriole and the cilium ... 27

1.3.2.1 Cilium conservation and the green algae Chlamydomonas reinhardtii as a model system ... 29

1.3.2.2 Cilium ultrastructure ... 31

1.3.2.3 Cilium functions in Homo sapiens ... 32

1.3.2.4 Centriole as basal body ... 35

1.4 Centriole Protein Composition ... 41

1.4.1 Proteome of Centriole ... 41

1.4.2 POC16/WDR90 ... 41

1.4.3 FAP20 homology ... 43

1.5 Hypothesis ... 44

2 Results ... 46

2.1 Identification of Chlamydomonas Central Core Centriolar Proteins Reveals a Role for Human WDR90 in ciliogenesis ... 46

2.2 Flagellar Microtubule Doublet in vitro Reveals a Regulatory Role of Tubulin C-terminal Tails ... 82

2.3 The inner scaffold protects from centriole fracture ... 110

2.4 POC16 characterization in Paramecium tetraurelia ... 176

2.4.1 Introduction ... 176

2.4.2 Results ... 176

2.4.2.1 POC16 is a conserved basal body protein ... 176 2.4.2.2 POC16 is not essential for Paramecium tetraurelia survival 177

2.4.2.3 POC16 depletion does not impair basal body ultrastructure 178

2.4.2.4 POC16 depleted cortical units can be purified ... 180

2.4.3 Preliminary conclusions and perspectives ... 181

2.4.4 Methods ... 182

3 Discussion ... 186

3.1 POC16/WDR90 and centriole assembly ... 186

3.1.1 POC16/WDR90 and the inner junction ... 186

3.1.2 WDR90 and centriole targeting ... 188

3.1.3 POC16/WDR90 and microtubule wall assembly ... 189

3.1.4 POC16/WDR90 and core assembly ... 190

3.2 POC16/WDR90 functions ... 192

3.2.1 POC16/WDR90 and microtubule wall stability ... 192

3.2.2 POC16/WDR90 and cilium formation ... 193

3.2.2.1 POC16/WDR90 and sensory cilium formation ... 194

3.2.2.2 POC16/WDR90 and motile cilia formation ... 194

4 Appendix. ... 196

5 References ... 199

D

Figure 1

A B C

(A) Drawing of a dividing Ascaris cell. The inset represents one of the two centrosomes. Centrioles are pointed by arrows (from Bovery 1901). (B) Electron micrograph of a longitudinal section in Chlamydomonas basal body apparatus (from Harold et al., 1983). (C) Schematic representation of centriole conservation and function across evolution (from Beisson and Wright, 2003). (D) Distribution of centriolar and centrosomal proteins among eukaryotes (from Hodges et al., 2011).

2

1 Introduction

1.1 Centriole, an evolutionary conserved organelle

The centriole is an organelle identified and named for the first time by Boveri in 1887, as part of the centrosome in dividing Ascaris eggs, where he detected by light microscopy two tight and dense structures per centrosome at each pole of the cell during mitosis (Figure 1A). This discovery, coupled to his observation that DNA segregation mistakes are correlating with an abnormal number of centrosomes and centrioles, led to the hypothesis that centrosomes and centrioles are critical for the fundamental process of cell division (Azimzadeh and Bornens, 2007). In addition, centrioles are not only important within the centrosome, but also depict a crucial dual function by templating cilium formation in many organisms. In this context, they are called basal bodies (Figure 1B, C) (Beisson and Wright, 2003).

Decades of research in centriole biology have lead to the discovery of several dozens of proteins constituting this organelle through genetic screens and mass –spectrometry approaches (Andersen et al., 2003; Gonczy et al., 2000; Keller et al., 2005; Kilburn et al., 2007). Importantly, through the growing number of genomes sequenced, bioinformatic analyses have been applied from an evolutionary perspective and have shown that genes coding for centriolar proteins are conserved among ciliates species. Interestingly, four core genes (coding for the proteins Centrin-2, WDR16, SAS-4 and SAS-6) have been found to be conserved in every ciliate, suggestive of the minimal set of crucial centriolar proteins (Hodges et al., 2011). A parallel comparative genomic study on six described centrosomal components (Cep192, Plk4, SAS-6, SAS-4, Cep135 and CP110) has confirmed that SAS- 4 and SAS-6 are conserved core components (Carvalho-Santos et al., 2010). Both studies show the molecular conservation of a subset of centriolar components across evolution, which opens doors for investigations of centriolar-related processes in multiple species (Figure 1D)

Through this evolutionary perspective, essential proteins for basal body function have been shown to be ancestral in comparison to the one involved in centrosome organization,

3

suggesting that the initial appearance of centrioles has been linked to cilium formation and that their implication within the centrosome is a secondary event (Hodges et al., 2011).

