A Dissertation Entitled Centriole Inheritance During Fertilization of Drosophila Melanogaster by Atul D. Khire Submitted To

Home , Centriole

A dissertation

entitled

Centriole Inheritance during Fertilization of Drosophila melanogaster

Atul D. Khire

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Biological Sciences

______Dr. Tomer Avidor-Reiss, Committee Chair

______Dr. William Taylor, Committee Member

Dr. Song-Tao Liu, Committee Member

______Dr. Rafael Garcia-Mata, Committee Member

______Dr. Ronny Woodruff, Committee Member

______Dr. Sue Hammoud, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo December 2017

Under copyright law, Chapter 1 and Chapter 5 of this document may not be reproduced without the expressed permission of the author. An Abstract of

Centriole Inheritance during Fertilization of Drosophila melanogaster

Atul D. Khire

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biological Sciences

The University of Toledo

December 2017

Centrioles, surrounded by peri-centriolar material (PCM), play an important role in cell division and signaling. The cell requires two centrioles for an effective cell division.

These first two centrioles will duplicate and make up the centrosome of an animal. The number of centrioles inherited by the Drosophila zygote has always been contentious.

The female centrosome is eliminated during oogenesis and thus, the zygote centrioles are paternally inherited. The existing hypothesis at the beginning of this project was that

Drosophila sperm cells have only one functional centriole called the giant centriole (GC), and it was postulated that this GC is the only functional centriole inherited by the zygote after fertilization. Here, we report that, not only GC but also PCL is also inherited from the sperm into the zygote post-fertilization. These observations were made using indirect methodology, like labelling the maternally recruited PCM proteins and microtubules around GC and the PCL. Direct detection of GC and PCL in the zygote was not possible due to a mechanism during spermatogenesis called centrosome reduction. Centrosome reduction is a conserved phenomenon, during which there is step wise loss of proteins

iii associated with the centrosome and subsequent modifications in its structure. However, the mechanism underlying centrosome reduction was not known and also its importance was unclear. Here, we show that during Drosophila spermiogenesis, centrosomal protein

Asterless (Asl) levels decrease as it becomes undetectable in mature sperm. Asl reduction is mediated by centriole duplication master regulator, Polo-like Kinase 4 (Plk4) and

Slimb ubiquitin ligase, which degrades Plk4. Forced increase of Asl in the mature sperm, whether through Asl overexpression, or through mutating Plk4 or Slimb ubiquitin ligase , reduced animal fertility and also caused delayed embryo development.

In parallel to centrosome reduction, the GC and the PCL also undergo remodeling throughout spermiogenesis. This is characterized by enrichment of specific proteins and modification in the structure of GC and PCL. These modifications result in the formation of an atypical GC and PCL in the mature sperm of the fly. Two isoforms of a centriolar protein Poc1, Poc1A and Poc1B, show enrichment in GC and PCL respectively. Further, this remodeling in both structure and protein configuration is essential for normal fly embryo development post fertilization. Once, the remodeled atypical centrioles enter the oocyte, where they are immediately reconstituted. Reconstitution is process where centrosome becomes functional by recruiting maternal PCM from the oocyte and nucleating astral microtubules. The mechanism of this centrosome reconstitution is not yet characterized. In Drosophila, maternal activated Plk4 plays a role in modulating centrosome reconstitution. Activation of Plk4 was found to be essential normal recruitment of PCM and astral microtubules to the PCL and GC. Further, maternal Plk4 also was essential for the development of Drosophila embryo.

To my wife, my sister and my parents. This would not have been possible without your unwavering support.

Acknowledgements

First and foremost, I would like to thank my mentor Dr. Tomer Avidor-Reiss for guiding me throughout my PhD journey. He helped me at every step, be it scientific or personal, he has been pillar of strength and support for the last five years. I am truly blessed to get an opportunity to work in his lab. It is because of Tomer, I am able to fulfill my dream of becoming a scientist. In addition to this, I am also thankful for the suggestions of all the committee members namely; Dr. William Taylor, Dr. Song-Tao

Liu, Dr. Rafael Garcia-Mata, Dr. Sue Hammoud and Dr. Ronny Woodruff. I also acknowledge the advice and inputs of Dr. Deborah Chadee and Dr. Richard Komuniecki.

Finally, I also appreciate the support of the Chair of the Biology Department, Dr. Bruce

Bamber.

I am also extremely thankful to Marcus Basiri, Dr. Stephanie Blachon, Dr.

Amitabha Mukhopadhya, Andrew Ha, Lilli Fishman, Kyoung Jo, Sushil Khanal, Alberto

Vizuet, Maryum Jawaid, Michela Roberts for all help and wonderful memories. I would also like to thank Dr. Alan Hammer for proofreading this dissertation.

Last but not the least I am extremely grateful to my parents, my sister Aparna and my wife Pooja for providing me invaluable support and encouragement. Thanks a lot for being my there for me.

Table of Contents

Abstract ...... iii

Acknowledgements…………………………………………………………………….. vi

List of Figures ...... xiii

List of Abbreviations ...... xv

List of symbols ...... xvii

Preface ...... xviii

Chapter 1 Introduction...... 1

1.1 Centriole biogenesis in Drosophila ...... 1

1.2 Drosophila centrosome architecture ...... 4

1.3 Drosophila melanogaster as an ideal model organism for centrosome research.. 7

1.4 Centrosome biology during Drosophila spermatogenesis ...... 8

1.4.1 Centrosome Reduction ...... 10

1.4.2 Centrioles during fertilization of sexually reproducing animals...... 12

1.4.3 Origin of centrioles in animal zyygote...... 16

Chapter 2 The Origin of the Second Centriole in the Zygote of Drosophila melanogaster ...... 21

vii

2.1 Abstract ...... 21

2.2 Introduction ...... 22

2.3 Materials and Methods ...... 26

2.3.1 Fluorescence microscopy ...... 26

2.3.2 Embryo development ...... 27

2.3.3 Statistical methods ...... 27

2.3.4 Transgenic Flies ...... 27

2.3.5 Antibodies ...... 27

2.3.6 Generating homozygote aslmecD embryos ...... 28

2.4 Results ...... 29

2.4.1 PCL and GC undergo centrosome reduction during late spermiogenesis ...... 29

2.4.2 Zygote has two centrioles immediately after fertilization ...... 29

2.4.3 Unlike their daughter centrioles, the first two zygotic centrioles do not

incorporate centriolar proteins...... 31

2.4.4 Homozygote aslmecD zygotes have two centrioles ...... 32

2.5 Discussion...... 33

2.6 Acknowledgments ...... 34

2.7 Author contributions ...... 34

Chapter 3 Asterless Reduction During Spermiogenesis is Regulated by Plk4 and is

Essential for Zygote Development in Drosophila ...... 42

3.1 Abstract ...... 42

3.2 Methods and Materials ...... 43

3.2.1 Transgenic Flies...... 43

viii

3.2.2 Antibodies...... 44

3.2.3 Fluorescence Microscopy...... 45

3.2.4 Testes Fluorescence Microscopy...... 45

3.2.5 Embryo Fluorescence Microscopy...... 45

3.2.6 Western blot ...... 46

3.2.7 Ubiquitination IP ...... 47

3.2.8 Phosphorylation IP...... 47

3.2.9 Larval hatching...... 47

3.2.10 Embryo development...... 47

3.2.11 Statistical Methods...... 48

3.3 Results ...... 48

3.3.1 Asl Protein Levels are Dynamically Reduced During Spermiogenesis ...... 48

3.3.2 Asl Reduction is Attenuated in Asl with Domain Deletions ...... 50

3.3.3 Plk4 and Slimb Regulate Asl Reduction ...... 51

3.3.4 Asl Reduction is Essential for Post-Fertilization Development ...... 52

3.4 Discussion...... 54

3.5 Acknowledgment ...... 58

Author contribution ...... 58

Chapter 4 Centriole Remodeling during Spermiogenesis in Drosophila ...... 73

4.1. Abstract ...... 73

4.2 Methods and Materials ...... 74

4.2.1 Transgenic Flies ...... 74

4.2.2 Fluorescence Microscopy ...... 75

4.2.3 Testes Fluorescence Microscopy ...... 76

4.2.4 Testes Super Resolution Microscopy...... 77

4.2.5 Antibodies...... 78

4.2.6 Western blot ...... 78

4.2.6 Tissue Processing and Transmission Electron Microscopy ...... 79

4.2.7 Electron Tomography ...... 80

4.2.8 Correlative Light and Electron Microscopy ...... 80

4.2.9 One Hour Embryo Development Assay ...... 80

4.2.10 Seventy-Two Hour Embryo Development Assay Analysis and Larval

Hatching ...... 81

4.2.11 Sperm Flagellum Detection in Embryo ...... 81

4.2.12 Statistical Methods ...... 81

4.3 Results and Discussion ...... 82

4.3.1 Poc1 enrichment during spermiogenesis ...... 82

4.3.2 Sperm centrioles are modified into atypical structures...... 82

4.3.3 Poc1 has a differential role in GC and PCL formation ...... 84

4.3.4 Sperm centriole remodeling is essential for the formation of zygotic

centrosomes...... 86

4.3.5 Poc1 enrichment in the spermatocyte and spermatid is essential for

embryogenesis...... 87

4.5 Acknowledgements ...... 90

4.6 Author Contributions ...... 90

Chapter 5 Maternal Plk4 is essential for Centrosome Reconstitution Post-

Fertilization ...... 106

5.1 Abstract ...... 106

5.2 Methods and Materials ...... 107

5.2.1 Transgenic Flies...... 107

5.2.2 Antibodies...... 108

5.2.3 Fluorescence Microscopy...... 108

5.2.5 Embryo Fluorescence Microscopy...... 108

5.2.6 Western blot ...... 109

5.2.7 Embryo development...... 110

5.2.8 Statistical Methods...... 110

5.3 Results and Discussion ...... 110

5.3.1 Maternal Plk4 is essential for Centrosome Reconstitution ...... 110

5.3.2 Maternal Plk4 is activated upon fertilization...... 111

5.3.3 Plk4 mediates centrosome reconstitution via Asl N-terminus ...... 112

5.3.4 Model for Centrosome Reconstitution...... 114

Chapter 6 Discussion ...... 122

6.1 General Conclusions ...... 122

6.1.1 The PCL as the second zygotic centriole of Drosophila melanogaster ...... 123

6.1.2 Plk4 regulates centrosome reduction and is essential for normal embryo

development ...... 125

6.1.3 Centrosome is remodeled during Drosophila spermiogenesis ...... 127

6.1.4 Maternal Plk4 is essential for centrosome reconstitution...... 128

6.2 Future Directions ...... 129

6.2.1 Development of phosphomimetic in Asl domain 1 ...... 129

6.2.2 Identification of downstream effectors in centrosome reconstitution pathway

...... 129

References ...... 130

Appendix A…………………………………………………………………………… 158

xii

LIST OF FIGURES

1-1 Cross section of a centriole ...... 1

1-2 The centriole duplication cycle ...... 1

1-3 The architecture of the centrosome ...... 6

1-4 Centrosome biology during Drosophila spermatogenesis ...... 10

1-5 Centrosome reduction in Drosophila ...... 11

1-6 The centrioles facilitate male and female pronucleus migration ...... 16

1-7 Hypotheses for centriole Inheritance ...... 17

2-1 The PCL and GC undergo centrosome reduction and recruit PCM ...... 35

2-2 Maternally GFP tagged centriolar proteins label DC of the GC and the PCL ...... 36

2-3 Two centrioles are observed in an oocyte mutant for centriole duplication ...... 37

2-4 The PCL in Drosophila fertilization ...... 38

3-1 Asl is overexpressed in the testes of uAslGFP and eAslGFP ……………………59

3-2 Asl is reduced during spermiogenesis……………………………………………60

3-3 Specific coiled-coil domains in Asl are essential for normal Asl reduction……..61

3-4 Asl truncation proteins ...... 62

3-5 Plk4 and Slimb regulate Asl reduction ...... 63

3-6 Plk4 regulates Asl reduction ...... 64

3-7 Attenuated Asl reduction reduces post-fertilization zygotic development ...... 65

xiii

4-1 Poc1 constructs, as well as PCL and GC structure ...... 91

4-2 Poc1 is enriched in the modified sperm centrioles ...... 92

4-3 Poc1A and Poc1B have differential roles in centriole and sperm ...... 93

4-4 Characterization of poc1 mutants ...... 94

4-5 Atypical centrioles are essential for normal fertility and embryogenesis ...... 95

4-6 poc1W45R males have normal fertility ...... 96

4-7 Poc1 isoforms have differential roles during spermiogenesis ...... 97

4-8 Paternal Poc1 enrichment essential for normal fertility & embryogenesis ...... 98

5-1 Maternal Plk4 is essential for centrosome reconstitution ...... 115

5-2 Maternal Plk4 is activated upon fertilization ...... 116

5-3 Plk4 mediates centrosome reconstitution via Asl N-terminus ...... 117

5-4 Model for centrosome reconstitution ...... 130

xiv

LIST OF ABBREVIATIONS

3D-SIM…………….Three-dimensional structured illumination microscopy

A……………………Almost Needle Spermatid Asl………………….Asterless Protein

BSA………………...Bovine Serum Albumin

C……………………Spermatocyte Cnn…………………Centrosomin

DAPI……………….4',6-Diamidino-2-Phenylindole, Dihydrochloride DC………………….daughter centrioles DPlp………………..Drosophila Pericentrin like Protein eAslGFP……………Endogenous promoter AslGFP EE…………………..elongated spermatid

FPN…………………Female Pronucleus

GC ...... giant centriole G…………………….Spermatogonium

HP-FFS……………..High-Pressure Freeze Fracturing Staining

IE……………………Intermediate elongated spermatid IN…………………....Initial Spermatid

MPN………………. Male Pronucleus MT…………………. Microtubule MTOC………………Microtubule Organizing Centre

P……………………..Presence PACT………………..Pericentrin-AKAP450-centrosomal-targeting PAGE………………. Polyacrylamide Gel Electrophoresis PBS………………….Phosphate Buffer Solution

PBST-B…………….. Phosphate Buffer Solution-Triton-BSA PBST……………….. Phosphate Buffer Solution-Triton PCL ...... Proximal Centriole Like PCM ...... Pericentriolar material Plk4 ...... Polo like kinase-4 Poc1………………… Protein of Centriole 1

R……………………. Round Spermatid RNAi………………...RNA interference

S………………...... Spermatozoa SEM…………………standard error of the mean

TEM…………………Transmission Electron Microscopy uAslGFP……………..Ubiquitin promoter AslGFP

xvi

LIST OF SYMBOLS

♂…………………….Male ♀...... Female (-)...... Undetectable

xvii

PREFACE

Centrosomes were first discovered in the context of reproductive biology. This dissertation delves into some of the fundamental aspects of centrosome biology using

Drosophila melanogaster as a model organism.

Chapter 1 starts by presenting a literature survey of centrosome biology, namely, its origins, its role in cell cycle and its architecture. Further, it also reviews the role of centrioles during sexual reproduction, centrosome biology in sperm cells and a phenomenon called centrosome reduction. The existing paradigm at the beginning of this project was that Drosophila sperm had only one functional centriole called the giant centriole (GC). It was hypothesized that this GC is the only functional centriole inherited by the zygote after fertilization. Our lab was the pioneer in discovering the presence of a second centriole in the Drosophila sperm cells, which was termed as the Proximal

Centriole-like (PCL) (Blachon et al., 2009)

Chapter 2 is a manuscript chapter that has been published as The Origin of the

Second Centriole in the Zygote of Drosophila melanogaster (Blachon and Khire et al.,

2014). Here we describe how both the PCL and GC are inherited post fertilization.

Chapter 3 is a manuscript chapter previously published as Asterless Reduction

During Spermiogenesis is Regulated by Plk4 and is Essential for

Zygote Development in Drosophila (Khire et al., 2015). Here, we describe a novel phenomenon called centrosome reduction. Centrosome reduction is a process wherein the

xviii centriole loses most of its proteins during spermiogenesis. It is known to occur in most animal spermatogenesis, however, the mechanism of centrosome reduction was not known till to this day. We have shown here, for the first time, that centrosome reduction is an active phenomenon mediated by a kinase. In addition, centrosome reduction is also essential for normal animal development.

Chapter 4 is a manuscript chapter previously published as Centriole Remodeling during Spermiogenesis in Drosophila (Khire and Jo et al., 2016). This chapter also explores a novel concept called centrosome remodeling. We show evidence that the centrosome is not simply reduced but that it is remodeled. The amount of some centriolar proteins decreases during spermiogenesis while that of others increases. Based on evidence presented in the thesis, we show that centrosome remodeling is essential for normal animal development

Chapter 5 introduces a concept called centrosome reconstitution. It states that immediately after fertilization, the centrioles provided by the sperm become functional by recruiting the maternal PCM (Pericentriolar material) proteins from the embryo. The mechanism of sperm centriole reconstitution was previously not known. Here, for the first time, we present data to show that centrosome reconstitution is mediated by the maternal kinase dependent pathway.

Chapter 6 summarizes and discusses the findings made in this dissertation.

xix

Chapter 1

Introduction

1.1 Centriole biogenesis in Drosophila

The pioneering work by Holenbeck, Fleming and van Beneden in the 19th century, led to the discovery of the centriole (Wheatly, 1982), which is a cylinder ∼200 nm in diameter and 500 nm long in animal cells (Avidor-Reiss T et al., 2012). In resting cells, a mature centriole can template cilia or flagella by attaching itself to the plasma membrane, thus playing a role in signal transduction and cell motility (Sluder et al.,2014). The most interior and the proximal-most part of the centriole is a cartwheel structure that has nine spokes, each linked to microtubule blades that form the microtubule wall made of triplet microtubules (Fig. 1-1). It is surrounded by an electron dense pericentriolar material

(PCM) that provides the nucleating center for spindle and astral microtubules.

Figure 1-1: Centriole cross section in the proximal end. The barrel shaped centriole. The centriole’s cross-section shows a cartwheel structure with 9-fold triplet microtubules.

Figure 1-2: The centriole duplication cycle: The S-phase marks the beginning of centriole duplication, wherein a procentriole is formed near the proximal end of the mother centriole. Next, in the G2 phase, the procentriole elongates, with both the centrioles recruiting PCM. This accumulation of PCM increases during mitosis M-phase) further leading to astral microtubule nucleation. Lastly, in the G1/G0 stage, the mature centriole develops appendages at the proximal end, where it anchors cilia (Adapted from Avidor-Reiss et al.,2012).

As a cell gets primed for cell division, the two centrioles begin to change immediately.

Firstly, the cilium is absorbed, leading to the internalization of the two centrioles

(Kitagawa D, et al. 2011). Each cell has a pair of centrioles, one attached orthogonally to the other (Avidor-Reiss et al., 2012). The attachment between two centrioles is lost as they disengage in early M phase, and remain linked by a loose fibrous connection

(Glover et al., 2015). Assembly of a procentriole perpendicular to the mother centriole begins in G1/S and the procentriole subsequently elongate throughout G2 until they are similar in size to their respective mothers (Bettencourt-dias et al., 2016). Before the cell enters mitosis, the older centriole is able to recruit PCM as well as nucleate astral microtubules for the spindle pole formation (Bettencourt-dias et al., 2016). The fibrous leash among centrosomes resolves, permitting centrosomes to decouple and move to opposite sides of the cell as the spindle poles (Fig. 1-2) (Kim TS, et al., 2013).

Centriolar proteins Polo like kinase-4 (Plk4), Sas-6 and Ana2, play a crucial role by localizing at the site of procentriole assembly at the G1–S transition. Plk4 is a serine/threonine kinase. At its N-terminal, it has a kinase domain which is followed by polo-box motifs. These motifs play an important role in recruitment of Plk4 to the centriole Ana2, another important centriolar protein plays an essential role in centriole biogenesis. Coiled-coil domains of Ana-2 was shown to interacting with C-terminal of

Plk4. In addition to this, Plk4 also phosphorylates Ana-2, which in turn facilitates recruitment of Sas-6 to the centriole (Arquint C, et al., 2014, Stevens NR, et al., 2010,

Stevens NR, et al., 2010). Sas-6 is a key centriolar protein which makes up the core of the centriole and is also an important player in imparting the nine-fold symmetry to the centriolar architecture (Kitagawa D, et al. 2011, Nakazawa Y, et al. 2007, van Breugel M, et al. 2011).

Sas-6 was thought to be the earliest protein at the site of procentriole assembly

(Strnad P, et al. 2007). However, recent reports suggests that Plk4/SAK, a polo-like kinase, localizes at a site on the procentriole, prior to Sas-6, suggesting that it recruits

Sas-6 (Kim TS, et al. 2013). In C. elegans, Sas-6 is recruited to the centriole by ZYG-1, a

3 functional orthologue of PLK4, thus this might be an evolutionarily conserved mechanism of centriole assembly (Lettman MM, et al. 2013). Plk4 makes the ring-like organization around the proximal region of the centriole in early G1 phase, and this organization coincides with the subsequent centriole duplication pathway, further implicating Plk4 in marking the origin of centriole duplication (Kim TS, et al. 2013).

More recent studies have shown that Plk4 phosphorylates Ana2, which triggers the recruitment of Sas-6, initiating procentriole assembly (NS Dzhindzhev, et al. 2014).

In Drosophila, the targeting of Plk4 to the centriole is mediated by a centrosomal protein Asterless (Asl) (Dzhindzhev NS, et al. 2010), while in C. elegans, another centrosomal protein, SPD-2 interacts with Plk4 (Delattre M, et al. 2006). Furthermore, in mammalian cell culture, the orthologues of these two proteins, CEP152 and CEP192, respectively, show similar interaction with PLK4 (Kim TS, et al. 2013, Sonnen KF, et al.