Both centriole functions in cell division and ciliogenesis are involved in human development and have been linked to ultrastructural descriptions based on observations made by transmission electron microscopy. However, the fact that centrioles are present in low copy numbers (two to four) in most human cells has limited their accessibility for structural and functional investigations. In parallel, scientists have used and continue to exploit the advantages of model organisms such as Chlamydomonas reinhardtii, Tetrahymena thermophila, Paramecium tetraurelia or different species like Trychonympha to describe in details the centriole function, protein composition and architecture. Even if some species such as Caernorharbditis elegans and Drosophila megalonaster display non-canonical centriolar structures, they have proven to be extremely useful to tackle fundamental questions thanks to genetics screens and live imaging.

Thanks to these extensive studies in several species and to advances in microscopy technics, mainly electron microscopy, we currently know that centrioles are evolutionary conserved microtubule-based cylinders, important for fundamental processes such as cell division, motility and signaling (Marshall, 2009), characterized by a radial arrangement of nine microtubules-triplets as well as by their robustness.

Whether it is in the context of cell division or during cilium formation, centrioles are exposed to strong mechanical forces that require structural resistance. The components responsible for centriole cohesion remain elusive. For this reason, deeper characterization of the centriole architecture is necessary and more precisely of its different sub-regions.

This involves the use of different microscopy technics as well as biochemistry and cell biology in order to map protein complexes within the centriole.

To introduce the context of my research, I will start by describing the current view of the centriole architecture according to microscopy limitations. I will then selectively restrict the description of its function in cell division to mammalian discoveries. On the other hand, I will introduce the role of basal bodies in ciliogenesis in different models, with a special attention on Chlamydomonas reinhardtii and human cells. Eventually, I will present proteomic approaches and will focus on the uncharacterized and evolutionary conserved protein POC16/WDR90.

A

Figure 2 C B

D

250nm

450nm

A B C

(A) Schematic representation and electron micrographs of the mammalian cycling centrosome (from Bettencourt and Glover 2007). (B) Schematic represensation of the centriolar microtubule organization. Arrow points triplet to doublet transition. (C) Schematic representation of microtubules in interphase and in mitosis (adapted from Wlaczak, Cai and Khodjakov 2010). (D) Schematic representation of individual microtubule structure and dynamic (from Lasser, Tiber and Lowery 2018).

Mother

centriole Daughter

centriole

Subdistal appendage

Interconnecting fibre Distal

appendage

PCM

Microtubule a

c

b

CBA

Basal body Basal body

Transition fibre Transitional zone

Axoneme

Axoneme

CW

Cell membrane

Nucleus-associated body The microtubule-organizing centre of D. discoideum is a nucleus-associated body that consists of a multilayered, box- shaped core embedded in an amorphous corona from which the microtubules emerge.

Coiled-coil

A region of low complexity formed by a number of α-helices wound around each other, which is common among structural and motor proteins.

Coiled-coil domains are also involved in protein interactions.

the recruitment of MT-organizing molecules during interphase and mitosis. It has long been clear that protein phosphorylation has an important role in this:

among other protein kinases, Polo-like kinase-1 and Aurora A promote MT nucleation, and this is opposed by protein phosphatase-1 (PP1), PP4 and other pro- tein phosphatases (reviewed in^{REFS 43,50}). A model for the coordinate functions of Polo and Aurora A in centrosome maturation has been proposed in which Polo promotes the recruitment of the MT-organizing γTuRC⁵¹. Polo also activates the abnormal spindle pro- tein (Asp), a conserved PCM-associated protein that itself has MT-organizing properties. Aurora A also promotes MT growth from centrosomes, and this seems to be achieved through the phosphorylation of a

conserved centrosome protein, TACC, and its recruit- ment to the centrosome inD. melanogaster^52–55. TACC in complex with the MT-associated protein minispindles (MSPS; XMAP215 in Xenopus laevis) modulates astral MT behaviour^52–55.

A surprising recent finding has been that ubiquityla- tion might also play a role in maturation. The ubiquitin- ligase activity of the tumour suppressor protein BRCA1 has been found to modifyin vitroγ-tubulin on residues Lys48 and Lys344, which leads to the inhibi- tion of MT-nucleating activity by the centrosome⁵⁶. Two other proteins, spindle-defective protein-2 (SPD-2) andSPD-5, are essential for centrosome maturation in Caenorhabditis elegans^27,57,58; however, their homologues have not yet been studied in other organisms.