2013).

1.2 Drosophila Centrosome Architecture

We use Drosophila as model organism as it has a well-defined tissue organization so that we can stage and show different centriolar developmental stage (González et al. 1998).

Using antibodies to label PCM proteins like tubulin, it became convenient to observe the ability of centrosome to recruit PCM and nucleate astral microtubules (McGill and

Brinkley, 1975; Connolly and Kalnins, 1978; Heidemann and Kirschner, 1975).

Identification of centriolar proteins including, Bld10, Sas-6, Sas-4, Ana2 and Ana1.

Bld10 was shown to be a centriolar marker in Chlamydomonas. Markers like Ana-2 and

Ana-1 were identified using RNAi methodologies in Drosophila S2 cells. Sas-4 as a centriolar microtubule protein was first discovered in C.elegans (Matsuura et al., 2004;

Goshima et al., 2007; Kirkham et al., 2003; Dammermann et al., 2004) was mainly done through immunofluorescence and genetic manipulations..

The use of electron microscopy provided a major boost to our understanding of the centrosome. This has enabled us to understand the cartwheel architecture of the centrioles much better. Embryonic and somatic cell centrioles have doublet microtubules

(MTs) that are 200 nm long and 200 nm wide with cartwheels (Debec et al., 1999; Moritz et al., 1995). In male germ stem cell, however the mother centriole has triplet microtubules before disengagement (Gottardo et al., 2015b). Wing cells and sensory bristles centrioles shows a triplet microtubule without a cartwheel (Debec et al., 1999).

Moreover, the structures that anchor the centrioles to the cells, called the subdistal appendages are not observed (Callaini et al., 1997).

The advent of super-resolution light microscopy has provided tremendous insight into the position and association of the multiple components of the centrioles and the surrounding PCM (Fu et al., 2016; Fu and Glover 2012; Dzhindzhev et al., 2014;

Mennella et al. 2012). As shown in Fig. 1-3, Sas-6 and Ana-2, are localized at the central zone (I) of the centriole. Sas-6 has been shown to be the core of cartwheel structure (

Cottee et al., 2015; Van Breugel et al., 2011; Kitagawa et al. 2011). Sas-6 not only colocalizes with Ana2 but also with Cep135/Bld10 (Hilbert at al. 2016; Matsuura et al.,

2004). Bld10 also shows a radial like symmetry with its C-terminal part in the central zone (I), with its N-terminus projecting out, required for the conversion of daughter centriole to mother centriole (Fu et al., 2016)

The main component of Zone II, as shown in Fig. 1-3, is Sas-4, which connects the microtubule and the cartwheel, and interacts with Ana-2/Sas5 (Hsu et al., 2008;

Tang et al., 2011). Similar to the centriole diameter seen by EM, the circular structure of

Sas-4 in cultured cells is 200 nm diameter, (Fu and Glover 2012). Even the immuno-EM studies show that Sas-4 is at the core of the centriole extending from the inner surface towards outer surfaces.

Figure 1-3 The architecture of the centrosome. First and second zone proteins represent core centriolar proteins. PCM proteins fall in third and fourth zone. Distal end of the centriole is represented by fifth zone. (Adapted from Lattao et al., 2017)

Polo and Spd-2, which are PCM components, are also found in the Zone II. The foundation of the PCM is built with the assembly of the third zone (III). The marker for this zone is D-plp protein. It is organized around the wall of the centriole with its C- terminal forming a loop, with a radius of 86 nm and its N-terminus protruding outside within the PCM at 138 nm (Mennella et al., 2012). Zone III also have other proteins like

Asl and Plk4 that coincide with D-plp.

During mitosis, PCM proteins like Cnn, Spd-2 and γ-tubulin, that help in nucleating astral microtubules are recruited and organized to form the fourth zone (IV).

CP110, which acts as a capping protein for regulating the centriole length occupy the fifth zone (V).

1.3 Drosophila melanogaster as an ideal model organism for centrosome research.

The fruit fly Drosophila melanogaster is a widely used model organism for various disciplines such as genetics, molecular biology, developmental biology, and cell biology.

Thomas Morgan was the first to use flies during his research at Columbia University

(Morgan, T.H et al., 1916). Drosophila has lots of advantages such as shorter life cycle

(10 days at 25 °C), high fertility, relatively cheaper to maintain fly stocks and its applications in broad biological field. Moreover, about 75% of known human disease genes have identifiable matches in the fruit fly genome (Reiter et al., 2001). Thus,

Drosophila can serve as a model organism to understand the molecular mechanisms of diverse human diseases and conditions including cancer, aging, infertility, neurodegenerative disorders and drug abuse (Brandt A, et al., 2013; Avidor-Reiss et al.,

2015). Finally, the genomes of most of the Drosophila species have been have been indexed in universally known database, Flybase.

This makes Drosophila a very attractive tool to study centriole biology as many centrosomal genes are conserved throughout evolution. In Drosophila, the major function of the centrosome is to form microtubule organization center (MTOC) during cell division, which further plays a role in syntactical embryo development. Thus, many maternal effect (genes from the mother side that determine phenotype) cell cycle mutants and paternal effect (genes from the father side that determine phenotype) centriolar mutants have been identified, which are responsible for Drosophila fertility and embryo development ( Khire at al., 2015, Khire and Jo et al., 2016, Freeman et al., 1986, Freeman and Glover 1987, Lee et al., 2003, Renault et al., 2003).

1.4 Centrosome biology during Drosophila spermatogenesis

The Drosophila testis is a common tool to study centriole inheritance. The testis is a long organ, and when stretched, its length is more that of the Drosophila itself (Basiri et al.,

2014). One of the reasons for the testis being widely studied is that there is a distinct layout of all cell cycle processes (stem cell hub region, spermatogenesis (mitosis), meiosis and spermiogenesis) along the length of the testis (Basiri et al., 2014).

Drosophila testes can also be used to study the evolutionarily conserved centrosome biology using fluorescence microscopy. Many proteins that are essential for centrosome in the Drosophila testes are evolutionarily conserved, and thus important for gaining information pertinent to human centrosome biology (Blachon S, et al., 2009; Blachon S, et al., 2008; Basto R et al., 2008). The centrosomes are distinctly long all through

Drosophila spermatogenesis, making them easy for analysis. One can chronologically study centrosome biology arranged along the Drosophila testes, beginning at the top with stem cell hub containing the centrosomes of sperm stem cells and culminating at the end

8 of the testes, where centrosome is different in size, structure and composition. This tip that is the end of the testis is called the seminal vesicle, which houses the spermatozoa

(mature sperm cells) (Basiri et al., 2014)

Every sperm cell is derived from the stem cell niche located at the apex of the testes. Stem cell division is asymmetrical, where each new stem cell inherits a pair of centrioles and a precursor sperm cell inherits the daughter centriole (Yamashita YM et al., 2007). When spermatogonia forms, it becomes enclosed by two cyst cells, which continue to form the spermatocytes and spermatids throughout spermatogenesis.

Spermatogonia then divide four times to produce sixteen spermatogonia, each containing two centrioles. During development of the spermatocyte, centrioles duplicate one more time, giving rise to four centrioles arranged in two pairs per cell and sixty-four centrioles per cyst. During early spermatocyte development, each centriole docks to the plasma membrane and further extends to form a primary cilium (Riparbelli MG et al., 2012;

Tates AD, 1971; Baker JD et al., 2004; Avidor-Reiss T et al., 2012). As spermatocytes mature, the length of the centriole reaches ~1.8 μm. Further, during meiosis I, the centriole pairs that are attached to the plasma membrane move towards the center of the cell and make membrane invaginations at their distal tip. The PCM around the centrioles expands, nucleates astral microtubules, and colocalizes with the spindle pole. Next, during the transition to meiosis II (see Fig. 1-4), the centriole pair separates so that just one centriole is present at each spindle pole. The ending of meiosis II, marks the beginning of spermiogenesis. Each early round spermatid cell has a single centriole attached to its plasma membrane. The centriole is more elongated than known centriole found in other animals, so it is called the giant centriole (GC). In addition to giant

9 centriole, one more centriolar structure was recently discovered in Drosophila. It was named as Proximal Centriole-Like (PCL) (Mottier-Pavie et al., 2009; Stevens et al., 2010;

Blachon et al., 2009, 2014). PCL formation takes place immediately after meiosis, in close proximity of the GC and is also independent of DNA duplication.

Figure 1-4 Centrosome biology during Drosophila Spermatogenesis During meiosis I and meiosis II, the centriole number remains the same, and each spermatid has only one centriole, the giant centriole (GC). Further, during spermiogenesis, GC then appears to bud off, giving rise to Proximal Centriole-Like (PCL). During spermiogenesis, nuclear morphology also changes distinctively. N and 2N indicate chromosome copy number. (Adapted from Avidor-Reiss et al., 2015)

1.4.1 Centrosome Reduction

When the spermatid cells differentiate into spermatozoa, the centrosome tends to lose many of its features in a phenomenon known as centrosome reduction (Manandhar et al.,

2005) Centrosome reduction occurs in most animals and can involve losing just the pericentriolar material (like in C. elegans) to an intermediate condition, in which the centriole loses its triplet microtubule symmetry and in a more severe case, both the

10 centrioles in the sperm are completely disintegrated (like in mice). During centrosome reduction, the centrioles cannot recruit microtubule asters, there is PCM loss (wherein many protein components are lost) and the centrosome loses its structure, leading to collapse of triplet microtubule assembly. The centrosome that remains is completely reduced and functions post fertilization (Manandhar et al., 2000, 2005).

Figure 1-5 Centrosome reduction in Drosophila. The PCL and GC first lose their ability to nucleate astral microtubules (MTs). Next, it leads to the loss of PCM proteins, and then finally, most centriolar proteins are eliminated, leaving a reduced bare GC and PCL in the spermatozoa (mature sperm). (Adapted from Avidor-Reiss et al., 2015) (Adapted from 2015) Centrosome reduction is conserved throughout the evolutionary tree, and appears in mammals, arthropods, and hexapods. However, these studies have only been conducted on rhesus monkeys, mice and Drosophila (Manandhar et al., 1998, Manandhar and Schatten, 2000 and Blachon et al., 2014). Centrosome reduction was studied by these techniques: antibody staining of protein components, electron microscopy and fluorescent labeling of centrosomal proteins. Immunofluorescence using antibodies against PCM proteins like γ-tubulin or pericentrin failed to detect any presence of proteins in the spermatids of rhesus monkey (Manandhar et al., 1998, Manandhar and Schatten, 2000).

Similarly, a reduction of Drosophila GFP tagged-centriolar proteins like Ana1, Sas-6,

Sas-4 and Bld10 were seen during spermatogenesis. There is also loss of PCM proteins like γ-tubulin and Cnn during Drosophila spermatogenesis (Wilson et al., 1997; Li et al.,

1998; Blachon et al., 2014) (see Fig. 1-5). Mass spectrometric analysis of Drosophila spermatozoa however detects centriolar proteins like Ana3, Bld10 and Ana2 suggesting that some amount of protein is still present in the centriole, just that it is below the detection levels of fluorescence microscopy (Wasbrough et al., 2010).

Electron microscopy of human sperm centrioles showed some structural modifications in the distal centriole (Woolley and Fawcett, 1973). Distal centriole shows partial organizational anomaly but the proximal centriole is structurally sound (Zamboni and Stefanini, 1971). Loss of some centriolar microtubules is one of the hallmarks of the structural modifications in the distal centriole of the human sperm (Manandhar and

Schatten, 2000).

1.4.2 Centrioles during fertilization of sexually reproducing animals.

Centrioles or asters were first studied in reference to sexual reproduction

(Wheatley et al., 1982; Scheer et al., 2014; Sluder et al., 2014). Yet, despite knowing that centrioles are essential to initiate animal embryo development, how centrioles are inherited during reproduction and the composition of these centrioles post fertilization has long remained a mystery (Avidor-Reiss et al., 2015). Compared to the canonical centrioles, centrioles in the zygote differ in their organization and protein composition

(Avidor-Reiss et al., 2015). The cell cycle during fertilization differs in many ways from the commonly known cell cycle, where Drosophila embryo undergoes thirteen rounds of synchronous division(Avidor-Reiss, et al., 2015). These differences may be the explained by the presence of different centriolar structure during fertilization and during the normal

12 cell cycle (Riparbelli and Callaini, 2010; Riparbelli et al., 2012; Gottardo et al., 2013).

Fertilization in most animals occurs when the ovum is arrested at meiosis II.

The fusion of male and the female gametes primes the zygote for cell division.

Zygotic centrioles play an essential role in this mechanism. Functional centrioles in the female gametes of most animals cease to exist, as all centrioles are inactivated or eliminated during oogenesis (Manandhar et al., 2005; Sun and Schatten, 2007;

Mikeladze-Dvali et al., 2012). It is also known that in animals like rhesus monkey, humans and Drosophila, the spermatozoa have centriolar structures (Sutovsky and

Schatten, 2000; Schatten and Sun, 2011). These centrioles carried by the sperm

(sometimes referred as paternal centrioles) are inherited in the zygote and are essential for zygote development (Sathananthan et al., 1996; O'Connell et al., 2001; Stevens et al.,

2007; Varmark et al., 2007). Immediately after fertilization, the human sperm, which has two centrioles, proximal centriole and distal centriole, provides proximal centriole to the zygote. In sea urchin, the distal centriole is detached from the sperm’s axoneme (Fechter et al., 1996), but remains attached to the centrosomal fossa of the male pronucleus

(Paweletz et al., 1987). However, in Drosophila, the zygotic centriole remains attached to the sperm’s flagellum (Riparbelli and Callaini, 2010). This helps in preventing the movement of the zygotic centrosome and further assists the migration of the female pronucleus, thus facilitating female and male pronuclei congression (see Fig 1-6). The fate of distal centriole in humans is not clear yet (Simerly et al., 1995). However, it was reported that, 26S proteasome is responsible for the detachment of the connecting piece from the human's proximal centriole (Rawe et al., 2008).

As stated above, paternal centrioles are the source of all the centrioles of the zygote, and thus, the origin of all the centrioles in an adult animal. For this to succeed, there is a phenomenon called maternal centrosome reduction, wherein the maternal centrioles are eliminated or inactivated, so as to disable them from participating in spindle pole assembly (Schatten, 1994). Oogenesis is primed as the germ cells undergo mitosis and thus forms an oogonium followed by an oocyte and finally into an ovum.

Every oocyte goes through meiosis eventually forming a mature egg. In mammals, centrioles are detected up to the pachytene stage of meiosis I, but are not detected during oocyte meiosis (Szollosi et al., 1972; Sathananthan et al., 2006; Luksza et al., 2013). In non-mammals, such as starfish and sea urchin, centrioles are detected later during female meiosis, but they are reduced when the polar bodies are formed (Sluder and Rieder,

1985a; Sluder et al., 1989; Nakashima and Kato, 2001; Uetake et al., 2002; Shirato et al.,

2006). Therefore, in animals like Drosophila, C. elegans, rhesus monkey, humans, sea urchin, starfish and many other insects and mammalian species, maternal centrioles are neither functional during zygote cell division nor do they play a role in embryo development. In the case of parthenogenesis, zygote centrioles originate from maternal contributions (Washitani-Nemoto et al., 1994). In addition to this, failure to reduce maternal centrioles results in multiple spindle poles in both Drosophila and C. elegans embryos; further making the case for elimination of maternal centriole during normal embryo development (Kim and Roy, 2006). As the zygote undergoes mitotic divisions, zygote centrioles duplicate in a typical manner (Callaini and Riparbelli, 1996;

Sathananthan et al., 1996; O'Connell et al., 2001). Thus, the zygotic centrioles, which originate from the sperm centrioles, are the source of all the animal centrioles.

The zygotic centrioles become functional as the paternal centrioles recruit pericentriolar material from the mother and start nucleating large microtubule aster

(Sluder and Rieder, 1985b; Stearns and Kirschner, 1994; Callaini and Riparbelli, 1996;

Terada et al., 2010; Schatten and Sun, 2011). The zygote centrosome not only facilitates the male and female pronuclei migration but also helps in zygotic cell division.

(Riparbelli et al., 2000; Schatten et al., 1986; Sutovsky and Schatten, 2000). In C. elegans, during pronuclei migration, the male pronucleus migrates towards the female pronucleus, a process facilitated by the centrosome (Kimura and Onami, 2005). In animals like sea urchin, Drosophila and bovines, migration of female pronucleus towards the male pronucleus is facilitated by the centrosome (Riparbelli et al., 2000; Fechter et al., 1996; Navara et al., 1994).

Interestingly, in somatic cells, the centrosome is not indispensable for proper cell division (Debec and Abbadie, 1989; Hinchcliffe et al., 2001; Khodjakov and Rieder,

2001; Basto et al., 2006). Conversely, there are studies which provide strong evidence that unlike somatic cells, centrosomes are absolutely vital for cell division of zygote in

Drosophila, C. elegans, and sea urchin (Sluder et al., 1989; O'Connell et al., 2001;

Stevens et al., 2007; Varmark et al., 2007). In addition to this, the mouse embryo shows de novo synthesis of centrioles, which are only detected at the 32- cell stage of an embryo

(Schatten et al., 1986; Gueth-Hallonet et al., 1993). This further substantiates that idea that in most species, paternal centrioles are the source of animal centrioles.

Figure 1-6 The centrioles facilitate male and female pronucleus migration. As the sperm enters the egg, it delivers the zygote with reduced centrioles along with its DNA (male pronucleus). The centrioles (brown) then assemble the PCM proteins from the mother and further nucleate astral microtubules (green lines), facilitating the congregation of pronuclei. After duplication takes place, the centrioles found at each spindle pole facilitate chromosome separation. N and 2N indicate chromosome copy number. (Adapted from Avidor-Reiss et al., 2015) (

1.4.3 Origin of centrioles in animal zygote

All the centrioles present in somatic cells have their origins in the zygotic centrioles. It was originally hypothesized that the animal zygote inherits two centriolar structures from the sperm (Baccetti and Afzelius, 1976). However, more recent studies in different animal groups like humans, insects and mammals, demonstrate that sperm appear to have a single or no centriole at all (Sun and Schatten, 2007; Manandhar et al., 2005; Fuller

1993). Due to lack of advanced microscopy techniques previous studies may have missed the presence of second centriolar structure like the PCL (Blachon et al., 2009). Thus, it is still possible that the second centriolar structure might be the elusive zygotic centriolar structure.

As the sperm centriole structure, number and composition varies from species to species across the animal kingdom, several hypotheses have been postulated to elucidate on origin of centrioles. Following are the hypotheses (Fig. 1-7):

A) The Classical Hypothesis.

B) The Maternal Hypothesis.

C) The De novo Hypothesis.

D) The Duplication Hypothesis.

E) The Regeneration Hypothesis.

Figure 1-7 Hypotheses for centriole Inheritance. (A) In the classical hypothesis, where, zygote inherits two centrioles from the sperm. (B) The maternal hypothesis/ de novo hypothesis states that sperm cells undergo complete centrosome elimination. The zygote is able to undergo division without any centrioles (C) Duplication Hypothesis states that, during spermiogenesis, one of the centriole is reduced, and post fertilization the intact centriole undergoes duplication. D) The Regeneration Hypothesis postulates that the distal centriole undergoes partial degeneration, but upon fertilization, this distal centriole is restored. (Adapted from Avidor-Reiss et al., 2015)

A) The Classical Hypothesis:

The classical hypothesis proposes that the sperm has two centrioles and these two centrioles are inherited by the zygote (see Fig. 1-7). In C. elegans, electron microscopy studies of the zygote convincingly show that there are two singlet microtubule centrioles

(Pelletier at al., 2006). In accordance to this, there have been reports where the centriolar proteins Sas-4 and Sas-6 from the sperm were detected in the worm zygote. Most of the data suggests that C. elegans inherit two intact centrioles. Similarly, in the mollusk, C. virginica, two centrioles (the distal and the proximal centriole) were detected in the spermatozoa (Daniels et al., 1971). During sea urchin embryo mitosis, two centrioles were detected at each of the spindle pole (Sluder and Rieder, 1985a). In addition to this, the classical model can also be attributed to X. laevis. Electron microscopy of the testicular sperm revealed an intact proximal and distal centriole (Bernardini et al.1986;

Felix et al., 1994).

B) The Maternal Hypothesis:

The maternal precursor hypothesis postulates that the mother nor father provide the centriolar structures during fertilization (Calarco, 2000). In rodents, it is believed that the embryo does not inherit any centrioles from the father. Centrioles have been detected only at the 32/64-cell stage. Structural analysis failed to find any centrioles immediately after fertilization (Woolley and Fawcett, 1973; Zambonu et al., 1972). Even fluorescence microscopy and live imaging could not reveal any functional centrioles nor microtubule asters post fertilization near the sperm head. Moreover, immediately after fertilization, only the sperm tail and nucleus were detected. (Schatten et al., 1985, 1986; Courtois et

18 al., 2012). All this evidence suggests that the mother may have a centriolar precursor which somehow gets activated sometime after fertilization.

C) The de novo Hypothesis:

The de novo hypothesis proposes that centrioles are synthesized de novo in the animal zygote (Courtois et al., 2012). Immediately after fertilization, random aggregation of

PCM proteins ensues in the zygote leading to the formation of microtubule mini-asters.

These mini-asters attach to the pronuclei membrane, leading to the formation of bipolar spindle (Courtois et al., 2012).

C) The Duplication Hypothesis:

The duplication hypothesis states that the sperm provides a single functional centriole post fertilization, which then duplicates in the zygote (Sutovsky and Schatten, 2000).