Figure 1 | Centriole and basal body structure. a | Schematic view of the centrosome. In each triplet, the most internal tubule is called the A-tubule; the one following it is the B-tubule; and this is followed by the most external one, the C-tubule. At its distal end, the centriole constitutes of doublets. Adapted with permission from REF. 63© (2005) Macmillan Magazines Ltd. b | Electron micrograph of the centrosome. The top inset indicates a cross-section of subdistal

appendages; the bottom inset indicates a cross-section of the proximal part of the centriole. Note the triplet microtubules (MTs) . Scale bar: 0.2 µm. Adapted with permission from REF. 32© (1992) Elsevier.c | Electron micrographs and schematic view of the flagella of green algae. There are different types of cilia and flagella, depending on the structure of the axoneme. The axoneme is a cylindrical array of nine doublet MTs that surround either zero MTs (called structure 9C0) or the two singlet MTs (structure 9C2), represented here. The two singlet MTs are called the central pair. Differences in the structure of axonemes might be reflected in their properties: for example, whether they are motile or not (reviewed in REF. 137). The transition fibres extend from the distal end of the basal body to the cell membrane. It has been suggested that they can be part of a pore complex that controls the entry of molecules into the cilia. Scale bar: 0.25 µm. CW, cartwheel (one of the first structures to appear in a forming centriole). Adapted with permission from REF. 138© (2002) Macmillan Magazines Ltd, and with permission from REF. 139 © (2004) Company of Biologists.

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NATURE REVIEWS|MOLECULAR CELL BIOLOGY VOLUME 8| JUNE 2007|⁴⁵³

Mother

centriole Daughter

centriole

Subdistal appendage

Interconnecting fibre

Distal appendage

PCM

Microtubule a

c

b

CBA

Basal body Basal body

Transition fibre Transitional zone

Axoneme

Axoneme

CW

Cell membrane

Nucleus-associated body The microtubule-organizing centre of D. discoideum is a nucleus-associated body that consists of a multilayered, box- shaped core embedded in an amorphous corona from which the microtubules emerge.

Coiled-coil

A region of low complexity formed by a number of α-helices wound around each other, which is common among structural and motor proteins.

Coiled-coil domains are also involved in protein interactions.

the recruitment of MT-organizing molecules during interphase and mitosis. It has long been clear that protein phosphorylation has an important role in this:

among other protein kinases, Polo-like kinase-1 and Aurora A promote MT nucleation, and this is opposed by protein phosphatase-1 (PP1), PP4 and other pro- tein phosphatases (reviewed in^{REFS 43,50}). A model for the coordinate functions of Polo and Aurora A in centrosome maturation has been proposed in which Polo promotes the recruitment of the MT-organizing γTuRC⁵¹. Polo also activates the abnormal spindle pro- tein (Asp), a conserved PCM-associated protein that itself has MT-organizing properties. Aurora A also promotes MT growth from centrosomes, and this seems to be achieved through the phosphorylation of a

conserved centrosome protein, TACC, and its recruit- ment to the centrosome inD. melanogaster^52–55. TACC in complex with the MT-associated protein minispindles (MSPS; XMAP215 in Xenopus laevis) modulates astral MT behaviour^52–55.

A surprising recent finding has been that ubiquityla- tion might also play a role in maturation. The ubiquitin- ligase activity of the tumour suppressor protein BRCA1 has been found to modifyin vitroγ-tubulin on residues Lys48 and Lys344, which leads to the inhibi- tion of MT-nucleating activity by the centrosome⁵⁶. Two other proteins, spindle-defective protein-2 (SPD-2) andSPD-5, are essential for centrosome maturation in Caenorhabditis elegans^27,57,58; however, their homologues have not yet been studied in other organisms.

Figure 1 | Centriole and basal body structure. a | Schematic view of the centrosome. In each triplet, the most internal tubule is called the A-tubule; the one following it is the B-tubule; and this is followed by the most external one, the C-tubule. At its distal end, the centriole constitutes of doublets. Adapted with permission from REF. 63© (2005) Macmillan Magazines Ltd. b | Electron micrograph of the centrosome. The top inset indicates a cross-section of subdistal

appendages; the bottom inset indicates a cross-section of the proximal part of the centriole. Note the triplet microtubules (MTs) . Scale bar: 0.2 µm. Adapted with permission from ^{REF. 32}© (1992) Elsevier.c | Electron micrographs and schematic view of the flagella of green algae. There are different types of cilia and flagella, depending on the structure of the axoneme. The axoneme is a cylindrical array of nine doublet MTs that surround either zero MTs (called structure 9C0) or the two singlet MTs (structure 9C2), represented here. The two singlet MTs are called the central pair. Differences in the structure of axonemes might be reflected in their properties: for example, whether they are motile or not (reviewed in

REF. 137). The transition fibres extend from the distal end of the basal body to the cell membrane. It has been suggested that they can be part of a pore complex that controls the entry of molecules into the cilia. Scale bar: 0.25 µm. CW, cartwheel (one of the first structures to appear in a forming centriole). Adapted with permission from REF. 138© (2002) Macmillan Magazines Ltd, and with permission from REF. 139 © (2004) Company of Biologists.