This single centriole is normally known as the proximal centriole. Thus, the second centriole that forms after duplication is thought to be the proximal centriole’s daughter centriole. Further, these two centrioles undergo duplication in the zygote, resulting in a total of two centrioles at each pole during the first mitotic spindle. A major caveat to this model is that electron microscopy studies have only detected three centrioles at the mitotic stage (Crozet et al., 2000). Until recent years, it was thought that Drosophila falls under this hypothesis (Riparbelli et al., 1997). Here, we have attempted to challenge this notion.

5) The Regeneration Hypothesis:

The regeneration hypothesis makes the case that, in many non-rodent mammals, the early spermatids have two intact centrioles, whereas the spermatozoa has only one functional centriole and the one is a degenerated centriole. Subsequently, the degenerated centriole,

19 which is also inherited during fertilization, regenerates to form the second zygotic centriole (Schatten and Sun, 2009).

In conclusion, in the zygote centrosomes facilitate the fusion of the male and female pronucleus and thus, cell division. Very little information is available regarding the mutations in sperm centrioles, and its implications on animal fertility. Chapter 3 and 4 will shed a light on the role of the centriole in male fertility. Chapter 5 will introduce the role of centrioles in female fertility. The subsequent chapters therefore will focus on the fundamental aspects of centrioles like inheritance of sperm centrioles in the Drosophila zygote and the role of these inherited centrioles in animal fertility and development.

Chapter 2

The Origin of the Second Centriole in the Zygote of Drosophila melanogaster

Previously published as Blachon S., Khire A., Avidor-Reiss T., 2014. The origin of the second centriole in the zygote of Drosophila melanogaster. Genetics 197: 199–205.

2.1 Abstract

Centrosomes are composed of two centrioles surrounded by pericentriolar material

(PCM). However, the sperm and the oocyte modify or lose their centrosomes.

Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila melanogaster spermatids contain a single centriole called the Giant Centriole (GC) and a Proximal centriole-like

(PCL) structure whose function is unknown. We found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins and are seen in

21 asterless mutant embryos that cannot form centrioles. The zygote’s centriolar proteins label only the daughter centrioles of the first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization.

2.2 Introduction

Centrioles, in the cytoplasm and basal bodies at the plasma membrane, are conserved microtubule-based organelles essential for cell division and cilium formation (Nigg and

Raff 2009). Centrioles are essential for fertilization, development, and animal physiological functions (Nigg and Raff 2009). In the newly fertilized egg (i.e., zygote), a centriole normally functions by recruiting pericentriolar material (PCM) and becoming the primary centrosome (Delattre and Gonczy 2004). This centrosome, in the zygote, acts as a microtubule-organizing center and nucleates the astral microtubules that mediate the migration of the female and male nuclei toward each other (Callaini and Riparbelli 1996).

A centriole forms by one of two pathways. In the “duplication pathway,” a pre-existing centriole acts as a scaffold to ensure that only a daughter centriole is formed per cell cycle. However, the pre-existing centriole does not appear to impart structural information to the daughter (Rodrigues-Martins et al., 2007). In the “de novo pathway,” a centriole forms without a pre-existing centriole and forms more than two centrioles. This pathway occurs when multiple centrioles are required in a cell or in the unusual situation where pre-existing centrioles are absent (Uetake et al., 2007).

Most resting cells have two centrioles. A cell preparing to divide duplicates its centrioles and consequently has four centrioles; each mother/daughter centriole pair forms a centrosome at opposite poles of the cell. Having precisely two centrioles before commitment to cell division and four centrioles during mitosis is particularly critical for proper cell division and an organism’s development (Fukasawa 2007). Having no centrioles interferes with the proper orientation of the spindle axis (Basto et al., 2006), while too many centrioles results in an increase in aneuploidy and defects in cilium formation (Basto et al. 2008; Mahjoub and Stearns 2012). Because a cell requires two centrioles to function, the zygote is expected to require two centrioles. In animals, during oogenesis centrioles are lost, and therefore, oocytes lack centrioles and do not contribute any centrioles to the zygote (Sun and Schatten 2007). Instead, it has been reported that in many animals, centrioles are inherited by the zygote from the sperm (Sun and Schatten

2007). However, in many other animals, a single functional centriole is observed.

Humans and most mammalian sperm have only a single centriole because during the last phase of spermatogenesis the spermatid centrioles are modified and degraded in a process known as centrosome reduction (Manandhar et al., 2005). In sea urchins, frogs, and Caenorhabditis elegans, the sperm provides two centrioles (Longo and Anderson

1968; Felix et al., 1994; Leidel 2005). The origin of this second zygotic centriole is uncertain. To help explain these uncertainties, four hypotheses have been put forth:

1. The “de novo/maternal-precursor hypothesis” attempts to explain the observation

that, due to a dramatic centrosome reduction, rodent sperm lack a recognizable

centriole, yet centrioles are observed when the embryo reaches the 64-cell stage

(Schatten et al., 1986; Gueth-Hallonet et al., 1993). It has been proposed that the

embryonic centriole forms de novo (Howe and Fitzharris 2013). Alternatively, it

has been claimed that the oocyte contains centriolar precursors that give rise to

the embryo’s centrioles (Calarco 2000). This hypothesis may also apply to some

insects that reproduce by parthenogenesis (Ferree et al., 2006).

2. The “regeneration hypothesis” proposes that non-rodent mammalian sperm cells

have an intact centriole (the proximal centriole) and, due to centrosome reduction,

a degenerated centriole (the distal centriole). After fertilization, the degenerated

centriole regenerates to form a second centriolar structure (Manandhar et

al. 2005; Schatten and Sun 2009). It is claimed that both the intact and the

“regenerated” centriole duplicate to form two pairs of centrioles, together

resulting in three centrioles and one regenerated centriole.

3. The “duplication hypothesis” postulates that a single functional centriole is

inherited from the sperm, which is duplicated soon after fertilization.

In Drosophila, only one functional centriole is known (Fuller 1993); the second

zygotic centriole is presumed to be the product of the sperm centriole duplicating

in the zygote (Callaini and Riparbelli 1996). In this hypothesis, a round of

centriole duplication happens prior to the zygote’s first cell division.

4. The “paternal precursor hypothesis” theorizes that a sperm provides both a

centriole and a centriolar precursor that originated during spermiogenesis, but did

not mature to a centriole; in the zygote, the precursor becomes the second

centriole (Crozet et al., 2000).

We have recently discovered that, in addition to the giant centriole (GC), which is attached to the plasma membrane and is equivalent to a basal body, Drosophila sperm

24 contain an unidentified centriolar structure that lacks the distinctive structural characteristic of a centriole; we named it the proximal centriole-like (PCL) (Blachon et al., 2009). The PCL is a centriole precursor. Initially, the PCL forms in a similar way to a centriole, as they both use the same molecular pathways. However, the PCL diverges from the centriolar formation pathway prior to when a centriole acquires centriolar microtubules, a defining characteristic of a centriole. The PCL may be the predicted precursor of the precursor hypothesis.

Here we report that, like the GC, the PCL also undergoes centrosome reduction. As a result, currently, there is no way to stably label the PCL of the Drosophila sperm to directly test the PCL hypothesis. However, the PCL hypothesis can be differentiated from the duplication hypothesis and the maternal-precursor/de novo hypothesis using three criteria. First, the PCL hypothesis predicts that the first two zygotic centriolar structures appear in the zygote simultaneously, immediately after fertilization. Second, in the zygote that is generated from an oocyte expressing GFP-tagged early centriolar proteins, only the daughter centrioles of the first two zygotic centrioles should be labeled by GFP proteins that initiate centriole formation. Third, in a zygote that is generated from a mutant oocyte that cannot support centriole duplication, two centrioles should be observed after fertilization. Here, we test these predictions and provide evidence for the

PCL/paternal precursor hypothesis.

2.3 Materials and Methods

2.3.1 Fluorescence microscopy

For embryo imaging, 50 males and 50 virgin female flies, <5 days old, were placed in an egg collection chamber with a grape agar plate with yeast paste. Chambers were used for

3 days and embryos were collected every 4 min. Immediately after collection, the embryos were placed in a mac-tech dish and washed with 100 μl of distilled water and then a wash buffer (0.7% NaCl + 0.05% Triton 100X). Afterward, 50% bleach solution was added onto the embryos until the appendages of the embryos disassociated. The embryos were rinsed twice with wash buffer, fixed in a 1:1 solution of heptane and methanol, and shaken vigorously by a vortex until the embryos settled down in the methanol layer. This was followed by removal of the fixative and suspending the embryos in acetone. At this stage, the embryos were usually stored at −20°. Then the embryos were rehydrated sequentially in 70, 50, 30, and 10% methanol in PBS and then in PBS alone. The embryos were then incubated in PBT (PBS+1% Triton 100X) for 30 min and blocked in PBST (PBT+3% BSA) for 1 hr. Then the embryos were incubated with primary antibodies in PBST for 1 hr. at room temperature. After three 5-min washes in PBT, the embryos were incubated with secondary antibodies and 1 μg/ml of DAPI for

1 hr. at room temperature. The embryos were washed three times with PBT for 5 min each and then washed in PBS for 5 min. The embryos were mounted on a slide using a mounting medium (PBS, 50% glycerol, 0.5% N-propyl-gallate) and imaged. Testis imaging was performed as described in Basiri et al., 2013. Images were taken by a Leica

SP5 or SP8 scanning confocal microscope as Z stacks. Maximal projection images were then modified using Adobe Photoshop and annotated using Adobe Illustrator.

2.3.2 Embryo development

To analyze the embryo development, 50 males and 50 female flies were allowed to mate overnight in a chamber. Then the agar plate with the laid embryos was incubated at 25° for 24 hr., and the number of larvae were counted.

2.3.3 Statistical methods

Experiments were repeated at least three times, and statistical analyses (±SEM) were done with GraphPad Prism 5. A two-tailed, unpaired Student’s t-test was used.

2.3.4 Transgenic Flies

All Drosophila stocks were cultured on standard media at 25°C. Ana1-GFP, Ana1- tdtomato, Bld10-GFP, Asl-GFP, Sas-6-GFP, and Sas-4-GFP are expressed to near physiological levels using their corresponding promoter, and were previously described in Blachon et al., 2008 and Blachon et al., 2009. Ana2-GFP is expressed under the strong ubiquitin promoter (Stevens et al 2008). Bloomington stock number 7 (P (hsFLP), y1 w1118;;DrMio/TM3, ry* Sb) and 2149 (w*;;P(neoFRT)82B P(ovoD1-18,W+)3R/TM3) were used to generate aslmecD mutant embryos. Bloomington stock 5748 (P(neoFRT)82B cu1 sr1 es ca1) and meiotic recombination was used to make the fly containing FRT82B and aslmecD. It was selected for the presence of neomycin resistance and lack of cu, and was confirmed by failure to complement aslmecD.

2.3.5 Antibodies

The following primary antibodies were used for immunofluorescence at the indicated concentrations: Guinea-pig anti-Cnn, 1:200 (a kind gift from Thomas C. Kaufman); mouse anti-β-tubulin,1:50 (Developmental Studies Hybridoma Bank); Rabbit Anti-Asl,

1:200 (Ap1193. Blachon et al 2008) mouse anti-beta-tubulin, 1:200 (Sigma); Rabbit anti-

27 alpha-tubulin, rabbit anti-DSpd-2 1:200 (a kind gift from Maurizio Gatti); Rabbit anti-

GFP (1:200; Fitzgerald Industries). The following secondary antibodies were used: Alexa

Fluor® 488-conjugated goat anti-mouse IgG, 1:800; Alexa Fluor® 647-conjugated goat anti-guinea pig IgG, 1:800; Cyanine Cy3-conjugated goat anti-rabbit IgG,1:800;

Rhodamine goat anti-mouse 1:200; Rhodamine goat anti-rabbit 1:200 (Jackson

ImmunoResearch). DAPI was used at final concentration of 1 µg/ml (Sigma).

2.3.6 Generating homozygote aslmecD embryos

Three crosses were performed. In the first cross, 50 males having the FRT (Flippase recognition target) were crossed with the 50 females having Flippase enzyme coding gene (FLP) on the first chromosome. The resultant F1 generation males were selected which had FLP gene on the first chromosome and FRT site on the third chromosome along with TM3 balancer. In the second cross, 50 males from the F1 generation were then crossed with 50 females, which were heterozygous for aslmecD having a FRT site along with aslmecD on the third chromosome. After 3 days, the flies were moved to a new vial and the vial with embryos was subjected to heat shock at 37°C for 1 hour. The heat shock was performed each day for the next 3 days, thus activating the Flippase enzyme and carrying out the recombination reaction at the FRT site and making the resultant F2 homozygous for aslmecD mutation. The females lacking TM6B (Humeral+) were selected from this F2 generation, and then were finally crossed with the wild type males and aslmecD mutant embryos were collected.

2.4 Results

2.4.1 PCL and GC undergo centrosome reduction during late spermiogenesis

At the end of spermiogenesis, mammalian centrosomes undergo a process by which they lose their PCM and centriole structure is degraded, resulting in sperm with modified centrioles (Schatten 1994; Manandhar and Schatten 2000; Manandhar et al., 2000, 2005).

Similarly, during Drosophila spermiogenesis, the PCM proteins γ-tubulin (Wilson et al.,

1997) and Cnn (Li et al., 1998) are eliminated from the GC, indicating that centrosome reduction also happens in Drosophila. The GC marker PACT-GFP, which can be intensely observed in the spermatid GC, is hardly observed in the sperm GC (Figure 2-

1A) (Martinez-Campos et al., 2004). To test if the PCL also undergoes centrosome reduction, and whether during centrosome reduction centriolar proteins are also eliminated from the GC, we studied the localization of GFP-tagged centriolar proteins during spermiogenesis and in sperm. We observed PACT-GFP, Ana1-GFP, BLD10-GFP,

Ana2-GFP, Sas-6-GFP, and Sas-4-GFP in intermediate or round spermatids, in the giant centriole, and in the PCL (Figure 2-1, A-F). We found that all of these centriolar proteins are missing from mature sperm, indicating centrosome reduction takes place in both the

GC and the PCL. This indicates that the PCL, which was formed during early spermiogenesis and without microtubules (Blachon et al., 2009), loses many of the proteins that formed it during centrosome reduction.

2.4.2 Zygote has two centrioles immediately after fertilization

After fertilization, the centrioles that lost their proteins via centrosome reduction can be detected by labeling the PCM components that they recruit from the oocyte’s cytoplasm

(Callaini and Riparbelli 1996). To image the zygote’s centrioles, we performed immunofluorescence using an antibody against the PCM protein Asterless (Asl) and an anti-α-tubulin antibody that labels microtubule asters (Blachon et al., 2008). We found that immediately after fertilization, when the twin meiosis II spindles of the female are observed, the two Asl-labeled centrioles are present in the zygote. One of the centrioles is longer and is likely to be the GC (Riparbelli and Callaini 2010), and the other one is a smaller centriole (Figure 2-1G). Because the two centrioles are observed immediately post-fertilization, this second smaller centriole is likely to be derived from the PCL

(Figure 2-1G, left; see below). Both centrioles are marked by Asl, which they must have recruited from the cytoplasm, together with the rest of the PCM, to form their own aster microtubules. This suggests that both the GC and PCL, which lose many of their components during centrosome reduction, are capable of recruiting PCM and anchoring astral microtubules. During the zygotic prometaphase, the two centrioles are at opposite spindle poles (Figure 2-1H) During anaphase, centriole duplication has already occurred, and daughter centrioles are present near each of the pre-existing centrioles (daughter centrioles (DC),Figure 2-1I). These data suggest that the PCL, like a typical centriole, can serve as a scaffold for daughter centriole formation and forms a second centriole.

Altogether, these data indicate that either the sperm transported both the GC and the PCL or that the GC duplicated immediately after fertilization and before the completion of maternal meiosis II.

2.4.3 Unlike their daughter centrioles, the first two zygotic centrioles do not incorporate centriolar proteins.

During their formation, centrioles incorporate centriolar proteins such as Sas-6 and Sas-4, which are available in the cytoplasm (Kirkham et al., 2003; Leidel and Gonczy 2003).

Therefore, to test whether the first two zygotic centrioles are formed in the oocyte, we used oocytes expressing the centriolar proteins Sas-6-GFP and Sas-4-GFP. These oocytes were fertilized with sperm centrioles, which can be observed once they recruit PCM. As expected, immediately after fertilization, there are two zygotic centrioles labeled by the

PCM protein Asterless (Figure 2-2 A-C). However, neither of these centrioles was labeled by Sas-6-GFP or Sas-4-GFP, indicating that they did not form in the zygote and that their origin must be from the sperm. One of these centriolar structures is large, suggesting that it is the GC, while the second is smaller, suggesting that it is the PCL.

Later, during mitosis, Asl staining identifies four centrioles. Of these, two are labeled only by Asl, and the remaining two are labeled by Asl and Sas-6-GFP or Sas-4-GFP

(Figure 2-2, B-D). The two centrioles labeled by only Asl are likely to be the GC and the

PCL. The two centriolar structures labeled by Sas-6-GFP or Sas-4 -GFP are likely to be the DCs of the GC and the PCL (Figure 2-2 B-C, left). Altogether, this experiment strongly argues against both the maternal precursor hypothesis and the duplication hypothesis. Indeed, maternal centrioles and centrioles duplicated in the zygote should have been labeled by Sas-6-GFP or Sas-4-GFP. These data, however, are consistent with the PCL hypothesis.

2.4.4 Homozygote aslmecD zygotes have two centrioles

Drosophila Asl and its vertebrate homolog Cep152 are essential proteins for centriole duplication (Blachon et al., 2008) and embryo development (Varmark et al., 2007).

Depletion of Cep152 prevents both centriole duplication and Plk4-induced de novo centriole formation (Cizmecioglu et al., 2010; Hatch et al., 2010), indicating that

Asterless/Cep152 is essential for centriole formation by both the duplication and de novo pathways. We recently showed that aslmecD completely blocks centriole duplication in flies (Blachon et al., 2008). Because the aslmecD flies die shortly after emerging from their pupal cases, these flies are not capable of mating. To remedy this, we generated aslmecD oocyte clones that lack the Asterless protein in heterozygous aslmecD females To test for the role of Asl in fertilization, these adult females were mated to normal males, and their zygotes were analyzed. As expected, we found that aslmecD embryos cannot develop into larvae (Figure 3A). Importantly, introduction of Asl-GFP using germline transformation reverted the aslmecD phenotype, allowing embryo development (Figure 2-3A). We then analyzed the phenotype of zygotes generated by aslmecD oocytes. Embryos collected immediately after they were laid were stained with the nuclear marker DAPI, an antibody for microtubules, and an antibody for the centrosomal protein Cnn. Two populations of embryos were found. The first population had two to four nuclei associated with the microtubule spindles, which are lacking centrosomes as judged by the absence of PCM markers (36% or 28/77), indicating that they were unfertilized oocytes. The second population had one paternal pronucleus that possesses Cnn labeling and one maternal pronucleus that lacks Cnn labeling, indicating that the second population were fertilized oocytes (i.e., an embryo) (64% 49/77) (Figure 2-3B). The maternal and paternal

32 pronuclei were not close to each other, indicating that the embryo’s development arrested before completion of pronuclei migration (data not shown).

Analysis of the Cnn labeling of the paternal pronuclei found that 32% of embryos (16/49) had two clearly separated Cnn foci; 30% (15/49) had two close, but distinct, Cnn foci that look like the “figure 8”; and 36% (18/49) have a single Cnn focus (Figure 2-3B).

Because the size of the single Cnn focus was larger than a clearly separated Cnn focus

(Figure 2-3D), it is likely that many of the single foci are two centriolar structures that are closely associated with each other. Similar to Cnn, γ-tubulin and DSpd-2 also label both centrioles of the zygote generated by the aslmecD oocytes (Figure 2-3E). Altogether, this analysis demonstrates that an embryo that cannot duplicate centrioles has two centriolar structures, consistent with the PCL hypothesis.

2.5 Discussion

We have shown previously that the Drosophila sperm contain a second centriolar structure, the PCL (Blachon et al., 2009). In this article, we demonstrated that two centrioles are present in the zygote after fertilization, that these two centrioles are not marked by maternally contributed centriolar proteins, and that, in a zygote unable to duplicate centrioles, there are two centrioles present. Therefore, the sperm must have brought two centrioles to the zygote.

We therefore propose the following model for centriole inheritance in Drosophila: the sperm provides a GC and a PCL, and both lack PCM and many centriolar proteins, while the oocyte is lacking any centrioles (Figure 2-4A). Immediately after fertilization, both the GC and the PCL recruit the PCM and nucleate astral microtubules, but they do not incorporate centriolar proteins (Figure 2-4B). Later, the centriole and PCL each template

33 a daughter centriole, which is made from maternally contributed proteins (Figure 2-4C).

Finally, the two centrosomes, one derived from the GC, and the other derived from the

PCL, migrate to the spindle poles of the embryo’s first spindle (Figure 2-4D). Because after fertilization the PCL does not incorporate maternal Sas-6 and Sas-4, it is possible that the PCL does not mature to a typical centriole.

2.6 Acknowledgments

We thank Alberto Vizuet-Torre, Suma Kolla, Marli Emad Gabriel, and Vidita Reddy for technical assistance; Sarah E. Hynek for editing this manuscript; and Jarema Malicki for commenting on the manuscript. We also thank Maurizio Gatti for the DSpd-2 antibody,

Thomas C. Kaufman for the Cnn antibody, and Jordan Raff for the Ana2-GFP and

PACT-GFP flies. This work was supported by National Science Foundation grant

1121176. The authors declare no competing financial interests.

2.7 Author contributions

T.A-R. conceived and supervised the project. A.K. performed the experiments of figure

1A-E and 3. S.B. performed the experiments of figure 1F-H and 2.