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NATURE REVIEWS|MOLECULAR CELL BIOLOGY VOLUME 8| ^{JUNE 2007} |⁴⁵³

5

1.2 Centriole Architecture

1.2.1 Microtubule-based structure

The centriole is a microtubule based-organelle measuring in average 450nm in length and 250nm in diameter in human cells (Paintrand et al., 1992) (Figure 2A). Its structure is a cylinder radially divided in nines entities of microtubules-triplets linked together by scaffolding structures. The centriole is polarized along its longitudinal axis. Starting from the proximal part, each microtubule-triplet is composed of an A-microtubule made of 13 tubulin-based protofilaments, followed by an incomplete B-tubule and C-tubule, each of them being composed of 10 protofilaments and closed by non-tubulin proteins (Wang and Stearns, 2017). A transition from triplet to doublet then takes place on the distal part of the centriole (Figure 2B).

Microtubules are part of the cell cytoskeleton and are responsible for essential processes such as motility, organelle positioning or intracellular transport in interphase (Etienne- Manneville, 2013). Moreover, there are crucial during cell division to organize the mitotic spindle that will allow the segregation of DNA into daughter cells (Figure 2C) (Vicente and Wordeman, 2015). Microtubules are cytoskeletal filaments resulting from the longitudinal and polarized polymerization of α- and β-tubulin heterodimers into linear protofilaments (Akhmanova and Steinmetz, 2015). In most species, thirteen protofilaments are necessary to form a complete microtubule (Figure 2D). They first assemble into a sheet-like structure templated by the γ–tubulin ring complexes (γ–TuRCs,) and close at a junction called

“seam” to form a semi-helical tubular structure of 25nm in width (Job et al., 2003; Nogales, 2000). Cytoplasmic microtubules are characterized by their dynamic instability. They undergo stochastic phenomena of growth, catastrophy and rescue upon GTP hydrolysis, which induces rapid reorganization of the cell morphology and organelles positioning (Desai and Mitchison, 1997). In contrast, centriolar microtubules are known to be long- lived stable structures (Wang and Stearns, 2017) with potentially no dynamic instability.

Several mechanisms are hypothesized to be responsible for centriolar microtubules stability. First of all, their peculiar organization in triplets might rigidify their structure and make it robust enough to resist forces, especially during mitosis. The centriole is the only

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cellular component where microtubules are organized as such and it is possible that their geometrical organization as a triplet unit strengthens them with regards to forces.

Second of all, centriolar microtubules are enriched in tubulin post-translational modifications (PTMs). They consist in a diverse subset of modifications, mostly happening on the C-terminal tail of α- and β-tubulin proteins (Song and Brady, 2015). Tubulin PTMs are described to be a regulatory code for microtubules stability, dynamics and functions selectivity (Yu et al., 2015). Typically, tubulins glutamylations marks recognized by anti- GT335 and anti-PolyE antibodies are conserved and common-used centriolar markers.

They have been proposed to be important for centriole cohesion and might play a role in maintaining their overall architecture at the microtubule level (Abal et al., 2005; Bobinnec et al., 1998).

Eventually, scaffolding proteins associated to the centriolar microtubule wall might maintain their intrinsic stability as well as their cohesion. At the cytoplasmic level, many Microtubules-Associated-Proteins (MAPs) have been shown to regulate their assembly and their dynamic (Gouveia and Akhmanova, 2010). Within the cilium, Microtubules Inner Proteins (MIPs) have been identified and hypothesized to be involved in ciliary stabilization (Nicastro et al., 2006). Therefore it is intuitive to assume that centriolar scaffolding proteins play similar roles within the centriole vicinity.

1.2.2 Centriole and advances in microscopy

Understanding centriole assembly and maintenance has been directly linked to advances in microscopy technics. In the last two decades, it has become possible to observe its architecture in greater details, opening doors to link functions to structural elements. So far, the centriole features have been described in both contexts of the centrosome as well as basal body associated to a cilium in different organisms.

1.2.2.1 Electron microscopy

1.2.2.1.1 Resin-embedded electron microscopy

The centriole ultrastructure has been described for the first time in 1956 by Bernhard and De Harven from studies of mice, tritons and chickens centrosomes using transmission

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(A) Schematic representation of longitudinal and transversal views of the basal body/cilium apparatus of flagellates (from Gibbons and Grimstone, 1960). (B) Electron micrographs of longitudinal and transversal basal bodies from Paramacium (from Dippel 1968). (C) 3D-model of the oviduct basal body (from Anderson et al., 1972). (D) Electron micrograph of the human centrosome with the mother centriole displaying appendages and the daughter centriole orthogonally oriented (from Paintrand et al.,1992).

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electron microscopy (TEM) on resin-embedded samples, showing that centrioles are cylinders made of nine tubes (Bernhard and De Harven, 1956). After that, most of the ultrastructural studies have been performed on basal bodies, which are mature centrioles docked at the base of a cilium.