Figure 2-1

Figure 2-2

Figure 2-3

Figure 2-4

FIGURE LEGENDS

Figure 2-1: The PCL and giant centriole undergo centrosome reduction and recruit

PCM after fertilization. (A) The giant centriole (GC, solid line) is intensely labeled by

PACT-GFP (that is over-expressed by the strong ubiquitin promoter in intermediate spermatids), but can be barely observed at the base of the sperm nucleus (see inset for magnification of this giant centriole). (B-F) The PCL (dashed line) and giant centriole

(GC, solid line) are observed in intermediate spermatids by Ana1-GFP (B) and BLD10-

GFP (C), as well as round spermatids by Sas-6 GFP (D), Sas-4-GFP (E), and Ana2-GFP

(F). The giant centriole is also marked by Ana1-td tomato (D-F). However, none of these proteins are observed in sperm found in the seminal vesicle. Note that Ana2-GFP

(Stevens et al 2010) is strongly expressed using the ubiquitin promoter, yet it is completely eliminated during centrosome reduction, N, nuclei. (G-I) Drosophila zygotes contain two centrioles immediately after fertilization and four during mitosis

(G). After fertilization, and during maternal meiosis II (twin meiosis II spindles are labeled red, ♀), the giant centriole (GC) and second centriolar structure, which is likely to be the PCL (PCL), are labeled by Asterless (Asl); these structures form microtubule asters (red) that associate with the male pronucleus (♂). (H). During prometaphase, the giant centriole and the PCL form distinct microtubule asters (I). During anaphase, the giant centriole and the PCL are each accompanied by an Asl-labeled daughter centriole, indicating that they each gave rise to a daughter centriole (DC).

Figure 2-2: Maternally GFP tagged centriolar proteins are incorporated into the daughter centrioles of the giant centriole and the PCL. (A and C) Immediately post-

39 fertilization, the first two zygotic centrioles are not labeled by maternal centriolar proteins, but are labeled by Asl (magenta). (B and D) During the zygote’s first mitosis, an antibody against Asl labels all centrioles, but Sas-6-GFP (B) or Sas-4-GFP (D) only labels two centrioles. This indicates that three types of centrioles are observed: a giant centriole (GC), the presumed PCL, and two daughter centrioles (DC).

Figure 2-3: Two centrioles are observed in an oocyte mutant for centriole duplication. (A) Embryo development is arrested in embryos generated by aslmecD oocytes, and is rescued in the presence of Asl-GFP. (B) A zygote generated by an aslmecD oocyte with a paternal pronucleus (♂) labeled by two Cnn labeled centrioles and a maternal pronucleus (♀) lacking centrioles. (C) Paternal pronuclei in zygotes generated by aslmecD oocytes have two centrioles that are superimposed (1), near each other (8-like), or apart from each other (2). (D) Graph depicting the length of the Cnn foci in embryos with one Cnn focus (single) as well as long and short CNN foci in embryos with two separate Cnn foci, demonstrating that most of the apparent foci are two superimposed foci. ***, P<0.0003. (E) Cnn, γ -tubulin, and Spd-2 are co-labeling the two zygotic centrioles.

Figure 2-4: The PCL in Drosophila fertilization. A. During fertilization, the oocyte lacks centrioles, whereas the sperm contains a giant centriole and a PCL. B. After fertilization, the centriole and the PCL form asters. C. During the pronuclear stage, the centriole and PCL are duplicated, resulting in two daughter centrioles. D. During mitosis,

40 each pole has a centrosome that consists of a centriole with its daughter, or a PCL with its daughter.

Chapter 3

Asterless Reduction During Spermiogenesis is Regulated by Plk4 and is Essential for Zygote Development in Drosophila

Previously published as Khire, A., Vizuet, A. A., Davila, E., & Avidor-Reiss, T. 2015.

Asterless reduction during spermiogenesis is regulated by Plk4 and is essential for zygote development in Drosophila. Current Biology, 25(22), 2956-2963.

3.1 Abstract

Centrosome reduction is the decrease in centrosomal components during spermatid differentiation (spermiogenesis). It is one of several dramatic subcellular reorganizations that leads to spermatozoa formation common to a wide range of animals. However, the mechanism underlying centrosome reduction is unknown and its functions are unclear.

Here, we show that in Drosophila melanogaster spermiogenesis, the quantity of centrosomal proteins is dramatically reduced, for example, Asterless (Asl) is reduced

~500-fold and is barely detected in spermatozoa. Asl reduction is regulated through a subset of its domains by the master regulator of centriole duplication Plk4 and by the

42 ubiquitin ligase that targets Plk4 for degradation: Slimb. When Asl reduction is attenuated by Asl overexpression, plk4 mutations, Plk4 RNAi, or Slimb overexpression,

Asl levels are higher in spermatozoa, resulting in embryos with reduced viability.

Significantly, overexpressing Plk4 and Asl simultaneously, or combining plk4 and slimb mutations, balances their opposing effects on Asl reduction, restoring seemingly normal fertility. This suggests that increased Asl levels cause the observed reduced fertility, and no other pleotropic effects. Attenuation of Asl reduction also causes delayed development and a failure to form astral microtubules in the zygote. Together, we provide the first insight into a molecular mechanism that regulates centrosome reduction and the first direct evidence that centrosome reduction is essential for postfertilization development.

3.2 Methods and Materials

3.2.1 Transgenic Flies.

All Drosophila stocks were cultured on standard media at 25°C. For in vivo expression of

Hills, CA), using w1118 flies. Plk4KDGFP flies (D156N point mutation in Plk4-wt) were generated in pUAS vector using a pMT/V5-HisCplasmid obtained from Dr. Gregory

Rogers. eAna1GFP, Ana1-td-tomato, Sas-4GFP and eAslGFP, were previously described and are expressed by their own promoter. UAslGFP, Plk4GFP, uAna2GFP, uSas-4GFP,

43 uSas-6GFP and uPACTGFP were expressed using a (strong) ubiquitin promoter and were provided by Jordan Raff. Bld10 was expressed using own promoter and was provided by

Tim Megraw. Asl-RNAi (Bloomington stock# 38220) was expressed using Bam-Gal4 promoter. Bam-Gal4 fly line was provided by Yokiko Yamashita. SlimbGFP was expressed using a UAS promoter and were provided by Dr. Daniel St Johnston. Piggybac

Transposase (stock #32070), Nos-GAL4 (stock #7303), slimb00295 (stock #11493), and plk4c06612 (stock #7774) were obtained from the Bloomington Stock Center.

3.2.2 Antibodies.

The following antibodies were used for immunofluorescence and western blots at the indicated concentrations:

Primary antibodies for immunofluorescence: E7-mouse anti-tubulin, 1:200

(AB_2315513, DSHB at The University of Iowa); rabbit anti-Asl, 1:200 (Tomer Avidor-

Reiss Lab)

Secondary antibodies for immunofluorescence: Cy5™AffiniPure Goat Anti-Rabbit IgG

(H+L), 1:200; Alexa Fluor® 488-conjugated donkey anti-rabbit IgG, 1:200 (Jackson

ImmunoResearch).

Primary antibodies for western blot: anti-actin (Abcam-ab8227), 1: 10000, anti-Asl,

1:5000, and anti-GFP (Thermo Scientific, MA5-15256), 1: 2000

Primary antibodies for immunoprecipitation: Ascites Mouse Anti-Ubiquitin (BD-

Pharmingen-550944), 1:25, and Mouse p-Threonine (42H4) (Cell Signaling-9386S),

1:50.

3.2.3 Fluorescence Microscopy.

Images were taken by Leica SP8 scanning confocal microscope as Z stacks. Maximal projection images were then modified using Adobe Photoshop and annotated using

Adobe Illustrator.

3.2.4 Testes Fluorescence Microscopy.

Testes imaging was performed as described in Basiri et al. (2013). Testes of pharate adult pupae were dissected in PBS, fixed in 3.7% formaldehyde in PBS for 5 min at room temperature, squashed with a coverslip, frozen in liquid nitrogen for 5 min, and washed with PBST (PBS+ 0.1% Triton 100-X). Antibodies were incubated in PBST-B (PBS +

0.1%Triton+ 1% BSA) for 1h at room temperature followed by three washes in PBS.

For photon counting, at least 5 testes were analyzed. In each testis, photons were counted from 5 centrioles at each stage. Pictures were taken in the photon counting mode using

63x objective with a zoom of 6, pin hole of 1.2, resolution of 512×512 pixel, 488 nm laser power of 1% of 20% and using measuring box of 1m by 2.5m that was placed on top of the centriole.

3.2.5 Embryo Fluorescence Microscopy.

Fifty male and fifty virgin females flies less than 5 days old were placed in an egg collection chamber with a grape agar plate supplemented with yeast paste. Chambers were used for 3 days and embryos collected every 4 min. Immediately after collection, the embryos were placed in a mac-tech dish and washed with 100μl of distilled water, and then washed in a wash buffer (0.7% NaCl+0.05% Triton 100-X). Afterwards, a 50% bleach solution was added to the embryos until the appendages of the embryos disassociated. The embryos were rinsed twice with the wash buffer, fixed in a 1:1

45 solution of heptane and methanol, and shaken vigorously by vortex, until the embryos settled into the methanol layer. This was followed by removal of the fixative, and suspending the embryos in acetone. At this stage, the embryos were usually stored at -

20°C. Prior to staining, embryos were rehydrated sequentially in 70%, 50%, 30%, and

10% methanol in PBS, and then in PBS alone. Embryos were then incubated in PBT

(PBS+1% Triton) for 30 min, and blocked in PBST (PBT+3%BSA) for 1 h. Then the embryos were incubated with primary antibodies in PBST for 1 h at room temperature.

After three 5-min washes in PBT, the embryos were incubated with secondary antibodies for 1 h at room temperature. The embryos were washed three times: first with PBT for 5 min, next with PBT Hoechst (1μg/ml) for 20 min, and finally in PBS for 5 min. The embryos were mounted on a slide using a mounting medium (PBS, 50% glycerol,

0.5%N-propyl-gallate) and imaged.

3.2.6 Western blot

Testes were collected in PBS buffer and boiled for 5 min in 95 °C with 1X Laemmli smaple buffer samples. Twelve testes were run per lane in 10% polyacrylamide gels and transferred into nitrocellulose membranes. The blots were incubated with primary antibodies overnight at 4°C followed by peroxidase-conjugated secondary antibodies at

RT for 1h. Super Signal West Pico (for tubulin staining) or Femto (for Asl and GFP staining) Chemiluminescent substrate (Pierce) was used to detect peroxidase activity.

Molecular masses were determined by comparison to Precision Plus Protein Standard

(Bio-Rad). Western blots were analyzed with ImageJ (National Institute of Mental

Health, Bethesda, Maryland, USA) and intensity of total area was calculated using an identical rectangular box.

3.2.7 Ubiquitination IP

Twenty testes were lysed in immunoprecipitation (IP) lysis buffer (50mM Tris-HCl (pH

7.5), 1% Triton X-100, 150mM NaCl, 0.1% 2-mercaptoethanol, 1mM Na3VO4, 1μM leupeptin, 1μM aprotinin, 1 mM PMSF) supplemented by Phosphatase inhibitor cocktail

3 (Sigma) diluted 1000-fold and N-Ethylmaleimide at a final concentration of 10 mM.

Immunoprecipitation was carried out by adding to the lysed Protein-G Sepharose Beads

(GE Healthcare) and the Anti-ubiquitin antibody. Anti-Asl antibody was used in the immunoblot.

3.2.8 Phosphorylation IP.

Fifty testes were lysed in Immunoprecipitation (IP) lysis buffer supplemented by

Phosphatase inhibitor cocktail 3(Sigma) diluted 1000-fold. Immunoprecipitation was carried out by adding to the lysed Protein-G Sepharose Beads (GE Healthcare) and the

Anti-Phospho-Threonine antibody. Anti-Asl antibody was used in the immunoblot.

3.2.9 Larval hatching.

Five males and five females were placed in humidified mating chamber at 25°C to lay eggs for 24hours. The parents were removed and embryos counted. Seventy-two hours later hatched eggs were counted and the percent of hatched eggs was calculated.

Experiments were repeated at least 5 times. Each experiment typically resulted in 80 or more embryos.

3.2.10 Embryo development.

Twenty-five males and twenty-five females were placed in humidified mating chamber at

25°C to lay eggs for 5 min. The parents were removed and embryos were placed for 1

47 hour in a MatTek dish containing wash buffer (0.7% NaCl+0.05% Triton 100-X). The embryos were processed for embryo fluorescence microscopy staining.

3.2.11 Statistical Methods.

Statistical analyses were done with Excel and Graph Pad Prism 5. A two-tailed, unpaired

Student’s t-test (with samples that do not have equal variances) was used.

3.3 Results

3.3.1 Asl Protein Levels are Dynamically Reduced During Spermiogenesis

During spermiogenesis, many centrosomal component proteins are diminished in a process known as centrosome reduction. It is unclear how and why centrosome reduction occurs even though it is ubiquitous throughout the animal kingdom and several indirect studies suggest it is necessary (Borges, et al. 2002, Comizzoli, et al., 2006, Tachibana,

Tet al., 2009) However, skeptics speculate that centrosome reduction is mediated by general sperm differentiation mechanisms and is inconsequential for male fertility, since centrosome reduction mechanism remains unknown. To gain insight into this universal phenomenon, we investigated centrosome reduction in Drosophila melanogaster, which serves as a manipulable model for human spermiogenesis. Drosophila exhibit dramatic diminishment of centrosomal proteins during spermiogenesis (Wilson, et al., 1997, Li, Xu et al. 1998, Mottier-Pavie and Megraw 2009, Blachon, et al., 2014); genetically-encoded fluorescent fusion tags show that Ana1, Ana2, Ana3, Sas-4, Sas6, Bld10, and Dplp’s Pact domain are reduced to nearly undetectable levels (Fig 3-1A). As in humans, centrosome reduction in Drosophila takes place in distinct and well-defined steps of sperm differentiation (Manandhar, et al., 2005) and the centrosome is critical for the function of the zygote after fertilization(Simerly, et al., 1995)

Like other centrosomal proteins, Asterless (Asl) is abundant in pre-reduced spermatid centrosomes (Varmark,et al., 2007, Blachon,et al., 2008); Asl levels decrease during spermiogenesis such that it is undetectable in mature sperm (spermatozoa) (Fig 2A). We named this phenomenon Asl reduction. To study Asl reduction, two GFP-tagged Asl’s were used: one expressed by the endogenous Asl promoter (Blachon, et al., 2008)

(eAslGFP) and another strongly expressed by the exogenous ubiquitin promoter (Stevens,

Dobbelaere et al. 2010) (uAslGFP). Western blots showed that eAslGFP and uAslGFP, together with endogenously expressed Asl, results in a 1.6- and 2-fold greater than normal Asl content in testis (Figs 3-1B-C). Using eAslGFP as an indicator of Asl, before meiosis, in spermatocytes, Asl is normally found along the two very long centrioles

(1.8µm long) that are named giant centrioles (GCs) (Fig 3-2B). After meiosis and centriole separation, in round spermatids, Asl’s presence is maintained along the GC (Fig

3-2C). As the spermatid differentiates, Asl shrinks gradually into a collar-like structure

(Fig 3-2D). Later, Asl is only detectable in the second centriolar structure: the proximal centriole like (PCL) (Blachon,et al. 2009) (Fig 3-2E). In almost-needle spermatids, Asl is found in the PCL and in another location near the nucleus, presumably the GC’s proximal end (Fig 3-2F). Finally, in spermatozoa, eAslGFP is barely detectable (Fig 3-2G).

Similar patterns of Asl reduction were observed with uAslGFP, yet with greater GFP- intensity, especially in almost-needle spermatids and spermatozoa (Fig 3-1D). These data show that Asl is dramatically and dynamically reduced, initially from the GC and later from the PCL of spermatids.

Photon counting of eAslGFP centrosomes was used to quantify Asl reduction during spermiogenesis. A~522-fold reduction in the GFP-intensity between the spermatid and

49 spermatozoa centrioles was observed, with a spermatid / spermatozoa GFP-intensity ratio of 0.002 ± 0.001. Similarly, in uAslGFP, a ~22-fold reduction was observed (Figs 3-1H-

J). Since Asl reduction occurs when Asl is expressed by the exogenous ubiquitin promoter, Asl reduction is unlikely due to transcriptional regulation. Altogether, these data suggest that the spermatid has sufficient capacity to reduce the amount of endogenous Asl and expressed eAslGFP, but it is unable to handle the excess Asl expressed as uAslGFP.

3.3.2 Asl Reduction is Attenuated in Asl with Domain Deletions

The mechanism of Asl reduction was then investigated. To determine which of Asl’s six coiled-coil domains (Blachon, et al., 2008) are essential for Asl reduction, transgenic flies were generated that expressed a GFP-fusion protein with a N-terminal or C-terminal deletion of Asl. Each transgene was built from Asl cDNA and expressed by the endogenous Asl promoter (Fig 3-3A). Fusion proteins lacking Asl’s coiled-coil domain 1 and having domains 2 to 6 (Asl2–6), 3 to 6 (Asl3–6), 4 to 6 (Asl4–6), or 5 to6 (Asl5–6) showed GFP localized to the centrosome at all spermiogenesis stages. In centrosomes bearing the Asl2–6, Asl3–6, or Asl4–6 fusion proteins, Asl reduction is attenuated.

Although there was some Asl reduction occurring early in spermiogenesis (between round and almost-needle spermatid stages), Asl reduction was greatly attenuated between the almost-needle spermatid and spermatozoa stages (Figs 3-3B–D; Fig 3-4). The Asl deletions showed variable GFP-intensity levels in the round spermatid centriole but all had lower intensity than eAslGFP, suggesting that attenuated Asl reduction is not due to

Asl overexpression. Asl reduction was strongly attenuated in centrosomes bearing the

Asl5–6 fusion protein, which showed similar GFP-intensity throughout spermiogenesis

(Figs 3- 3D-E). Together, Asl’s coiled-coil domains 1 and 4 appear to be critical for

Asl’s reduction, with two mechanisms mediating Asl reduction: one dependent upon

Asl’s domain1, which relates to late Asl reduction, and another dependent upon Asl’s domain 4, which relates to early Asl reduction.

3.3.3 Plk4 and Slimb Regulate Asl Reduction

Asl’s domain 1 interact with Polo-like kinase 4 (Plk4) (Bettencourt-Dias, et al., 2005,

Habedanck, et al., 2005, Ko, et al., 2005, Klebba, et al., 2015), the master regulator of centriole formation. Like Asl, Plk4 is reduced during spermiogenesis (Fig 3-5A). Plk4’s contribution to Asl reduction was then tested using a plk4 knockdown and a plk4 mutant.

In late spermiogenesis, centrosomes having UAS-GAL4 knocked-down plk4 showed attenuated Asl reduction (Figs 3-5B and Figs 3-6A). Like the knockdown, homozygous or heterozygous partial loss of function P-element allele of plk4 (plk4c06612) (Bettencourt-

Dias, et al., 2005) showed attenuated Asl reduction in late spermiogenesis (Figs 3-5C and Figs 3-6B-D). When Asl reduction is attenuated, Asl is present in the spermatozoa as two foci that likely correspond to the PCL and a portion of the GC. This attenuation is reversed by overexpressing a GFP-tagged Plk4 (Plk4OE) driven by the ubiquitin promoter (Basto et al., 2008) or excising the P-element (Figs 3-5B and Figs 3-6E-G), except in Asl2-6 spermatozoa, which has Asl levels that are not affected by Plk4OE (Fig

3-6H). Together, these data suggest that Plk4 regulates Asl reduction via Asl domain 1.

Furthermore, since Plk4OE enhances eAslGFP and uAslGFP reduction (Fig 3-6I-K) and

Plk4 is reduced in parallel with Asl during spermiogenesis, it is likely that the amount of available Plk4 protein is a limiting factor in Asl reduction. In contrast, Asl does not appear to be essential for Plk4 reduction (Fig 3-6L).

Plk4 is targeted for degradation by ubiquitin ligase SCF-Slimb/βTrCP-E3 via Slimb activity (Cunha-Ferreira et al., 2009, Rogers, Rusan et al. 2009); therefore, we assessed whether Slimb regulates Asl reduction. For this, a GFP-tagged Slimb was overexpressed using the Nos-GAL4 driver in Asl-td-Tomato flies. Slimb overexpression attenuated Asl reduction, resulting in increased Asl levels in spermatozoa (Fig 3-5D), except in Asl2-6 spermatozoa, which lacks Asl’s domain 1 (Fig 3-6M). Furthermore, the slimb mutation counteracted the plk4c06612mutation’s impact on Asl reduction, resulting in undetectable eAslGFP levels in spermatozoa (Fig 3-6N). Together, these data suggest that Slimb controls Asl reduction by regulating Plk4.

To help clarify Plk4’s role in regulating centrosome reduction, we investigated

Plk4-induced posttranslational modifications. Consistent with observations that

Asl/Cep152 is phosphorylated by Plk4 in vitro (Hatch, et al., 2010), we found that Asl is phosphorylated in the testes (Fig 3-5E). Furthermore, Plk4 overexpression enhances Asl phosphorylation and Plk4kinase-dead (Plk4kd) overexpression attenuates Asl phosphorylation (Fig 3-5E). Since Plk4kdoverexpression attenuated Asl-td-Tomato reduction (Fig 3-5F), it is likely that Plk4-induced Asl phosphorylation regulates Asl reduction. We also found that Asl is polyubiquitinated in testes and Plk4 overexpression enhanced Asl ubiquitination (Fig 3- 5G), suggesting that ubiquitination also regulate Asl reduction.

3.3.4 Asl Reduction is Essential for Post-Fertilization Development

The role of Asl reduction in mature sperm function was studied using embryos fathered by males having increased Asl levels in their spermatozoa. In control experiments, 95%

52 or more embryos hatched when fathered by flies with or without GFP-tagged sperm proteins (Fig 3-7A), whereas only 90% and 79% of embryos fathered by eAslGFP and uAslGFP flies hatched (Fig 3-7A), an inverse correlation with Asl levels in the testes

(Fig 3-1A-B) and spermatozoa (Fig 3-2J). As expected from Plk4’s role in Asl reduction, Plk4overexpression rescued uAslGFP’s embryo-hatching phenotype (Fig 3-

7A). There was also decreased larval hatching of embryos fathered by Plk4-knockdown flies or by Slimb-overexpressing flies (Figs 3-7B-C). These observations suggest that increased Asl levels in spermatozoa interferes with embryonic development.

To help determine the mechanism underlying the embryo-hatching phenotype, one hour-old embryos were studied. As expected, all control embryos progressed beyond the 32-nuclei stage, whereas all uAslGFP-fathered embryos showed significant delays in cleavage cycle progression and embryo development, as evident by reduced numbers of nuclei and no embryo reaching the 32-nuclei stage (Figs 3-7D-E). Similarly, developmental delay was observed in embryos fathered by plk4c06612 heterozygous flies or by Slimb-overexpressing flies (Fig 3-7F). As expected from Slimb’s role in Asl reduction, the slimb00295 mutation rescued plk4c06612’s embryo-hatching phenotype (Fig 3-

7G). These data suggest that increased paternal Asl does not interfere with fertilization and instead interferes with early steps of embryo development.

Next, functions of zygotic centrosomes in uAslGFP-fathered embryos were studied. All zygotic embryos from control fathers had robust microtubule asters surrounding their centrosomes (Fig 3-7H), whereas all embryos fathered by uAslGFP or plk4c0661 had undetectable or reduced microtubule asters immediately after fertilization

(Fig 3-7I and J). At later stages, uAslGFP-fathered embryos had normal microtubule

53 asters and bipolar spindles (Fig 3-7K and 3-7B); this explains why many of these embryos eventually developed into larvae. The fact that all uAslGFP-fathered embryos had the paternal giant centrioles indicates that the uAslGFP sperm can fertilize an ovum; therefore, the embryonic phenotypes are due to post-fertilization defects. Apparently, high amounts of paternal Asl interferes with zygotic centrosome function and embryo development. In other words, centrosome reduction (at least regarding Asl) is essential for normal embryonic development.

3.4 Discussion

Centrosome reduction is a conserved process in Drosophila and mammalian sperm

(Manandhar, et al., 2005, Avidor-Reiss, et al., 2015). Elegant work in mammals by

Schatten, Simerly, Manandhar, and colleagues showed that centrosome lose many of their components in a stepwise fashion and identified some proteins that are reduced

(Manandhar, et al., 1998, Manandhar and Schatten 2000). Additional studies provided indirect evidence for an essential role of centrosome reduction in embryo development

(Borges, et al., 2002, Comizzoli, et al., 2006, Tachibana, et al., 2009). The data disclosed here provide the first insight in to the molecular mechanisms regulating centrosome reduction and the first direct evidence that centrosome reduction is essential for post fertilization development.

Using several independent approaches, we show that one aspect of centrosome reduction, Asl reduction, is essential for normal zygotic aster formation, post-fertilization development, and, ultimately, embryo viability: (i) Asl overexpression, (ii) RNAi knockdown of plk4, (iii) plk4 mutants, and (iv) Slimb overexpression. Furthermore, we show that reduced fertility, caused by increased spermatozoon Asl, is specific in two

54 ways: (i) reduced larva hatching caused by Asl overexpression, which is rescued by Plk4 overexpression, and (ii) delayed embryo development caused by plk4 mutant, which is rescued by slimb mutation. Our independent approaches, strongly argue that the described embryonic phenotypes are due to increased Asl levels rather than to other problems stemming from overexpression or mutations. These observations are consistent with indirect observations that centrosome reduction is essential for embryonic development in mammals; for example, in vitro fertilization using human, rabbit, or cat early spermatids, having unreduced centrosomes, provide reduced rates of zygotic aster formation and elevated miscarriage rates (Borges, et al. 2002, Comizzoli,et al. 2006,

Tachibana, et al. 2009).

We found that Asl reduction is a regulated process (Fig 3-5J). First, Asl reduction occurs in two distinct steps: the initial step taking place early in spermiogenesis, when

Asl is stripped from the GC, and the subsequent step in late spermiogenesis, when Asl is stripped from the PCL. Second, Asl reduction depends on specific domains in Asl: Asl domain 1 regulates late Asl reduction and Asl domain 4 regulates early Asl reduction.

Third, Asl reduction is attenuated by plk4c06612, plk4kd, and Plk4-RNAi. It is likely that

Plk4 regulates Asl reduction via Asl’s domain 1, since Plk4 is known to bind to Asl’s N- terminal domain (Dzhindzhev, et al., 2010) and reduction of Asl lacking domain 1 is not enhanced by Plk4; data shown here indicates that Slimb regulates Asl reduction, likely via Slimb’s regulation of Plk4 (Cunha-Ferreira, et al., 2009, Rogers, et al., 2009).

Finally, regulation of Asl reduction is independent of other processes occurring during spermiogenesis. As examples, conditions affecting Asl reduction do not affect nucleus differentiation or ovum fertilization. Also, a plk4 mutant that attenuates Asl reduction

55 does not attenuate other centrosomal proteins’ reduction (Figs 3-6O-P). Such specificity in Asl reduction regulation argues against a common belief that centrosome reduction is simply due to loss of sperm protein production machinery (Gur and Breitbart 2006,

Chenoweth and Lorton 2014). Since Plk4 only mediates late Asl reduction and Asl’s domain 4 appears to be essential for a complete Asl reduction block, and Plk4 is nonessential for reduction of other centrosomal proteins, there exist other complementary mechanisms that together control centrosome reduction.

The role of Plk4’s interaction with Asl/Cep152 differs between centrosome reduction and centriole duplication. In dividing cells, Asl serves as a receptor recruiting

Plk4 to the centriole for centriole duplication (Cizmecioglu, et al., 2010, Dzhindzhev, et al., 2010, Hatch, et al., 2010, Park, et al., 2014). However, Asl’s interaction with Plk4 is complex and mediated via two distinct domains (Asl C-terminus and N-terminus), which have distinct functions during centriole duplication (Klebba, et al., 2015). Here, we show that Plk4’sN-terminus regulates Asl attachment to the centriole during spermiogenesis.

This regulation may be specific to spermiogenesis, since Asl/Cep152 levels are constant in centrioles of dividing cells (Cizmecioglu, et al., 2010, Hatch, et al., 2010). Therefore, the interaction of Plk4 with Asl seems to be modulated to achieve distinct outcomes depending on a cell’s developmental state.

It remains unknown why paternal Asl reduction is essential for timely assembly of microtubules around the zygotic centriole; indeed, why must the parental Asl be lost and replaced with maternal Asl in the zygote? Asl, in itself, is clearly not harmful to the zygote since an ovum contains large amounts of maternal Asl, which is sufficient to form

~1000 centrioles (Rodrigues-Martins, et al., 2007). The embryonic arrest we observe may

56 be because much of the maternal Asl is unavailable after fertilization. This is consistent with the idea that the loss of centrosomal proteins inactivates the paternal centrosome until it recruits maternal centrosomal proteins in the zygote and at the appropriate time

(Schatten and Sun 2011, Mikeladze-Dvali, et al., 2012). Nonetheless, excess paternal Asl interferes with zygotic aster formation. This raises the possibility that paternal Asl is

“toxic” to the zygote, perhaps because paternal centrosomal proteins act differently from maternal ones. Normally, such a “toxic” effect must be rapid, since we cannot detect paternal Asl in zygotic centrioles, suggesting that any residual paternal Asl is soon striped from the centriole once in the zygote (data not shown). Another possibility is that the sperm centriole, like other sperm organelles, need to be remodeled, and Asl reduction is essential to complete this remodeling. For example, during spermiogenesis, remodeling of the nucleus involves eliminating histones and replacing them with protamines (Fabian and Brill 2012). Possibly, the centriole, after striping its original proteins, may require a yet unknown sperm-specific centriolar protein. Regardless of the pathological mechanism induced by increased paternal Asl, it appears that the inhibitory effect on astral microtubules formation is temporary; as the paternal centrioles regain their ability to assemble microtubules at later stages.

Together, we show that during spermiogenesis, Asl is gradually reduced from centrosomes and Plk4 and Slimb regulate its reduction. Additionally, we find that abnormally high amounts of Asl in spermatozoa are deleterious to embryonic development by interfering with the ability of the paternal centriole to form astral microtubules. This phenotype demonstrates that Asl reduction is an essential aspect of centrosome reduction and abnormalities in Asl reduction cause paternal effects.

3.5 Acknowledgment

We thank Andrew Ha and Allen Church for technical assistance; Dr. Edmund

Koundakjian, Sarah E. Hynek, and Emily Fishman for editing this manuscript; Dr.

Richard Komuniecki and, Dr. Timothy Megraw for commenting on the manuscript; Dr.

Deborah Chadee for advice. We also thank Dr. Jordan Raff for the uAslGFP, and

Plk4OE flies, Dr. Daniel St Johnston for SlimbGFP, Dr. Gregory Rogers for the PlkKD plasmid, and Bloomington Stock Center for theplk4 mutants, nos-GAL4 lines and

Piggybac-transposase line. This work was supported by grant 1121176 (MCB) from the

National Science Foundation and R01GM098394 from the National Institute of General

Medical Sciences.

Author contribution

T.A.R. conceived and supervised the project. T.A.R. and A.K. wrote the manuscript.

A.K. performed most of the experiments described.

Figure 3-1

Figure 3-2

Figure 3-3

Figure 3-4

Figure 3-5

Figure 3-6

Figure 3-7

Figure Legends

Figure 3-1 Asl is overexpressed in the testes of uAslGFP and eAslGFP.

(A) Quantification of centrosomal protein reduction in Drosophila at three stages (R: round spermatid, A: almost-needle spermatid, or S: spermatozoa). Ana1 was expressed under the control of the endogenous Ana1 promoter. All other proteins were expressed under the control of the exogenous ubiquitin promoter. (B-C) Testes of control (cont), uAslGFP, and eAslGFP were analyzed by western blot using anti-Asl and anti-actin

(loading control). Quantification of Asl intensity N=3, Mean±s.d., two-sided t-test,

***P<0.0001. (D) Attenuated Asl reduction in uAslGFP and AslGFP is consistently observed in almost-needle spermatid (left two panels and spermatozoa (right two panels).

The size and location of Asl in the spermatozoa suggest that uAslGFP label both the GC and the PCL.

Figure 3-2 Asl is reduced during spermiogenesis

(A) An Asl antibody (Blachon, et al., 2008) identifies Asterless (Asl; in red) in spermatid centrioles (top two panels) but not in spermatozoan centrioles (bottom two panels; the centriole location is identified by arrowhead). Nuclei are shown in blue. (B–G) Asl reduction during spermiogenesis is a continuous process. Each top panel shows low magnification and each of the bottom three panels magnifies the centriole. The stages of spermiogenesis are: (B) spermatocyte, (C) round spermatid, (D) elongated spermatid, (E) leaf spermatid, (F) almost-needle spermatid, and (G) spermatozoon. The giant centriole

(GC) and PCL are marked by Ana1-tdTomato (Blachon, Cai et al., 2009), eAslGFP is shown in green, and nuclei are in blue or indicated by N; mitochondrion, M. In (G), the

66 spermatozoan centrioles, identified by arrowhead in top panel, no longer have detectable levels of Ana1 or Asl. (H-J) Quantification of Asl reduction in Drosophila expressing eAslGFP or uAslGFP at three stages (R: round spermatid, A: almost-needle spermatid, or

S: spermatozoa). Note that graphs in H and I have a logarithmic scaled (***, P<0.0001,

N≥25, Mean±s.d.).

Figure 3-3 Specific coiled-coil domains in Asl are essential for normal Asl reduction

(A) Asl coiled-coil domain organization and Asl truncations (fused to GFP) used in the present study. Coiled-coil domain numbers and their amino-acid positions are indicated.

Asl truncations having GFP signal in round spermatid (R), almost-needle spermatid (A), or spermatozoa (S) are identified with “y” and those without GFP signal are identified with “n”. (B–D) Representative figures showing GFP signal for (B) a full-length AslGFP fusion having normal Asl reduction and two Asl truncation proteins, (C) an N-terminal

Asl truncation lacking coiled-coil domain 1 (Asl2-6) and having abnormal Asl reduction, and (D) an N-terminal Asl truncation lacking coiled-coil domains 1 to 4 (Asl5-6) and having no Asl reduction. Shown is GFP signal in centrioles during three stages of spermiogenesis (round spermatid (left), almost-needle spermatid (middle), and spermatozoa (right)). GC, giant centriole; N, nucleus; M, mitochondria; points to the spermatozoon centriole. Right panels include graphs quantifying the GFP signal at each of the three stages (R: round spermatid, A: almost-needle spermatid, or S: spermatozoa).

Scale bar, 5m (round spermatids) and 1m (almost-needle and spermatozoa). (E) The ratio of GFP-intensity in spermatozoon centrioles and spermatid centrioles shows

67 significantly abnormal Asl reduction in the Asl truncations. Two-sided t-test, ***,

P<0.001; N≥5; Mean±s.d

Figure 3-4 Asl truncation proteins.

(A–E) Representative figures of Asl truncation proteins (Asl1, Asl1-2, Asl1-4, Asl1-5, and Asl6) that do not appear to express during spermiogenesis. (F-G) Representative figures of Asl truncation proteins Asl3-6 and Asl4-6 that express during spermiogenesis and quantification of their reduction. N, nucleus; M, mitochondrion; an arrowhead points to where Asl would be expected to be in spermatids or spermatozoa lacking Asl. Scale bar, 5m (round spermatids) and 1m (almost-needle and spermatozoa). N≥5; Mean±s.d.

Note that the fusion protein having only coiled-coil domain 6 lacked GFP localization even in the round spermatid stage (Fig 2A), suggesting that domain 5 is essential for centriole localization. Also note that of the fusion proteins having C-terminal deletions, only the fusion protein having Asl’s coiled-coil domains1 to 5 (Asl1–5) retained GFP localization by the round spermatid stage; when compared to wild-type Asl, Asl1-5’s

GFP localization was prematurely lost by the almost-needle spermatid stage. Because this fusion protein’s signal in round spermatid is weak, when compared to others, its absence at later stages likely represents the outcome of centrosome reduction that beginning at a lower signal intensity. (H) Plk4GFP level is not affected by expression of

Asl deletion construct Asl2-6, and Asl5-6. Western blot using anti-GFP and anti-actin

(loading control). Asl, were analyzed by Quantification of GFP intensity N=3, Mean±s.d., two-sided t-test, ***P<0.0001. cont, control

Figure 3-5 Plk4 and Slimb regulate Asl reduction

(A) Like Asl, Plk4 is subject to centrosome reduction during spermiogenesis as demonstrated by a Drosophila line having a ubiquitin promoted, GFP-tagged Plk4. (B)

Plk4 knockdown by Nos-GAL4/UAS-Plk4 RNAi results in attenuated Asl reduction

(Nos+Plk4i); normal Asl reduction is shown by data from Nos-GAL4flies (Nos) without a UAS activator and by data from UAS-Plk4 RNAi (Plk4i) flies without a GAL4 activator. (C) Similarly, spermatozoa homozygous or heterozygous forplk4c06612 (a P- element insertion, partial loss of function allele) have attenuated Asl reduction; normal

Asl reduction is shown by flies bearing the eAslGFP transgene. Overexpression of Plk4

(Plk4OE) in plk4c06612homozygotes (hom) or heterozygotes (het) rescues the plk4c06612– dependent attenuated Asl reduction. (D) Attenuation of Asl reduction is observed by overexpression of slimb using Nos-GAL4 + UAS-SlimbGFP (Asl + Nos + Slimb) in the background of Asl-td-Tomato, which labels Asl in red. Nuclei are stained in blue. Scale bar, 1m; the spermatozoon centrioles are identified by arrowhead. In the right panel, normal Asl reduction is shown by data from Asl-td-Tomato files (Asl) or Asl-td-Tomato flies bearing Nos-GAL4 and without an UAS activator (Asl + Nos). (E)

Immunoprecipitation with anti phospho-threonine antibody shows that Asl is phosphorylated. Asl phosphorylation is enhanced when Plk4 is overexpressed (Plk4OE) and is attenuated when Plk4KD is overexpressed. (F) Overexpression of Plk4KD attenuates Asl-td-Tomato reduction. (G) Immunoprecipitation with anti-Ubiquitin antibody found that Asl is Ubiquitinated. Asl ubiquitination is enhanced when Plk4 is overexpressed (Plk4OE). Two-sided t-test, ***P<0.001, **P<0.01, *P<0.05; N≥5;

Mean ±s.d.

Figure 3-6 Plk4 regulates Asl reduction.

(A) Plk4 knockdown by Nos-GAL4/UAS-Plk4 RNAi results in attenuated Asl reduction

(Nos+Plk4i). Two lines point to the two Asl foci observed in the spermatozoa. (B) Plk4 domain organization and the plkc066122 mutation. An arrow points to the insertion site of the transposon in the plk4c06612 mutation. PB, polo-binding domain. (C-F) Spermatozoa homozygous (C) or heterozygous (D) for plk4c06612 have attenuated Asl reduction.

However, overexpression of Plk4 (Plk4OE) in plk4c06612 homozygotes (hom) (E) or heterozygotes (het) (F) rescues the plk4c06612–dependent attenuated Asl reduction. Two lines point to the two Asl foci observed in the spermatozoa. In some cases, as in “C” one of the foci is longer and likely represents a GC labeled with Asl along its length. In other cases, both foci are small and may be a PCL and a partial labeling of the GC. (G)

Excising the plk4c06612 transposon restores Asl reduction in eAslGFP flies. Two-sided t- test, ***P<0.001; N≥5; Mean±s.d. (H) Plk4OE does not affect the level of Asl2-6 in spermatozoon centrioles. (I-K) Plk4 overexpression enhances eAslGFP (I) and uAslGFP reduction. (J) Two-sided t-test, ***P<0.001; N≥5; Mean±s.d. Plk4 overexpression does not affect expression of uAslGFP in the testes (K). (L) Overexpression of Slimb using

Nos-GAL4 does not affect the level of Asl2-6 in spermatozoa. (M) Asl RNAi (Asl) does not affect Plk4GFP reduction. (N) Flies heterozygous for both slimb and plk4 mutations have undetectable uAslGFP in spermatozoa. (O-P) Heterozygous mutants of plk4 have attenuated Asl reduction (eAslGFP), but not attenuated Ana1 reduction (O) or Sas-4 reduction (P). N, nucleus; M, mitochondrion, A, almost-needle spermatids; S, spermatozoa. Scale bar, (round spermatids) and (leaf and almost-needle spermatids as well as spermatozoa).

Figure 3-7 Attenuated Asl reduction reduces post-fertilization zygotic development

(A–C) Embryos produced from spermatozoa with increased Asl have reduced larval hatching. (A) Compared to control flies (w1118), embryos fathered by eAslGFP or uAslGFP, which have attenuated Asl reduction, produced significantly fewer embryos that hatched. This phenomenon was rescued when Plk4 is overexpressed (PLK4OE).

(B) Embryos fathered by Plk4 knockdown males (via Nos-GAL4/UAS-Plk4 RNAi; “Nos

+ Plk4i”) produced significantly fewer embryos that hatched when compared to control flies: Nos-GAL4 flies without an UAS activator (Nos) or UAS-Plk4 RNAi flies without a

GAL4 activator (Plk4i). (C) Embryos fathered by males overexpressing Slimb, which bear Nos-GAL4/UAS-Slimb-GFP (SlimbOE) produced significantly fewer embryos that hatched when compared to control flies: Nos-GAL4 flies without a UAS activator (Nos) or UAS-Slimb-GFP flies without a GAL4 activator (Slimb). Two-sided t-test,

***P<0.001; N≥5; Mean±s.d. (D–F) One-hour-old embryos fathered by control flies

(w1118) or uAslGFP flies; all embryos from control fathers and no embryo having an uAslGFP father had greater than 32 nuclei (D, E); nuclei stained with Hoechest blue. (F)

Embryos fathered by Plk4 heterozygotes or fathers overexpressing Slimb had delayed embryo development compared to control males. (G) Embryos fathered by plk4 heterozygotes but not slimb or fathers having both plk4 and slimb had delayed embryo development compared to control fathers. (H–K) Microtubule asters are observed in zygotes fathered by control males (w1118) (H), but asters are not seen in zygotes fathered by uAslGFP (I) or plk4(J)males. Microtubule asters are eventually present in embryos fathered by uAslGFP males once they have passed the zygote stage (K). Scale bar, 25m

(egg panel) and 10m (nucleus, N, panel). (L) Model for Asl reduction. At least two

71 mechanisms regulate Asl reduction: one in early spermatogenesis and another in late spermatogenesis. Asl reduction in late spermiogenesis is mediated by the interaction of

Asl’s first coiled-coil domain with PLk4. Additional Asl reduction pathways depends on

Asl’s domain 4. When centrosome reduction (including Asl reduction) is attenuated, spermatozoa can fertilize an ovum, but the embryo develops abnormally.