Optimizing staining conditions on sperm tails has revealed that each of the nine entities are triplets of microtubules (Afzelius, 1959). This has been confirmed in three species of flagellates and has defined for the first time the current nomenclature. Within the triplet unit, tubes have been respectively named A-, B- and C-tubules from the lumen to the exterior. Moreover, based on differences observed on tubules-associated densities, it has been shown that the centriole is polarized along its longitudinal axis and two main regions have been defined (Figure 3A) (Gibbons and Grimstone, 1960).

On the proximal end, the lumen is composed of the cartwheel structure that displays a central hub from which nine radial spokes emanate, linking each microtubules-triplet. More externally, between the microtubules triplets, a connection named A-C linker bridges consecutive triplets together. On the other hand, the distal part has initially been described to be filled with unorganized fibers (Gibbons and Grimstone, 1960).

Work on Paramecium centrioles has confirmed the conservation of the centriole architecture (Figure 3B). Moreover this study described for the first time that microtubule triplet formation starts with the nucleation of a singlet A-tubule prior to the nucleation of the B-tubule from the lateral surface (Dippell, 1968). This information will be re-enforced along the manuscript.

The architecture of the mammalian centriole has been further described from resin- embedded samples from monkey oviduct, where multiple cross sections have allowed discriminating between sub-regions along the longitudinal axis. It has also highlighted peripheral structures on the distal side as well as the twist of the microtubule-triplets, resulting in the reconstruction of the first centriole 3D model (Figure 3C) (Anderson, 1972).

From work on tissue-cultured cells, the distal peripheral densities have been called for the first time “appendages” and it has been shown that the C-tubule does not cover the total length of the centriole (Vorobjev and Chentsov, 1980).

Eventually, the current referring study has been performed on centrosomes from human cycling cells, showing that the two centrioles are oriented orthogonally to each other and are surrounded by the pericentriolar matrix (PCM). Moreover, this work has emphasized

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Figure 4 B

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(A) Cryo-electron micrograph of purified centrosome from human cell, displaying the pair or centrioles oriented orthogonally to each other (from Chretien et al., 1997). (B) Representative image extracted from a tomogram reconstructed from a cryo-fixed human centrosome. Yellow boxed region represents the stoke between the mature and the procentrioles as well as the cartwheel within the procentriole (from Guichard et al., 2010). (C) Representative image extracted from a tomogram reconstructed from a cryo-fixed Triconympha centriole. Green reconstructions highlight cartwheel periodicities (from Guichard et al., 2012). (D) Representative image with inverted contrast from a tomogram reconstructed from a cryo-fixed Chlamydomonas basal body. Yellow starts represents the start of the central core. Blue reconstruction highlights the microtubule triplet and associated protein densities (from Li et al., 2012).

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the asymmetry between the mother and the daughter centriole: appendages are specific to the mother centriole and the daughter centriole is approximately twenty percent shorter than its mother (Figure 3D) (Paintrand et al., 1992).

1.2.2.1.2 Cryo-electron microscopy

Another important step in centriole structure characterization has been based on the development of cryo-fixation coupled to electron microscopy (Dubochet and Mcdowall, 1981). Mostly applied to purified samples in solution, the advantage of cryo-fixation is the preservation of 3D structures in their native state, in opposition to fixation artefacts induced during sample preparation for resin embedding.

This method has been applied for the first time to purified centrosome from human cells, preserving the overall structure and centrioles dimensions (Figure 4A) (Chretien et al., 1997). However, due to the thickness of the centriole, a limited number of electrons from the microscope beam are transmitted through the sample, which highly limits the amount of information collected. So if the comparison with TEM data could be done regarding length and diameter, more complex structures could not be retrieved in this case.

Deeper characterization of the centriole architecture could be revealed after applying cryo- electro-tomography (cryo-ET). This method developed in 1995 is based on the action to physically tilt, from -60° to +60° angle, the grid containing the sample within the microscope (Dirksen, 1971). This allows to image multiple sides of the object and to reconstruct 3D volumes. Exploiting computation further to improve the resolution and contrast, these volumes can be subdivided in different boxes (sub-tomograms), classified based on their different features, aligned to a reference and averaged, so that the different densities become visible and the resolution of structures is affined. This second approach is called sub-tomogram averaging.

Cryo-ET has been applied the first time to study the human procentriole assembly, revealing the preliminary periodicity of the cartwheel, the nucleation of the A-microtubule from a γ-TuRC-like structure and the nucleation of the B-microtubule from its surface, confirming Dippel’s observations (Figure 4B) (Guichard et al., 2010). Next, using Trichonympha, which had been reported to display an extremely long cartwheel (Gibbons and Grimstone, 1960), cryo-ET and sub-tomogram averaging have been instrumental to

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Figure 5 B

(A) Representative image extracted from a tomogram reconstructed from a cryo-fixed isolated Paramecium basal body. Quantification and length conservation of the different centriolar subregions from multiple species (from Le Guennec et al., in press). (B) 3D-reconstruction of Paramecium basal body central core. Blue structures represent the microtubule triplets and yellow densities the helical inner scaffold (from Le Guennec et al., in press).

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reveal the 3D structure of the cartwheel and its characteristic periodicities (Figure 4C) (Guichard et al., 2012).

The same year, the overall architecture of Chlamydomonas reinhardtii basal body has been described, delimiting boarders for the different centriolar sub-regions. Of particular interest, this work has emphasized the central core region that starts 100nm from the proximal side and covers 250nm in length. More precisely, it has revealed luminal structures associated to the microtubules triplets, including a stem that localizes at the inner junction between A- and B- microtubules and that is part of a molecular complex named Y-shaped linker (Figure 4D) (Li et al., 2012).

The following year, precisions have been added regarding the proximal end of the centriole, where the cartwheel has been shown to bind to microtubule triplets through the pinhead structure. Moreover, the A-C linker that bridges consecutive triplets has been reconstructed from native Trichonympha centrioles (Guichard et al., 2013).

Importantly, recent work from our lab on cryo-ET and sub-tomogram averaging on four evolutionary distant species has revealed that the Y-shaped linker is not specific to Chlamydononas reinhardtii but is conserved among eukaryote species as we have retrieved it in centrioles isolated from Paramecium tetraurelia, Naegleria gruberi and human KE37 cells in culture (Figure 5A). In parallel, the analysis of in-situ cryo-ET data from Chlamydomonas, which revealed for the first time the native architecture of centrioles in their cellular context, has been performed and confirmed Li’s work from 2012.

Strikingly, this study has also revealed that the Y-shaped linker is actually the root bound to the microtubule triplets for an entire helical inner scaffold that covers the total length of the central core and spanning about 70% of the human centriole. This inner scaffold is a very dense network of protein densities, hypothesized to be important for overall microtubule cohesion (Figure 5B, Le Guennec et al., in press).

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(A) Confocal and 3D-SIM images representing centrioles stained for SAS-4, either in interphase or in mitosis (from Gopalakrishnan et al., 2011). (B) 3D-SIM images of human human cells stained for POC5, POC1B, CPAP or Hs- SAS-6 together with Centrin (from Sydor et al., 2018). (C) STED reconstructed images of centriole (Centrin, Cep164) and transition zone (Cep290, RPGRIP1L, MKS1, TMEM67, TCTN2) markers in RPE1 basal body (from Yang et al., 2015). (D) EM and STORM reconstructed images for distal appendages mapping in RPE1 basal body (from Bowler et al., 2019)

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14 1.2.2.2 Fluorescence light microscopy

1.2.2.2.1 Conventionnal light microscopy

Historically, structural descriptions have been based on the observation of densities organized in a specific manner. To address functional and phenotypical questions, these densities have to be correlated with their protein composition.

If TEM allows ultra-structural investigation, the staining with heavy metals that is used is unspecific to any kind of proteins. Regarding the centriole composition, it has required the establishment of immunofluorescence to understand that centriolar triplets-tubes are made of tubulin (Connolly and Kalnins, 1978). However, the diffraction for conventional light microscopy is limited to 200nm in the lateral axis and 600nm in the axial axis. Due to their size of 450nm in length and 250nm in diameter, centrioles appear as small bright dots in fluorescence-light microscopy and no structural correlation is possible.

Therefore, there is a gap in scale between conventional light microscopy and transmission electron microscopy that restrains the correlation between the sophisticated structures described and their protein composition.

1.2.2.2.2 Super resolution fluorescence microscopy

A subset of centriolar proteins has been localized using EM after immuno-gold labeling, but sample preparation and image acquisition remains fastidious (Geimer and Melkonian, 2005; Graser et al., 2007; Kilburn et al., 2007). For this reason, the development of super- resolution light microscopy technics has been a key strategy to bridge the gap between EM and conventional light microscopy scales (Sieben et al., 2018).