Chapter 4

Centriole Remodeling during Spermiogenesis in Drosophila

Previously published as Khire, A., Jo, K. H., Kong, D., Akhshi, T., Blachon, S., Cekic, A.

R., Avidor-Reiss, T. 2016. Centriole Remodeling During Spermiogenesis in Drosophila. Current Biology: CB, 26(23), 3183–3189

4.1. Abstract

The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM). In some animals, the sperm centrioles with typical ultrastructure are essential for sperm motility and are the origin of the first centrosomes in the zygote. In other animals, sperm centrioles lose their proteins and are thought to be degenerated and non-functional. Here, we show that the two sperm centrioles (the giant centrioles, GC, and Proximal Centriole-

Like structure, PCL) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and structural modification in parallel to previously

73 described, centrosomal reduction. We found that the ultrastructure of the matured sperm

(spermatozoa) centrioles are modified dramatically and that PCL has no resemblance to a typical centriole. We also describe a new phenomenon, “centrosome enrichment”, by which the two isoforms of the centriolar protein Poc1 (Poc1A and Poc1B) are enriched at the two atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, we found that the enrichment of paternal Poc1 is essential for formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote.

Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and structure, yet they are functional and essential for normal embryogenesis in Drosophila.

4.2 Methods and Materials

4.2.1 Transgenic Flies

All Drosophila stocks were cultured on standard media at 25°C. In gPoc1ABGFP, the poc1 promoter, intron and exons were subcloned into a pattB vector with an in-frame C- terminal GFP tag attached to the last exon of poc1B and were docked at attP1 landing site. Germline transformations were performed by BestGene Inc. (Chino Hills, CA).

Ana1GFP, Ana1tdTomato, and AslGFP were previously described and are expressed by their respective promoters (Blachon, et al., 2008, Blachon, et al., 2009). Ana2GFP was expressed using an ubiquitin promoter, and was provided by Jordan Raff (Stevens et al.

2007, Basto, et al. 2008). Poc1-RNAi (Vienna Drosophila Resource Center # v38353) was expressed using the Bam-Gal4 promoter. The fly line with Bam-Gal4 on the X chromosome was provided by Yokiko Yamashita. Deficiency for Poc1: w1118; Df(3L)

ED4502, P{3'.RS5+3.3’} ED4502/TM6C was obtained from the Bloomington Stock

Center (stock #8097). poc1k245 and poc1c06059 were previously described (Blachon, Cai et al., 2009). poc1W87X (Zuker line 3-3416) is an EMS mutation from the Zuker collection

(Koundakjian, Cowan et al., 2004). poc1W45R (University of Toledo line 1446) is an EMS mutation from the Avidor-Reiss collection that was generated in a similar manner to that of the Zuker collection (unpublished).

4.2.2 Fluorescence Microscopy

For embryo imaging, 25 males and 25 virgin flies, no older than 5 days old, were placed in an egg collection chamber with a grape agar plate with yeast paste. Chambers were used for 3 days and embryos collected every 4 minutes. Immediately after collection, the embryos were placed in a MatTek dish and washed with 100μl of distilled water, and then a wash buffer (0.7 %(w/v) NaCl+0.05%(v/v) Triton 100X). Then, wash buffer was removed, and 50%(v/v) bleach solution was added to the embryos in the MatTek dish, until the appendages of the embryos disassociated. Next, the embryos were rinsed twice with wash buffer. Finally, the embryos were transferred to a 1.5ml microcentrifuge tube where they were fixed in a 1:1 solution of heptane and methanol and vortexed vigorously until the embryos settled down in the methanol layer. This was followed by removal of the fixative and suspension of the embryos in acetone. At this stage, the embryos were usually stored at -20°C. Embryos were rehydrated sequentially in 70%, 50%, 30%, and

10% (v/v) methanol in PBS, and then in PBS alone. The embryos were then incubated in

PBT (PBS + 1% (v/v) Triton) for 30 minutes, and blocked in PBST (PBT+3% (w/v)

BSA) for 1 hour. The embryos were incubated with primary antibodies in PBST for 1 hour at room temperature. After three 5-minute washes in PBT, the embryos were

75 incubated with secondary antibodies and 1 μg/ml of DAPI for 1 hour at room temperature. The embryos were washed three times with PBT for 5 minutes each, and then washed in PBS for 5 minutes. The embryos were mounted on a slide using a mounting medium (PBS, 50% (v/v) glycerol, 0.5% (w/v) N-propyl-gallate) and imaged.

4.2.3 Testes Fluorescence Microscopy

Testes imaging was performed as described previously (Basiri, Blachon et al. 2013). For

GFP/TdTomato imaging, testes of pharate adult pupae were dissected in PBS and imaged directly.

For antibody staining, testes of pharate adult pupae were dissected in PBS, squashed with a coverslip, frozen in liquid nitrogen for 5 min, fixed in 3.7% formaldehyde in PBS for 5 min at room temperature, and washed with PBST (PBS + 0.1% Triton). Antibodies were incubated in PBST-B (PBS + 0.1% Triton + 1% BSA) for 1 hour at room temperature, followed by three washes in PBS.

For photon counting, at least 5 testes were analyzed. In each testis, photons were counted from 5 centrioles at each stage. Pictures were taken in the photon counting mode using a

63x objective with a zoom of 6, pin hole of 1.2, resolution of 512×512 pixels, 488 nm laser power of 1% of 20%, and using a measuring box of 1µm by 2.5µm that was placed on top of the centriole.

Antigen accessibility to antibody can impact the analysis of protein enrichment. To overcome this concern, direct imaging of Poc1 tagged with GFP with no antibody staining was used.

Images were taken by Leica SP8 scanning confocal microscope, Z stacks at room temperature, and analyzed using Leica software. Maximal projection images were then modified using Adobe Photoshop and annotated using Adobe Illustrator.

4.2.4 Testes Super Resolution Microscopy

3DSIM data was acquired using an ELYRA PS.1 microscope from Carl Zeiss equipped with a Plan-Apochromat 63x/1.4 Oil immersion objective lens with an additional 1.6x optovar. Images were collected with an Andor iXon 885 EMCCD camera, resulting in a raw data pixel size of 79 nm. Z-stacks were acquired with a spacing of 101 nm/pixel.

The fluorophores were excited with a 200 mW 488 nm laser. Images were acquired with a laser power at the objective focal plane of 52.6 mW attenuated to 5%. Exposure times were between 50-200 ms and EMCCD camera gain values between 5-20. Five phases at each of three rotation angles (-75o, -15o, +45o) of the grid excitation pattern were acquired. A 495-550 band-pass filter was used to collect fluorescence from Alexa 488 antibodies labeled samples. The data were processed using the SIM module of the Zen software version 8.1 with Weiner filter between 10-3 and 10-5.

PCL diameters were determined using Zen software version 8.1, by drawing a line across the PCL in three different directions. The distance between two ends of the base line of the intensity graph was determined for each direction, and the average distance for three directions was calculated for each PCL. Identical measurements were performed for 45

PCLs expressing the individual GFP-fusion PCL proteins, and the average value was determined for each marker. The graph and the statistical analysis (unpaired student T- test) were performed using GraphPad Prism software.

4.2.5 Antibodies.

Anti-N-Poc1, Anti-C-Poc1, and Anti-M-Poc1 were made in rabbits by Pacific

Immunology Inc. Anti-N-Poc1, designed to detect both Poc1 isoforms, recognizes part of the amino acids 6 to 20 (RDPALERHFTGHSGG), and Anti-C-Poc1 recognizes amino acids 355 to 373 (LSEENFQVLDSKHSTQPEK). In western, Anti-Poc1 N-terminus

(3218) was used at 1:500 in 5% BSA, and Anti-Poc1 C-terminus (3763) was used at

1:4000 in 5% BSA. Anti-M-Poc1, designed to detect both Poc1 isoforms, recognizes amino acids 88 to 106 (EPKLRGVSGEFVAHSKAVR). For immunofluorescence, Anti-

Poc1 M-terminus was used at 1:500.

The following antibodies were used for immunofluorescence at indicated concentrations:

Primary antibodies for immunofluorescence: E7-mouse anti-β-tubulin, 1:200

(AB_2315513, DSHB at The University of Iowa); rabbit anti-Asl (Blachon,

Gopalakrishnan et al. 2008), Secondary antibodies for immunofluorescence:

Cy5™AffiniPure Goat Anti-Rabbit IgG (H+L), 1:200; Alexa Fluor® 488-conjugated donkey anti-rabbit IgG, 1:200 (Jackson ImmunoResearch). For super resolution microscopy, the antibodies used were anti-GFP (Chicken GFP 1:2000, Abcam) and secondary antibodies were Alexa Fluor® 488-conjugated goat anti-chicken IgG, 1:500

(Invitrogen).

4.2.6 Western blot

Equivalent amounts of testes lysates from wild type, poc1k245, poc1c06059, and poc1W45R were resolved in SDS-PAGE and immunoblotted with anti-Poc1-C and anti-Poc1-N antibodies. Testes were collected in PBS buffer and boiled for 5 min in 95 °C with 1X

Laemmli sample buffer. Twelve testes were run per lane in 10% polyacrylamide gels and

78 transferred into nitrocellulose membranes. The blots were incubated with primary antibodies overnight at 4°C, followed by peroxidase-conjugated secondary antibodies at

RT for 1h. Super Signal West Pico Chemiluminescent substrate (Pierce) was used to detect peroxidase activity. Molecular masses were determined by comparison to

Precision Plus Protein Standard (Bio-Rad). Western blots were analyzed with ImageJ

(National Institute of Mental Health, Bethesda, Maryland, USA), and the intensity of the total area was calculated using an identical rectangular box.

4.2.6 Tissue Processing and Transmission Electron Microscopy

For TEM analysis of PCL and centriole ultrastructure, the testes and seminal vesicles of flies were dissected and immediately processed using the High-Pressure Freezer system

(Leica EM HPM100) in 20% BSA. The frozen tissue was dehydrated and en bloc stained using a Freeze Substitution preprocessor (Leica EM AFS2) in 96% acetone with 1.5%

OsO4 (Osmium crystals were dissolved in acetone and 4% water). The freeze substitution started at -90ºC for 6 hours, then warmed up from -90ºC to -10ºC over 15 hours (5.3- degree slope). Then, the tissue was warmed to -3ºC over 1 hour (7-degree slope) while washed with 96% acetone (at -7ºC). Then it was warmed from -3ºC to 4ºC over 1 hour

(7-degree slope) while washed twice by 100% acetone. Lastly, it was infiltrated and embedded in EMBed 812 resin while warming up to room temperature. The ultrathin sectioning (70 nm) was performed using ultramicrotome (Leica EM UC6), and sections were post-stained with 6% Uranyl Acetate (in 1:1 70% ethanol and 100% methanol), and

Reynolds Lead citrate (3-4%) (in pre-boiled ddH2O). Then, the sections were imaged using TEM (JEOL 1400-plus) operating at 80 kV.

4.2.7 Electron Tomography

For TEM tomography, we used the high tilt probe for TEM (JEOL 1400-plus) to obtain serial electron micrographs at ±60 degree angles with 1.5 degree increments operating at

120 kV. The 3-D reconstruction was performed using ETomo, IMOD, and UCSF

Chimera.

4.2.8 Correlative Light and Electron Microscopy

Testes from 1-2 day old males, expressing Ana1-GFP or Poc1B-GFP, were prepared and fixed with 0.5% glutaraldehyde and 1% formaldehyde in PBS. The tissue was then embedded in 4% agarose. 200 nm thick Z-sections through the testes were recorded immediately to register the position of the centrioles within the tissue using a Nikon

Eclipse Ti inverted microscope, equipped with a 6.45 µm pixel CoolSNAP HQ2 camera

(Photometrics) and Intensilight C-HGFIE illuminator, using 60x NA 1.42 Plan Apo objective. The tissue was post-fixed with 2.5 % glutaraldehyde for 30 min. The agarose block was extensively washed in PBS, pre-stained with 2% osmium tetroxide and 1% uranyl acetate, dehydrated and embedded in EMbed-812 resin. 80 nm thick serial sections were then sectioned, additionally stained with uranyl acetate and lead citrate, and imaged using a transmission electron microscope (Hitachi), operating at 80 kV. Image analysis and alignment of the serial sections were performed using ImageJ and Photoshop.

4.2.9 One Hour Embryo Development Assay

Twenty-five males and twenty-five females were placed in a humidified mating chamber at 25°C to lay eggs for 5 minutes. Embryos were collected and placed for 1 hour in a

MatTek dish containing wash buffer, and were processed for embryo fluorescence microscopy staining.

4.2.10 Seventy-Two Hour Embryo Development Assay Analysis and Larval Hatching

Twenty-five males and twenty-five females were placed in a humidified mating chamber at 25°C to lay eggs for 24 hours. The parents were removed. Seventy-two hours later, unhatched eggs were stained for Hoechst to observe the cleavage stage, blastoderm stage, and post-blastoderm stage. For larval hatching, we used only five males and five females, and the percent of hatched eggs was calculated. Experiments were repeated at least 5 times. Each experiment typically resulted in 80 or more embryos.

4.2.11 Sperm Flagellum Detection in Embryo

Embryos were collected as described in the “Fluorescence Microscopy” section.

However, the males were transgenic flies with tubulin RFP (Basto, Brunk et al. 2008), and embryos were collected every 4 minutes. Antibodies were not used, but the embryos were incubated in PBT (PBS+1% Triton) for 30 minutes, and then incubated in 0.6 μg/ml

DAPI for 30 minutes at room temperature. The embryos were washed three times with

PBT for 5 minutes each, and then washed in PBS for 5 minutes. The embryos were mounted on a slide using PBS and imaged.

4.2.12 Statistical Methods

Experiments were repeated at least three times, and statistical analyses (SEM+/-) were done with GraphPad Prism 5. A two-tailed, unpaired Student’s T-test was used.

4.3 Results and Discussion

4.3.1 Poc1 enrichment during spermiogenesis

Poc1 is a conserved centriolar protein essential for centriole elongation and stability

(Hames, et al., 2008, Pearson, et al., 2009, Fourrage, et al., 2010). Humans have two gene orthologs (poc1A and poc1B) (Venoux, et al., 2013); however, in Drosophila, differential splicing of a single poc1 gene produces two protein isoforms (Poc1A and Poc1B). The correspondence between the human genes and the Drosophila splice isoforms is unclear.

To study the localization of Poc1 in Drosophila, we made a gPoc1ABGFP genomic construct, consisting of the Poc1 promoter, introns, exons, and GFP, which expresses an untagged Poc1A and GFP-tagged Poc1B (Poc1BGFP) (Fig 4-1A). The expression level of Poc1B was low in spermatogonia and increased in spermatocytes, and by the last stages of spermiogenesis, Poc1B intensely labeled the PCL and weakly labeled the GC in spermatids and spermatozoa (Fig 4-2A-B). This increase in Poc1B was unlike the behavior of centrosomal proteins Ana1 and Asl, which are reduced during spermiogenesis. The reduction in expression levels of these centrosomal proteins during spermiogenesis is referred to as “centrosome reduction” (Schatten 1994, Khire, et al.

2015). Therefore, we named the increase in the expression level of Poc1B during spermiogenesis “Poc1 enrichment.” Altogether, these data suggest that the sperm centrioles’ protein composition is modified by both centrosome reduction and Poc1 enrichment.

4.3.2 Sperm centrioles are modified into atypical structures.

To understand the correlation between centrosome reduction, Poc1 enrichment, and centriole ultrastructural modification, we analyzed the ultrastructure of GC and PCL

82 throughout spermiogenesis. We performed transmission electron microscopy (TEM) analysis with High Pressure Freezing-Freeze Substitution (HPF-FS), which better maintains structural integrity when compared to the chemical fixation that was used in the past (Blachon,et al. 2009).

Within each set of serial sections of early-elongated spermatids with round nuclei, we observed a novel structure in a single 70 nm-thin section near the GC in the centriolar adjunct (CA) (Fig 4-2C) (Fabian and Brill 2012). In longitudinal sections, the structure was composed of an electron dense material that was 119±9 nm (N=3) in diameter and a central tubule parallel to the electron dense material that was 24±2 nm (N=3) in diameter

(Fig 4-2Ci-ii). In cross-sections, it was composed of a thick, electron-dense ring that was

124±14 nm (N=9) in diameter and an internal ring at its center that was 24±3 nm (N=6) in diameter (Fig 4-2Ciii-iv). Based on its location, size, and Correlative Light and

Electron Microscopy (CLEM) with Ana1GFP, we concluded that this structure was the

PCL (Fig 4-2D). These results suggest that the PCL is an atypical centriole made of an interstitial material between an electron-dense wall and a central tubule, measuring ~120 nm and ~24 nm in diameter, respectively (Fig 4-2E).

The PCL of intermediate elongated spermatids had an ultrastructure similar to that of the early elongated spermatid PCL (Fig 4-2F). However, the diameter of the PCL central tubule was decreased to 21±1 nm (N=8, P<0.001). This is a specific modification, as the diameter of the axoneme central microtubules was unchanged (23±2 nm) (Fig 4-1B-C).

In late elongated spermatids, the PCL ultrastructure lacked the electron-dense wall and was made of a central tubule with a diameter of 16±4 nm surrounded by translucent interstitial material (Fig 4-2G). In spermatozoa, CLEM using gPoc1ABGFP showed that

83 the PCL resided near the GC and nucleus (Fig 4-2H). Using this PCL position and HPF-

FS-TEM, we identified the PCL as consisting of only the central tubule in spermatozoa

(Fig 4-2I). These findings suggest that the PCL is modified throughout spermiogenesis, resulting in a PCL made up of only a central tubule and enriched by Poc1 in spermatozoa.

Like the PCL, GC proteins were also reduced during spermatogenesis (Fig 4-2) (Wilson, et al. 1997, Li, et al. 1998, Blachon, et al. 2014). We found that the GC in spermatids had a translucent central lumen surrounded by triplet microtubules organized in 9-fold symmetry, as previously reported (Fig 4-1D) (Tates 1971, Tokuyasu 1975). During the maturation from spermatids to spermatozoa, the outer diameters of the GC and axoneme were reduced by 23% (from 205 nm to 156 nm) and 15% (from 175 nm to 149 nm), respectively (Fig 4-1D-F), indicating that the overall structure of the sperm became condensed. In spermatids, the GC central lumen was round, with a diameter of

127±12 nm (N=10); but in spermatozoa, it was elliptical, with the major axis measuring

63±20 nm long (~41% reduction) and the minor axis measuring 31±18 nm long (N= 5,

P<0.001; ~86% reduction), indicating that the GC was unevenly deformed. This modification was centriole-specific, as the central lumen diameter of the axoneme was reduced only by ~14% from 137±11 nm (N=10) in spermatids to 118±8.0 nm (N=10,

P<0.001) in spermatozoa (Fig 4-1D-F). Altogether, these findings indicate that centriolar ultrastructure is modified in parallel to centrosome reduction and enrichment, and suggest that typical centriole structure is dispensable for their functions during fertilization.

4.3.3 Poc1 has a differential role in GC and PCL formation

Since Poc1 is uniquely enriched in the spermatozoa centrioles after centrosome reduction, we studied its role using poc1 mutants (Fig 4-3A). Previously identified poc1k245 has a

84 splice site mutation and is predicted to result in premature termination at the 1st intron, and poc1c06059 has a p-element insertion that reduces Poc1 expression (Blachon, et al.

2009). Newly identified poc1W87X (Zuker line 3-3416 (Wakimoto, et al. 2004)) has a nonsense mutation, replacing Trp 87 with a stop codon, suggesting it is a severe loss-of-function mutant. Another newly identified mutant, poc1W45R (University of

Toledo line 1446), has a missense mutation which mutates the conserved hydrophobic

Trp 45 of the first WD repeat with positively charged arginine, eliminating two of the predicted pentad hydrogen bonds that stabilize its structure (Wang,et al. 2015). This suggests that poc1W45R is a partial loss-of-function mutant (Fig 4-4A). Antibodies that recognize the N-terminus (anti-Poc1N) and the C-terminus (anti-Poc1C) of Poc1 indicated that the poc1 mutants differentially affect Poc1 expression (Fig 4-4B).

Antibody that recognizes both Poc1A and B in situ (anti-Poc1M) labeled the spermatozoa centrioles of control and poc1k245, but not poc1c06059, poc1W45R, or poc1W87X suggesting that the mutants also have distinct effects on localization of Poc1 (Fig 4-4C).

Centrioles are required for cilium formation; therefore, many centriolar proteins are essential for the sensory cilia which mediate fly locomotor behavior, as well for the motile cilia (flagella) that propel the sperm (Avidor-Reiss, et al. 2012). Flies homozygous for any of the four-poc1 mutants had apparently normal locomotor behavior, suggesting that Poc1 is not essential for sensory cilium function. However, all poc1 mutants exhibit abnormal sperm phenotypes. poc1W87X had immotile spermatozoa with abnormal axoneme architecture, suggesting that it is a severe loss-of-function mutant and that Poc1 is essential for motile cilium function (Fig 4-4D-E). poc1W87X, poc1c06059 and poc1k245, but not poc1W45R, had shorter GCs (Fig 4-3B). Most importantly, in all of these mutants,

85 while Asl and Ana2 were recruited to both the GC and PCL, Ana1GFP was recruited only to the GC and not to the PCL (Fig 4-3B-C). Interestingly, unlike canonical centrioles, the poc1 PCL has Asl in the absence of Ana1, suggesting a molecular difference between canonical centriole and PCL assembly (Fu, et al. 2016). These findings suggest that Poc1 is involved in the modification of centriole protein composition during spermiogenesis. Currently, no Poc1 function has been reported in

Drosophila outside the male germ line.

Because Poc1B intensely labels the PCL in spermatids and spermatozoa, we studied the localization of Poc1B and Ana1 using 3D Structured Illumination Microscopy. Our data suggests that Poc1B is found in the PCL center along with N-terminal Ana1, while the outer periphery of the PCL is occupied by C-terminal Ana1 (Fig 4-3D). Furthermore, in poc1W87X, the PCL of the intermediate elongated spermatid has an amorphous electron dense structure, but the central tubule is missing (Fig 4-3E). These findings indicate that

Poc1B is localized in the PCL center.

4.3.4 Sperm centriole remodeling is essential for the formation of zygotic centrosomes

To study the role of Poc1 enrichment during fertilization, we examined the role of paternal Poc1 in the zygote (Fig 4-5). In controls, the GC and PCL were found near the decondensed male pronucleus and recruited Asl, Cnn, and D-PLP to form astral microtubules, which are essential for centrosome function in the zygote (Fig 4-5A-B). In

W45R W45R zygotes fathered by poc1 (poc1 pat-embryos), the spermatozoon tail was present in the cytoplasm of the embryo, suggesting that fertilization took place (Fig 4-6A). In these zygotes, Asl and Cnn were recruited to both the GC and PCL, but D-PLP was only recruited to the GC. These data suggest that Poc1 enrichment in the PCL has a role in

86 recruiting proteins to the paternal centriole to form a fully functional centrosome immediately after fertilization.

During the first mitosis, all of the control zygotes (N=46) had bipolar spindles, and each pole had both a microtubule aster and two Asl-labeled centrioles: at one pole, the GC, and its daughter centriole, and at the other pole, the PCL, and its daughter centriole (Fig

W45R 4-5C). In contrast, all poc1 pat-mitotic embryos had monopolar spindles (N=64) that lacked the microtubule aster but had two centrioles at this pole: a long Asl-focus (GC) and a daughter centriole, which was marked by maternal Sas-6GFP (Fig 4-5C-D)

(Blachon, et al. 2014). Expressing Poc1 (gPoc1ABGFP) in poc1W45R males rescued this monopolar spindle phenotype (N=35) (Fig 4-5C). These data suggest that defects in

W45R paternal Poc1 lead to abnormal zygotic division. However, in some poc1 pat-mitotic embryos, astral microtubules eventually form (Fig 4-6B), suggesting that some compensatory mechanism is present. Ultimately, these embryos suffered from dramatic developmental delay

(Fig 4-5E), developmental arrest (at the cleavage stage and at the blastoderm stage) (Fig

4-5F), and ~50% reduction in larval hatching (Fig 4-5G). Altogether, these findings suggest that Poc1 enrichment is essential for normal embryo development.

4.3.5 Poc1 enrichment in the spermatocyte and spermatid is essential for embryogenesis

To study the specific role of Poc1 enrichment in sperm cells, we expressed Poc1 specifically in late spermatogonia and early spermatocytes using the BamGal4 promoter with UASPoc1A, UASPoc1B, or a combination of both UASPoc1A and UASPoc1B

(cPoc1ABGFP) (Fig 4-7A). We found that spermatocyte Poc1A and B are both essential

87 for GC elongation (Fig 4-8A). Poc1A is essential and sufficient for sperm motility, and

Poc1B is essential and sufficient for regulating PCL protein composition (Fig 4-8B-C).

These findings indicate that Poc1 isoforms have differential and essential roles in the spermatocyte centrioles.

To understand the effect of Poc1 spermatocyte enrichment during embryogenesis, we mated the poc1W87X mutant males expressing the various Poc1 transgenes with control females (Fig 4-8D). Expressing both Poc1A and B (Poc1ABGFP or cPoc1ABGFP) in poc1W87X rescued the embryonic phenotypes. However, expressing only Poc1A did not rescue the phenotypes. These observations suggest that Poc1B enrichment in the spermatocyte is essential for forming the second spindle pole and for embryo development. Finally, to study the role of Poc1 later in spermiogenesis (and independently of its role in the spermatocyte), we used BamGal4 and UASPoc1RNAi, as their effect is executed later, due to the time it takes to sufficiently accumulate RNAi and for natural degradation of endogenous protein to occur (White-Cooper 2012). As expected, the Poc1 RNAi had no effect on centriole elongation in the spermatocyte, but it inhibited Ana1 recruitment to the PCL in the spermatids (Fig 4-8E). Importantly,

RNAi poc1 pat-embryos suffered from delayed development (Fig 4-8F), further demonstrating that Poc1 enrichment in the male is essential for normal embryo development.

In summary, our results suggest that during spermatogenesis and in parallel to centrosome reduction, the sperm centriolar ultrastructure is modified and that Poc1 is enriched in sperm centrioles. Poc1 enrichment is essential for spermatocyte centriole elongation, normal PCL formation in spermatids, and sperm motility. Furthermore,

88 paternal Poc1 is essential for the formation of normal zygotic centrosomes. Since sperm centrioles that are modified in both protein composition and ultrastructure are functional, we propose that during spermatogenesis, the centrosome is remodeled rather than degenerated and that this remodeling phenomenon is essential for its function in normal embryo development (fig 4-8G). Centrosome remodeling may be a universal phenomenon in all animals, as atypical sperm centrioles are found in many other animals, including humans.

4.5 Acknowledgements

We would like to thank, Dr. Alan D. Hammer for editing, Dotty Sorenson, Sasha

Meshinchi, Jeff Harrison, Chris Edwards for assistance at the Microscopy & Image

Analysis Laboratory (MIL) in the University of Michigan Medical School; EPIC facility

(NUANCE Center-Northwestern University, supported by NSF DMR-1121262) the

CryoCluster equipment (received support from the MRI program, NSF DMR-1229693); the International Institute for Nanotechnology (IIN); and the State of Illinois, through the

IIN and Charlene Wilke at the Biological Imaging Facility in the Northwestern

University; Emily Simone and Michaela Roberts for technical assistance. We would like to thank Dr. Jordan Raff for the D-PLP antibody, Dr. Timothy Megraw for the Cnn antibody, Dr. Barbara Wakimoto for Drosophila male sterile mutant, Dr. Yukiko

Yamashita for Bam/Gal4 fly line, Dr. Clemens Carbard for the cPoc1AGFP and BGFP lines and Bloomington Stock Center for the poc1c06059 mutant flies, Dr. David Glover for the GFPAna1 line. This work was supported by grant 1121176 (MCB) from the National

Science Foundation and R01GM098394 from the National Institute of General Medical

Sciences.

4.6 Author Contributions

Atul Khire performed Figure 1A, Figure 2A-B, Figure 3(A-C), Figure 4(C-D), Figure 5,

Figure 6B and Figure 7 and Kyoung H. Jo performed Figure 1B-C, 1D-F and Figure 2.,

Figure 6A. 3D-SIM experiments were performed in collaboration with Dr. Vito Menella.

CLEM was done in by Dr. Jadranka Loncarek

Figure 4-1

Figure 4-2

Figure 4-3

Figure 4-4

Figure 4-5

Figure 4-6

Figure 4-7

Figure 4-8

Figure Legends

Figure 4-1 Poc1 constructs, as well as PCL and GC structure

A) To gain insight into the functions of Poc1A and B in the sperm centrioles, we used a gpoc1ABGFP construct that codes for both Poc1 isoforms, with only Poc1B tagged with

GFP.

B) The PCL central tubule is thinner in intermediate elongated spermatids (IE) (N=8) and late spermatids (LS) (N=4), compared to early elongated spermatids (EE) (N=6) (***,

P<0.001). However, axoneme central microtubule diameter is similar in early spermatids

(EE) (N=10), intermediate spermatids (IE) (N=10), and late spermatids (LS) (N=6).

Also, the diameter of intermediate spermatid PCL central tubules is smaller than that of the axoneme central microtubules (**, P<0.01). Thus, the PCL central tubule is distinct from the microtubules.

C) The GC central microtubules are composed of canonical or non-canonical numbers of protofilaments and have diameters that do not change over time. However, the PCL central tubule diameter is dynamic; therefore, it is unlikely to be a microtubule. Also, unlike a centriole cartwheel, the central tubule does not have spokes, and it is present after Sas-6 reduction; therefore, the PCL central tubule is unlikely a cartwheel. Scale bars, 50nm.

D-F) The lumen of the GC shrinks more than the axoneme during the transition from elongating spermatid (ES) to spermatozoa (SZ). Scale bars, 100 nm. Mean ± SD; ***,

P<0.001; **, P<0.01.

Figure 4-2 Poc1 is enriched in the modified sperm centrioles

A) Poc1BGFP and Ana1tdTomato labels early spermatids (EE) but only Poc1BGFP labels spermatozoa (S) centrioles.

B) Poc1BGFP is enriched during spermatogenesis while AslGFP and Ana1tdTomato are reduced. G, Spermatogonia; C, Spermatocyte: R, Round spermatid; A, almost needle spermatid: S, spermatozoa.

C) In early-elongated spermatid with round nucleus (N), the PCL with a central tubule

(white arrow) is found near the GC inside the CA.

D) Correlative light (left panel) and electron microscopy (right panels) find that

Ana1GFP labels the microtubule base GC and a novel PCL structures without microtubules in an intermediate elongated spermatid.

E-F) A model (E) and TEM (F) of PCL in intermediate-elongated spermatid.

G) TEM and a model of PCL in late spermatid.

H) Correlative light (last panel) and electron microscopy find that Poc1BGFP

(gPoc1ABGFP) labels a modified PCL structures in the spermatozoon.

I) TEM and a model of PCL in spermatozoa.

J) Summary of GC and PCL proteins during spermiogenesis. P, presence; R, reduced; “-

“, undetectable. IN, initial spermatid; R, Round spermatid; EE, elongated spermatid; IE,

EE, intermediate elongated spermatid; S, spermatozoa.

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Figure 4-3 Poc1A and Poc1B have differential roles in centriole formation and sperm motility A) Poc1 codes for two isoforms, Poc1A and B, that share seven N-terminal-WD domains coded by exon 1 and a middle segment coded by exon 2, but differ in their C-termini; coded by the end of exon 2 in Poc1A or exon 3 in Poc1B. Poc1 gene structure (black), the two Poc1 protein isoforms (green), the location of poc1 mutations (blue), and two anti-Poc1 antibodies (red).

B) poc1W87X and poc1c06059 have the shortest GC labeling, poc1k245 having mild shortening, and poc1W45R have no effect (N=20). In these mutants, AslGFP and Ana2GFP labeled both the GC (square brackets) and PCL (line), but Ana1GFP/tdTomato selectively labeled the GC.

C) The GC (square brackets) and PCL (line) of poc1W45R sperm expressing gPoc1ABGFP have Ana1tdTomato-labeled PCL. ***, p<0.001; ** p<0.01.

D) Maximum intensity projections and quantification of 3DSIM micrographs of sperm centrioles containing GFPAna1, Ana1GFP, and Poc1BGFP. P=0.0001. Poc1BGFP and

GFPAna1 (N-terminal Ana1) are found in the PCL center, while Ana1GFP (C-terminal

Ana1) is found in the PCL periphery.

E) A TEM and model of PCL in intermediate-elongated spermatid of poc1W87X that has disorganized electron dense wall and is missing the central tubule.

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Figure 4-4 Characterization of poc1 mutants

A) The amino acids predicted to form the pentad hydrogen bond of Poc1’s WD1 are marked in blue. Only three of these amino acids form the triad hydrogen bond in WD1 of the W45R allele. The red-labeled arginine (R) is the mutated residue; it causes the loss of the hydrogen bond in the preceding Threonine (T).

B) poc1 homozygote mutants have variable effects on Poc1 protein expression in the testes. A western blot using both anti-Poc1N and anti-Poc1C antibodies recognizes Poc1 at the expected molecular weight of ~43KD in both control and poc1W45R testes lysates.

However, these antibodies did not detect the same band in poc1W87X, and weaker bands are detected in poc1k245 and poc1c06059. In contrast, poc1k245 is detectable only by anti-

Poc1N at a lower molecular weight (indicated as K245), because it is a mutation that truncates Poc1. This indicates that the antibodies are specific to Poc1.

Western blot and quantification (N=3) of N-terminal (anti-Poc1N) and C-terminal (anti-

Poc1C) anti-Poc1 antibodies staining of testicular extract K245 mark the location of the truncated Poc1 in the K245 allele; “*” Indicates a non-specific band; “tub” Indicates anti- tubulin staining. Mean ± SD; ***, P<0.001; **, P<0.01

C) poc1 mutant testes have variable effects on Poc1 protein expression.

Immunofluorescence using anti-Poc1M antibodies label spermatozoa centrioles in both control and poc1k245. However, the antibody did not label spermatozoa centrioles in poc1c0605, poc1W45R, or poc1W87X. Also, the antibody did not label spermatozoa centrioles in the presence of blocking peptide. Pre-immune serum also did not detect the centrioles, indicating that the antibodies are specific to Poc1. Scale Bars, 500nm

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D) Only poc1W87X has immotile sperm.

E) TEM revealed that the sperm tail of poc1W87X has an abnormal axoneme (yellow arrowhead). However, the GC appears to have normal 9-fold triplet microtubules, suggesting that the phenotype is not due to a defect in the centriolar microtubules. Bars,

200 nm (left top and lower panels) and 100 nm (right top and lower panels)

Figure 4-5 Atypical centrioles are essential for normal fertility and embryogenesis

A) 0-3-minutes-old poc1W45Rpat-embryos have abnormal PCL function at the decondensed male pronuclear (PN) stage. While Cnn and Asl labels both the GC and

PCL of poc1W45Rpat-embryos D PLP labels only the GC.

B) 0-3-minutes-old poc1pat-embryos have abnormal centrosome function. Microtubules are not formed around the GC and PCL.

C) 0-3-minutes-old poc1W45Rpat-embryos have monopolar spindles, while control and poc1W45Rpat-zygotes expressing gPoc1ABGFP have bipolar spindles.

D) The pole of 0-3-minutes-old poc1W45Rpat-zygotes has an Asl labeled GC and maternally expressed Sas-6 tdTomato labeled daughter centriole (DC).

E) Unlike 1-hour-old control embryos (cont) or poc1W45Rpat-embryos expressing gPoc1ABGFP, poc1pat-embryos have delayed embryo development.

F) 72-hour-old poc1W45Rpat-embryos are arrested at cleavage stage with 2-4 nuclei and post-blastoderm stages (see representative figure).

G) Poc1pat-embryos have reduced larval hatching.

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A-G) In all these experiments the female had WT Poc1.

Figure 4-6 poc1W45R males have normal fertility.

W45R A) The cytoplasm of poc1 pat-embryos (N=20) at various stages contains paternal

Tubulin-RFP sperm, indicating that the eggs are fertilized. Sperm in the yellow box is magnified on the right. Scale bars, 25 μm

B) In the 0-3 minutes fertilized eggs, astral microtubules are abnormally absent in the monopolar spindle of the poc1W45R-fathered embryos. However, astral microtubules are observed in all 4 spindle poles seen in poc1W45R-fathered embryos with two spindles, suggesting that astral microtubules are activated at some time after monopolar spindle formation.

Figure 4-7 Poc1 isoforms have differential roles during spermiogenesis

A) cPoc1AGFP and cPoc1BGFP constructs each have a UAS promoter that is activated by GAL4, a cDNA sequence (CDS), and an in-frame GFP tag. To express both cPoc1AGFP and cPoc1BGFP, a chromosome containing both transgenes was formed using meiotic recombination.

104

Figure 4-8 Paternal Poc1 enrichment is essential for normal fertility and embryogenesis

A) gPoc1ABGFP, cPoc1AGFP, or the combination of cPoc1AGFP and cPoc1BGFP

(cPoc1ABGFP), but not cPoc1BGFP, expressed in poc1W87X restored sperm motility, indicating that Poc1A is essential for sperm axoneme architecture.

B) In poc1W87X mutants background, Poc1A and B labels specifically the base of the GC and PCL, respectively, in the of round spermatids; and Poc1B also labels the base of the

GC in almost needle spermatids.

C) Both Poc1A and B are essential for normal GC length.

W87X D) poc1 pat-embryos expressing various Poc1 construct have differential effects on embryo development and spindle formation. Paternal Poc1B is essential for embryo development.

E) BamGAL4 expression of UASPoc1 RNAi affects the PCL but not GC length.

F) Paternal BamGAL4 and UASPoc1 RNAi reduce early embryo development.

G) Interpretive model: during spermatogenesis, the centrosome composition changes with the levels of some proteins decreasing (centrosome reduction) and others increasing

(Poc1 enrichment). This remodeling eliminates the pericentriolar material (PCM) and produces centrioles with atypical structure, which after fertilization, form atypical centrosomes by recruiting PCM. The atypical centrosomes are essential for the formation of typical daughter centrioles, pronuclear migration, zygotic cell division, and embryo development. The typical daughter centrioles form normal centrosomes at later stage of embryogenesis.

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Chapter 5

Maternal Plk4 is essential for Centrosome Reconstitution Post-Fertilization

The findings in this chapter are preliminary in nature and more experiments need to be done to substantiate the hypothesis.

5.1 Abstract

Centrosome reconstitution is the phenomenon by which centrioles recruit maternal PCM proteins and nucleate astral microtubules, after fertilization. However, the mechanism underlying centrosome reconstitution is unknown. Here, we show that in Drosophila, maternal Plk4 (Polo-like Kinase 4) is essential for centrosome reconstitution. For example, immediately after fertilization, in a P-element heterozygote mutation of Plk4, plk4c0661-mothered embryos, centrioles show no labeling of PCM proteins like Asl and

Cnn to the centrioles and also do not show any astral microtubule formation. The labelling is restored to the GC and PCL during zygote mitosis stage. Here also show that this effect is regulated through an interaction between Asl domain 1 and Plk4. plk4 mutations, Plk4 RNAi, Plk4KD (Kinase dead) mothered embryos show reduced viability.

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Significantly, overexpressing Plk4 in plk4 mutant background restores normal embryo development, with normal PCM recruitment and astral microtubule formation. This suggests that reduced maternal Plk4 levels cause the observed embryo defects. Together, these observations provide the insight into a molecular mechanism that regulates centrosome reconstitution and the provide direct evidence that centrosome reconstitution is essential for post-fertilization development.

5.2 Methods and Materials

5.2.1 Transgenic Flies.

All Drosophila stocks were cultured on standard media at 25°C. For in vivo expression of

Asl deletions, the asl promoter and cDNA were subcloned into a pUAS vector with an in- frame C-terminal GFP tag (for N-terminal deletions) or N-terminal GFP (for C-terminal deletions) tag such that Asl deletions were expressed using the Asl promoter in the absence of GAL4. AslD1-GFP flies (Ser/Thr-Ala) with the asl promotor and cDNA were subcloned in a pATTB vector with an in-frame C-terminal GFP tag such that it was expresses using Asl promoter in absence of GAL4. Germline transformations were performed by BestGene Inc. (Chino Hills, CA), using w1118 flies. Plk4KDGFP flies

(D156N point mutation in Plk4-wt) were generated in pUAS vector using a pMT/V5-

HisC-plasmid obtained from Dr. Gregory Rogers. Plk4OE fly line was provided by

Jordan Raff. cnnMI08383, plps2172, γTub37C3 and bld10c04199 mutant flies were obtained from

Bloomington fly stock centre. Plk4-RNAi was expressed using the Bam-Gal4 promoter.

Bam-Gal4 fly line was provided by Yokiko Yamashita.

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5.2.2 Antibodies.

The following antibodies were used for immunofluorescence and western blots at the indicated concentrations:

Primary antibodies for immunofluorescence: E7-mouse anti-tubulin, 1:200

(AB_2315513, DSHB at The University of Iowa); rabbit anti-Asl ,1:200 (Tomer Avidor-

Reiss Lab)

Secondary antibodies for immunofluorescence: Cy5™AffiniPure Goat Anti-Rabbit IgG

(H+L), 1:200; Alexa Fluor® 488-conjugated donkey anti-rabbit IgG, 1:200 (Jackson

ImmunoResearch).

Primary antibodies for western blot: goat anti-vinculin (Santacruz Biotechnology), 1:

500, anti-p-Plk4 T172 (Phosphosolutions Inc.) 1:5000. Secondary antibodies for western blot: Donkey anti-goat IgG (Santacruz Biotechnology) 1:1000, Donkey anti-Rabbit IgG

1:5000 (Santacruz Biotechnology)

5.2.3 Fluorescence Microscopy.

Images were taken by Leica SP8 scanning confocal microscope as Z stacks. Maximal projection images were then modified using Adobe Photoshop and annotated using

Adobe Illustrator.

5.2.5 Embryo Fluorescence Microscopy.

108 removed, and 50%(v/v) bleach solution was added to the embryos in the MatTek dish, until the appendages of the embryos disassociated. Next, the embryos were rinsed twice with wash buffer. Finally, the embryos were transferred to a 1.5ml microcentrifuge tube where they were fixed in a 1:1 solution of heptane and methanol and vortexed vigorously until the embryos settled down in the methanol layer. This was followed by removal of the fixative and suspension of the embryos in acetone. At this stage, the embryos were usually stored at -20°C. Embryos were rehydrated sequentially in 70%, 50%, 30%, and

10% (v/v) methanol in PBS, and then in PBS alone. The embryos were then incubated in

PBT (PBS + 1% (v/v) Triton) for 30 minutes, and blocked in PBST (PBT+3% (w/v)

BSA) for 1 hour. The embryos were incubated with primary antibodies in PBST for 1 hour at room temperature. After three 5-minute washes in PBT, the embryos were incubated with secondary antibodies and 1 μg/ml of DAPI for 1 hour at room temperature. The embryos were washed three times with PBT for 5 minutes each, and then washed in PBS for 5 minutes. The embryos were mounted on a slide using a mounting medium (PBS, 50% (v/v) glycerol, 0.5% (w/v) N-propyl-gallate) and imaged.