Its principle consists in developing physical and computational imaging approaches that allow overcoming the diffraction limit and address protein localization at nanoscale resolution. It currently encompasses a group of different technics developed simultaneously in individual labs (Betzig and Chichester, 1993; Dickson et al., 1997; Hell and Wichmann, 1994). Among the most used, Structured Illumination Microscopy (SIM), is based on multiple illuminations of the object to obtain a maximum of information that are integrated into algorithms to perform reconstruction (Gustafsson, 2000; Gustafsson et al.,

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2008). This method improves theoretically the resolution to 120nm in the longitudinal axis and 300nm in the radial axis, and does not require additional sample preparation in comparison to confocal microscopy and allows illumination with multiple wavelengths at the same time (Figure 6A). SIM has been used to precisely map centrosomal proteins within the PCM vicinity and to compare their respective localization (Fu and Glover, 2012;

Gopalakrishnan et al., 2011; Lawo et al., 2012; Mennella et al., 2014; Mennella et al., 2012; Sir et al., 2011; Sonnen et al., 2012). Regarding its use to map proteins within the centriole, the proximal marker SAS-6 protein, responsible for the cartwheel structure described by EM, has initially been shown as a luminal dot in comparison to PCM markers in Drosophila centrioles (Dzhindzhev et al., 2014; Gartenmann et al., 2017). This method has later on been used to precise the putative localizations of centriolar proteins such as Centrin, POC1B or POC5 in human U2OS cells (Figure 6B) (Sydor et al., 2018), refining information obtained by confocal microscopy, nevertheless without the sufficient resolution to be correlated with EM structures.

A second type of super-resolution light microscopy is STimulated Emission Depletion (STED) microscopy, based on the direct deactivation of out-of-focus neighboring fluorophores (Hell and Wichmann, 1994). This approach increases the contrast between signal and noise, hence improving resolution without computational approaches. The current longitudinal resolution for 3D-STED is about 50nm for the radial is about 130nm, which is the necessary range for centriole imaging. Nonetheless, STED microscopy is limited by the necessity of high laser intensity that are susceptible to photobleach weak signals. Still, STED has been applied to centrioles (Lau et al., 2012; Lukinavicius et al., 2014; Lukinavicius et al., 2013) and has been potent to map for example the transition zone components in different ciliated mammalian cell lines (Figure 6C) (Yang et al., 2015).

However, the localization of luminal centriolar proteins has not been addressed with this technic yet.

One of the latest and more precise super-resolution method is STochastic Optical Resolution Microscopy (STORM) which consists in the stochastic activation of photo- switchable fluorophores (Bates et al., 2013). The blinking of each fluorophore is individually visualized, including in dense environments like the centriole, inducing high spatial resolution (20nm in lateral, 50nm in axial). The major drawback is currently the necessity to use specific imaging dyes for optimal blinking of the fluorophores and a

Figure 7 A

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(A) Confocal and U-ExM images of isolated Chlamydomonas basal body stained for tubulin and PolyE.

Comparison between 2D-STORM and U-ExM on Chlamydomonas basal body apparatus stained for tubulin (from Gambarotto 2019). (B) U-ExM data of human U2OS cells stained for tubulin and HsSAS-6, FAM161 or CP110 to identify the different centriolar subregions. Mapping and quantification for POC1B, FAM161A, POC5 and Centrin-2 respective localization at the human central core and superimposed on Paramecium inner scaffold reconstruction (from Le Guennec, in press).

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dedicated microscope. Similarly to STED microscopy, STORM for centriolar components mapping has initially been focused on the localization of the appendages protein Cep164 (Sillibourne et al., 2011) but has also been compared to 3D-SIM on isolated Chlamydomonas reinhardtii basal bodies (Hamel et al., 2014; Hamel et al., 2017). This method has then been extended to the mapping of the entire distal appendages complex (Yang et al., 2018). Notably, STORM has lately been correlated to EM for the precise localization of appendages components at different stages of the cell cycle (Figure 6D) (Bowler et al., 2019).

Even if these different technics have been insightful to localize centrosomal proteins, they all exhibit singular limits: the computation required for SIM induces the risks to create artefacts; STED necessitates high intensity laser that damage samples overtime; and STORM is relying on the efficacy of imaging dyes.

Overall, these technics require a strong expertise in imaging and sophisticated microscopes that limit their use on the daily-basis within biology laboratories.

1.2.2.2.3 Physical expansion of biological samples and U-ExM

A recent alternative to super-resolution for imaging small structures such as centrioles with conventional microscopes has been the development of expansion microscopy (Chen et al., 2015). It consists in the isotropic physical expansion of biological samples within an extendable gel that is stained and imaged (Wassie et al., 2019).

Our lab has adapted this principle to centrioles and developed Ultra-structure Expansion Microscopy (U-ExM) for the nanoscale mapping of centriolar proteins, outreaching the current resolution of STORM. We have notably used it on Chlamydomonas basal bodies and on centrioles from cultured cycling U2OS human cells (Figure 7A,B) (Gambarotto et al., 2019) (Le Guennec et al., in press). We are currently able to image centrioles expanded up to four times in cellulo and to address both the mapping of centriolar components as well as phenotypical analysis.

Strikingly, we have localized a complex of four interacting proteins at the central core of the human mature centriole and we have correlated their respective fluorescence signal to the inner scaffold discovered by cryo-ET (Figure 7B). We have shown that POC1B, POC5, FAM161 and Centrin-2 endogenously span about 70% of the human centriole length and are located inside its lumen, close to the microtubule wall.

Figure 8

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(A) Electron micrographs of human basal body post immuno-labeling of POC1B, with gold particles at the transition zone, the cylinder wall and the cartwheel (from Pearson et al., 2009). (B) Electron micrographs of human centrosome post immuno-gold labelling of POC5, which localizes in the lumen at the distal part of the centriole (from Azimzadeh et al., 2009). (C) Electron micrographs of human centrosome post immuno-gold labelling of Centrin, which localizes in the lumen and close to appendages (from Paoletti et al., 1997).

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This finding is reminiscent of previous structural observations using immuno-gold labeling.

Indeed, POC1B has been described in human basal bodies near the transition zone, near the cartwheel but also along the internal side of the microtubule wall (Figure 8A) (Pearson et al., 2009). Similarly, POC5 has been localized in the distal part of the lumen, close to the microtubule wall in cycling cells (Figure 8B) (Azimzadeh et al., 2009), similarly to Centrin-2 in isolated human centrioles (Figure 8C) (Paoletti et al., 1996). FAM161A has not been mapped yet.

However, how the four proteins are localized with respect to one another, how they bind the microtubule wall and how they ensure triplet cohesion remain to be investigated and to be integrated in regards to centriole assembly and maintenance. Thank to the development of U-ExM, we can now test the function of this inner scaffold protein complex in cultured human cells.

1.3 Centriole Functions

1.3.1 Centriole and the mammalian centrosome

The centrosome is the main microtubule-organizing center (MTOC) in animal cells. This non-membranous organelle is composed of two centrioles surrounded by a protein organization termed pericentriolar matrix (PCM) (Paintrand et al., 1992). The PCM is a platform for proteins concentration and signaling (Fry et al., 2017). It contains large anchoring coiled-coil proteins as well as an enrichment of γ -tubulin and γ-TuRCs that nucleate cytoplasmic microtubules (Moudjou et al., 1996; Tassin et al., 1998). Additionally, small non-membranous protein condensates named centriolar satellites are associated to the centrosome and are involved in trafficking centrosomal components for its homeostasis (Kubo et al., 1999).

1.3.1.1 Mammalian centrosome functions in cycling cells

In physiological conditions, there is one centrosome per cell in G1-phase, inherited from the previous cell division. In cycling cells, the centrosome is duplicated in S-phase

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concomitantly to DNA duplication. The two newly formed centrosomes separate and undergo migration towards opposite poles of the cell for mitotic entry, where they will nucleate the microtubule-based mitotic spindle to ensure cytokinesis (Azimzadeh and Bornens, 2007). Moreover, the centrosome is a signaling platform highly enriched in cyclin-dependent kinases (CDKs) and associated phosphatases, providing an additional level of cell cycle regulation (Doxsey et al., 2005).

Since its identification, several studies have been performed to tackle the exact function of the centrosome in cell division. More specifically, it has been of great interest to uncouple the different centrosomal compartments in order to identify the minimal components necessary for mitosis.

Using microsurgery in BSC1 African green monkey kidney cells, it has initially been shown that centrioles are dispensable to enter the first mitosis but are crucial to generate a bipolar spindle in the next cell cycle (Hinchcliffe and Sluder, 2001). Even though cells can generate alternative MTOCs without centrioles, they cannot enter S-phase without a centriolar template (Maniotis and Schliwa, 1991). This result has then been confirmed using more precise methods such as laser ablation, highlighting a G1 to S-phase checkpoint (Khodjakov and Rieder, 2001). The evidence of an asymmetric contribution between mother and daughter centriole has followed, suggesting that appendages would play a role in microtubule-nucleation and cell cycle progression (Piel et al., 2000).

Nevertheless, centrioles are not sufficient to ensure cytokinesis. Displacement of the PCM component AKAP450 and disengagement from centrioles have shown that signaling at the centrosome is equally important for progression from G1 to S-phase in RPE1 cells, as well as for proper cytokinesis in HeLa cells (Keryer et al., 2003). The function of individual PCM components, especially of γ-tubulin, has then been addressed using small interfering RNA (Zimmerman et al., 2004).

Additionally, activation of p21 and p53 checkpoints has been identified under centrosome disruption, explaining the G1 arrest (Mikule et al., 2007; Srsen et al., 2006). Deactivating the p53 checkpoint has since been a tool to study the functionality of centrosomal proteins beyond G1-phase in mammalian cells (Fong et al., 2016).

Eventually, due to their heterogeneity in size and composition, less extensive work has been performed on the function of centriolar satellites in cell division. Depletion in U2OS cells for the major satellite molecular player, the scaffolding protein PCM1, has shown selective reduction in the recruitment of centrosomal components such as the proteins