5.2.6 Western blot

3 minute embryos were collected and immediately homogenized and boiled for 5 min in

95 °C with 1X Laemmli buffer samples. 30 embryos were run per lane in 6% polyacrylamide gels and transferred into nitrocellulose membranes. The blots were incubated with primary antibodies overnight at 4°C followed by peroxidase-conjugated secondary antibodies at RT for 1h. Super Signal West Pico (was used to detect peroxidase activity. Molecular masses were determined by comparison to Precision Plus

Protein Standard (Bio-Rad). Western blots were analyzed with ImageJ (National Institute

109 of Mental Health, USA) and intensity of total area was calculated using an identical rectangular box.

5.2.7 Embryo development.

Twenty-five males and twenty-five females were placed in humidified mating chamber at

25°C to lay eggs for 5 min. The parents were removed and embryos were placed for 1 h in a MatTek dish containing wash buffer (0.7% NaCl+0.05% Triton X-100). The embryos were processed for embryo fluorescence microscopy staining.

5.2.8 Statistical Methods.

Statistical analyses were done with Excel. A two-tailed, unpaired Student’s t-test (with samples that do not have equal variances) was used.

5.3 Results and Discussion

5.3.1 Maternal Plk4 is essential for Centrosome Reconstitution

During fertilization, as the sperm enters the egg, the centrioles in the sperm head region immediately recruit maternal PCM protein and nucleate astral microtubules. This phenomenon is termed centrosome reconstitution. It is an evolutionarily conserved across the animal kingdom. It was previously shown that an in vitro phosphorylation of a protein epitope on the human sperm plays a critical role in centrosome reconstitution (Simerly et al., 1999). But, the mechanism regulating the centrosome reconstitution in vivo is still unknown. Here, we use Drosophila melanogaster to study the mechanism of centrosome reconstitution. There have been previous studies implicating the role of maternal Plk4 in nucleating astral microtubules in mouse embryo (Coelho et al., 2013). Therefore, we hypothesize that maternal Plk4 in Drosophila plays a role in centrosome reconstitution.

To help determine the underlying mechanism, functions of zygotic centrosomes in P-

110 element heterozygote mutant of Plk4, plk4c0661, Plk4-KD (mutation in the kinase domain, making Plk4 non-functional) and Plk4 RNAi embryos were studied. All zygotic embryos from control males and control females had robust microtubule asters and Asl labeling on the GC and the PCL (Fig. 5-1A), whereas all embryos mothered by heterozygote plk4c0661 in the decondensed male pronucleus during the pronuclei migration stage had undetectable or reduced microtubule asters and failed to recruit Asl (Fig. 5-1A).

Interestingly, when we overexpressed Plk4 in heterozygote plk4c0661 or Plk4 KD mothers,

Asl labeling was restored to the centrioles, with astral microtubule formation. Together, these findings demonstrate that maternal Plk4 is essential for sperm centriole function and reconstitution in the zygote. Further, an embryo development assay was performed to test the role of maternal Plk4 in animal development.

While studying the one hour-old embryos, all control embryos progressed beyond the 32- nuclei stage as expected, whereas all heterozygote plk4c0661 mothered embryos showed significant delays in cleavage cycle progression and embryo development, as evident by reduced numbers of nuclei the 32-nuclei stage (Fig. 5-1B). Together, these data suggest that maternal Plk4 might be playing a role in recruiting PCM proteins post fertilization and in fact may be necessary for proper animal development.

5.3.2 Maternal Plk4 is activated upon fertilization.

To obtain insight into the function of Plk4 in the zygote, we monitored its activation.

Plk4 function as a dimer that trans-autophosphorylates T172 resulting in its activation

(Lopes et al, 2015). We used commercial antibodies that recognize this phosphorylation to monitor Plk4 activation. We found that while oocytes (unfertilized eggs) had low levels of phospho-T172-Plk4 immunoreactivity, zygotes formed after fertilization had

111 increased T172 autophosphorylation, suggesting that Plk4 is activated by fertilization

(Fig 5-2A). Phospho-T172-Plk4 immunoreactivity in the embryo was reduced when

Plk4KD was expressed during oogenesis (Fig 5-2B), demonstrating that this phospho-

T172-Plk4 immunoreactivity require maternal Plk4. Phospho-T172-Plk4 immunoreactivity was also reduced in heterozygote plk4c0661 mothered embryos and further, the phospho-T172 pPlk4 immunoreactivity was restored by maternally overexpressing Plk4 in the heterozygote plk4c0661.Thus, maternal Plk4 seems to be activated upon fertilization and this activation may be essential for centrosome reconstitution.

5.3.3 Plk4 mediates centrosome reconstitution via Asl N-terminus

During centriole duplication, it is known that Plk4 binds to the N-terminal domain of

Asterless (Asl). Also, Plk4-Asl N-terminus interaction is implicated in the centrosome reduction phenomenon (Khire et al, 2015). Therefore, we hypothesized that Asl N- terminus plays a role in centrosome reconstitution phenomenon. To investigate the role of

Asl N-terminus in Plk4 activation, we studied a N-terminal deletion construct of Asl.

Domain 1 was deleted and the construct was called Asl 2-6 (Khire et al, 2015). We found that when we stain Asl 2-6 zygotes, we observed decreased phospho-T172-Plk4 immunoreactivity compared to the control zygotes, suggesting that the Asl N- terminus is essential for the Plk4 activation (Fig 5-3B). In addition, when we studied a mutant allele of Asl called asl1, which is a C-terminus truncation in Asl protein (Blachon et al 2008) oocyte, we observe a low level of phospho-T172-Plk4 immunoreactivity. After fertilization however, the asl1 zygotes show an increase in immunoreactivity, suggesting that the Asl C- terminus is not essential for Plk4 activation (Fig 5-3A).

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We also studied the ability of zygotic centrioles to recruit PCM and nucleate astral microtubules. In the zygote, at the decondensed pronucleus stage, both the GC and the

PCL were not labeled by Asl and did not nucleate astral microtubules. This suggests that

Plk4 interaction with Asl N-terminus essential for its role in centrosome reconstitution.

To further understand the mechanism, we made a fly where we mutated four phosphorylation sites (two serine and two threonine to alanine) in domain 1 of Asl. (Asl

D1 phospho mutant). We found that in zygotes mothered from these females, antibodies do not detect Asl nor nucleate astral microtubules in either the GC and PCL. Further, we also made a fly wherein Plk4 binding sites in the domain 1 of Asl were mutated (Asl D1 binding mutant). We found that in zygotes mothered from these females, antibodies do not detect Asl nor nucleate astral microtubules in either the GC and PCL. Both the Asl

D1 phospho mutant and Asl D1 binding mutant was overexpressed flies with endogenous

Asl background. Moreover, embryos mothered from flies expressing endogenous Asl did not show any defects in localization of PCM proteins to GC and the PCL (Fig 5-3C).

This suggests that phosphorylation of Asl via its first domain may play a role in centrosome reconstitution post-fertilization. In addition, we also stained embryos mothered by PCM and centriolar protein mutants. mutant mothers. Embryos from heterozygote mutants of PCM proteins cnnMI08383, plps2172 or Tub37C3 demonstrated normal labeling of Asl and tubulin (Fig 5-3D). Previously, it was reported that Plk4 interacts with Cep135 (Galletta et al, 2016). Therefore, we tested the role of the Cep135 homologue (Bld10) and Plk4 in the centrosome reconstitution pathway. In P-element heterozygote mutation of Bld10, bld10c04199 mutant mothered embryos show no labeling for Asl or tubulin. Moreover, Asl and tubulin labeling is restored when Plk4 was

113 overexpressed in the bld10c04199 background (Fig 5-3E). These observations indicate that there is an interaction between Asl, Plk4 and Bld10 during centrosome reconstitution post fertilization.

5.3.4 Model for Centrosome Reconstitution.

Post fertilization, maternal PCM and centriolar proteins interact with each other, whereby the atypical centrioles gains functionality, which further leads to centrosome reconstitution. (Fig 5-4). During spermiogenesis, PCL is formed next to the GC. Levels of Asl and other centriolar proteins in PCL and GC decrease, while Poc1B levels increase leading to its enrichment in spermatozoa PCL. In parallel, centriole is also undergoing structural modifications. Thus, at the end of spermatogenesis, mature sperm is left with remodeled atypical centrioles. After fertilization, maternal Plk4 is activated which is essential for these atypical centrioles to recruit maternal PCM and nucleate astral microtubules, further priming the centrioles for embryonic cell division.

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Figure 5-1

115

Figure 5-2

116

Figure 5-3

117

Figure 5-4

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Figure Legends

Figure 5-1 Maternal Plk4 is essential for centrosome reconstitution

A) 0-3-minutes-old plk4c0661 mothered-embryos stained with Asl antibody to label the GC and PCL and tubulin antibody to label the microtubules. Compared to 0-3-minute old control embryos, no Asl and tubulin labelling is seen both at the GC and PCL. Similar phenotype is observed, when 0-3 minutes old Plk4 RNAi or Plk4KD mothered embryos are examined. Interestingly, 0-3-minutes-old, overexpressing Plk4 in plk4c0661 mothered- embryos rescues the phenotype, where GC and PCL are labeled with Asl and also nucleate astral microtubules, at the decondensed male pronuclear (PN) stage. Scale bar

1m

B) One-hour-old embryos of control male and female flies (w1118). Also, control male and plk4c0661or Plk4 RNAi or Plk4KD female flies were mated to collect embryos. All the embryos were collected after every five minutes and aged for sixty minutes and then stained by Hoechst that labels nuclei. Most of the embryos from control males and females had greater than 32 nuclei but embryos mothered by plk4c0661, Plk4 RNAi or

Plk4KD showed arrest in nuclear division.

Figure 5-2 Maternal Plk4 is activated upon fertilization

All the oocytes and zygotes are collected after every 0-3 minutes and lysed in 1X Lamelli sample buffer. A) Oocytes (unfertilized eggs) They show low level of phospho-T172-

Plk4 immunoreactivity, while in the zygotes formed after fertilization, this immunoreactivity increases, suggesting that Plk4 is activated by fertilization. B)

Phospho-T172-Plk4 immunoreactivity in the embryo collected after 0-3 minutes is

119 reduced when Plk4KD are expressed during oogenesis. Phospho-T172-Plk4 immunoreactivity is also reduced in plk4c0661 mothered 0-3 minutes embryos and further, the p-T172Plk4 immunoreactivity is restored by maternally overexpressing Plk4 in the plk4c0661. Western blot using anti-phospho-T172-Plk4- and anti-vinculin (loading control) phospho-Plk4 was analyzed by measuring intensity N=3, Mean±s.d., two-sided t-test

Figure 5-3 Plk4 mediates centrosome reconstitution via Asl N-terminus

All the oocytes and zygotes are collected after every 0-3 minutes and lysed in 1X Lamelli sample buffer. A) Oocyte (unfertilized eggs) shows low level of phospho-T172-Plk4 immunoreactivity, while in the zygotes of asl1 collected after 0-3 minutes, this immunoreactivity increases, suggesting that Plk4 is activated by fertilization.

B) Asl 2-6 mothered zygotes are collected after 0-3 minutes and lysed in 1X Lamelli sample buffer. They show increased phospho-T172-Plk4 immunoreactivity, compared to the control zygotes.

C) In the Asl 2-6 mothered zygote collected after every 0-3 minutes, Asl and tubulin antibodies are used to label GC and PCL as well as microtubules. At the decondensed pronucleus stage, both the GC and the PCL are not labeled by Asl and do not nucleate astral microtubules. Also staining of 0-3-minute old Asl D1 phospho mutant mothered zygotes did not detect any Asl or tubulin labelling. Similarly, staining of 0-3-minute old

Asl D1 binding mutant mothered zygotes failed to detect any Asl or tubulin labelling. 0-

3 minute old embryos mothered from flies expressing endogenous Asl show normal localization of PCM proteins to GC and the PCL.

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D) Staining of 0-3 minute old embryos using Asl and tubulin antibodies. cnnMI08383, plps2172 and γTub37C3 mutant mothered embryos show normal labeling of Asl and microtubules.

E) 0-3-minute old bld10c04199 mutant mothered embryos show no labeling for Asl and tubulin. Asl and tubulin labeling is restored when Plk4 is overexpressed in the bld10c04199 background.

Figure 5-4 Model for Centrosome Reconstitution

Post-fertilization, the atypical centrioles recruit maternal PCM proteins (Asl) and interact with other centriolar proteins including Plk4 and Bld10.

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Chapter 6

Discussion

6.1 General Conclusions

This study provides insights into the inheritance of paternal centriolar structures post fertilization and the highly-specialized processes and modifications that centrioles undergoes before the sperm fertilizes the oocytes. Moreover, this study also demonstrates that these modifications in centrioles are essential for normal animal development.

Lastly, this study also touches upon the role maternal contribution, which supports the hypothesis that paternal centrosomes are required to gain complete functionality post fertilization. The existing hypothesis at the start of this project was that Drosophila sperm have only one functional centriole, called the giant centriole (GC) and it was hypothesized that this GC is the only functional centriole inherited by the zygote after fertilization. Our lab was the first to discover a second centriolar structure in the

Drosophila sperm cells. We call this structure Proximal Centriole Like (PCL) (Blachon et al., 2009).

Chapter 2 provides evidence that Drosophila zygote inherits both the GC and the

PCL 122

Chapter 3 describes a novel and evolutionarily conserved phenomenon called centrosome reduction. Centrosome reduction is a process wherein the centriole loses all its proteins during spermiogenesis. Until recently, the mechanism of centrosome reduction was not known. We have shown that centrosome reduction is an active phenomenon mediated by a kinase, namely Plk4. In addition to this, we have also shown that centrosome reduction is also essential for normal animal development.

Chapter 4 introduces the concept of centrosome remodeling. We show evidence that the sperm centrosome is not actually reduced but that it is remodeled, wherein levels of some centriolar protein increase while levels of some centriolar proteins decrease.

Based on the observations discussed in this dissertation, we conclude that centrosome remodeling is essential for normal bipolar spindle pole assembly at the zygote metaphase stage and in turn is also essential for normal embryo development.

Chapter 5 introduces the concept of centrosome reconstitution and hypothesizes that immediately after fertilization, the centrioles provided by the sperm become functional by recruiting the maternal proteins from the embryo. This mechanism of sperm centriole reconstitution was previously unknown. Here, for the first time, we provide preliminary evidence that demonstrate centrosome reconstitution is mediated by a maternal kinase-dependent pathway.

6.1.1 The PCL as the second zygotic centriole of Drosophila melanogaster

Zygotic centrioles give rise to all the somatic cell centrioles. As all the somatic cells have two centrioles, it makes sense that the zygote also had two centrioles to begin with; however, the zygote inherits its centrioles from the sperm. The reigning hypothesis in the

123 field was that the Drosophila sperm provides only a single functional GC to the zygote, wherein it duplicates, resulting in the two centriolar structures like the somatic cells

(Sathananthan et al., 1996; O'Connell et al., 2001; Stevens et al., 2007; Varmark et al.,

2007). Many previous studies on humans, insects and mammals, demonstrate that sperm appear to have a single or no centriole at all (Sun and Schatten, 2007; Manandhar et al.,

2005; Fuller 1993). In mice and rats, it is hypothesized that the zygote does not inherit any centrioles from the father. Centrioles are detected only at 32/64-cell stage. Electron microscopy could not detect any centrioles immediately after fertilization (Woolley and

Fawcett, 1973; Zambonu et al., 1972). It was hypothesized that even in humans and

Drosophila, the sperm provides a single functional centriole post fertilization, which then duplicates in the zygote (Sutovsky and Schatten, 2000). It is possible that previous studies may have missed the second centriolar structure in the Drosophila sperm cells and thus, the number of centrioles inherited by the zygote was always debatable. It was showed that the Drosophila sperm has not one but two centriolar structures. The second centriolar structure was named as the PCL (Proximal Centriolar Like) (Blachon et al.,

2009). One of the primary findings of this study is that the PCL is indeed the second zygotic centriolar structure. Thus, the Drosophila zygote inherit both the GC and the PCL post fertilization (Blachon and Khire et al., 2014). In C. elegans, it was shown that the zygote inherits two functional centrioles (Pelletier et al., 2006). Similarly, in the mollusk have two centrioles in their spermatozoa (Daniels et al., 1971). During sea urchin embryo mitosis, two centrioles were detected at each of the spindle pole (Sluder and Rieder,

1985a). In addition to this, our lab demonstrated that even insect species like Dung

Beetles, which are far away in the evolutionary tree from Drosophila, also have second

124 centriolar structure in its sperm cells (Fishman et al., 2017). Further, our lab has also shown that, like Drosophila, even the humans have two functional centrioles in their sperm (Fishman et al., submitted). We can also hypothesize that the rodents indeed may have two PCL like structures in their mature sperm that have remained undetected due to use of traditional methods.

6.1.2 Plk4 regulates centrosome reduction and is essential for normal embryo development

During the process of spermiogenesis, the centrosome tends to lose its characteristic features in a step wise fashion. This phenomenon is known as centrosome reduction

(Manandhar et al., 2005). It is known to occur in all the animals studied; namely insects, mammals and mollusks. The process is characterized by three steps: the sperm centrosome cannot nucleate astral microtubules, followed by the loss of PCM and, sometimes the centrioles may also lose some of their components (Manandhar et al.,

2005). This process was widely studied in human and rhesus monkeys. It was found centrioles lose their structural integrity, where whole microtubules and nine-fold symmetry collapses. Immunofluorescence also did provide any labelling for centriolar proteins in the mature sperm of humans and rhesus monkey (Simerly et al., 1993, Deattre and Gonczy, 2004, Manandhar et al, 2005). During Drosophila spermiogenesis, several centriolar and PCM proteins like Ana1, Sas-4, bld10, Ana-2, Asl, γ-tubulin have been shown to undergo centrosome reduction (Blachon and Khire et al., 2014). Even after studying centrosome reduction for so many decades, there was no information regarding the mechanism of centrosome reduction and its role in animal development. In this study, we have used Asl as a marker to elucidate the mechanism of centrosome reduction.

125

During the normal spermiogenesis process, centriolar protein Asl undergoes complete reduction and remains undetectable in the mature sperm. But, we observed that when we express Asl in the Plk4 heterozygote background, Asl does not undergo reduction. This was the first clue, that Plk4 mediated Asl reduction. Plk4 is a master regulator of centriole duplication pathway and is a kinase, which phosphorylates its downstream substrates for building up the centriole. More importantly, Asl through it N-terminus acts as a receptor for Plk4 during centriole duplication. We observed that, even centrosome reduction phenomenon is mediated by Plk4, through phosphorylation of Asl N-terminus.

The fundamental question that needs to be answered is why does the animal go through this process of reduction. One might speculate that, during spermatogenesis, centrioles are built by proteins that are at rate limiting amount. Extra amounts of proteins need to be degraded, in order to avoid any problems during centriole biogenesis. So as new proteins are added to the centriole, their needs to be a mechanism by which the older proteins on the centrioles are reduced. For that purpose, the cell may utilize a kinase based mechanism to phosphorylate and dephosphorylate the centriolar proteins, where by priming them for reduction. Thus, at the end of spermiogenesis, this kinase based mechanism may be essential for unmasking the essential sites on the centrioles. Thus, the sperm with these unmasked centrioles fertilizes the ovum, another set of proteins from the maternal side may bind to centriole, priming it for recruiting maternal PCM proteins and nucleating astral microtubules and further facilitating for zygotic divisions.

This dissertation makes another critical observation that highlights the importance of centrosome reduction. Flies that show attenuation in Asl reduction also show reduction in fertility and delayed embryo development. These findings may have its

126 applications in human infertility treatment and diagnostics (Schatten et al., 2015) Infertile men have been known to harbor centrosomal abnormalities. Similar to the therapy targeting mitochondrial DNA mutations (Hamilton G, 2015), it is possible to target malfunctioned centrosomes and mitigate any infertility issues (Scahtten et al., 2015).

Another novel application can be in the development of male contraceptives by using centrosomal proteins as a marker instead of current female hormone based contraceptives

(Schatten et al., 2015).

6.1.3 Centrosome is remodeled during Drosophila spermiogenesis

Previously we showed that during spermiogenesis, Drosophila centrosome undergoes a step wise reduction process (Khire et al., 2015). After further investigation, we actually found that not all centriolar proteins undergo reduction. We found a novel centriolar protein, namely Poc1B, which enriches in the PCL of the mature sperm. In parallel to this, we also observed the centrosome undergoing several structural modifications during spermiogenesis. Thus, this process, where some protein levels decrease, while others increase, is termed as “centriole remodeling”. In addition to this, we also observed that this enrichment of Poc1B is essential for fly fertility and normal embryo development.

Similar to Drosophila sperm, Fishman et al (manuscript submitted) show that ejaculated human sperm has enrichment of Poc1B in its distal centriole. Thus, we can speculate that during animal spermiogenesis, levels of some centriolar proteins need to decrease and levels of other centriolar proteins need to increase, so that the centrioles in the fertilizing sperm are primed for normal animal development. This discovery can open up several avenues in human reproductive biology. We can use Poc1B as a diagnostic marker for male infertility. We hypothesize that a sperm sample with abnormal or lower levels of

127

Poc1B is dysfunctional, and men with such sperm may have difficulty conceiving a child or may lead to an untimely termination in pregnancies.

6.1.4 Maternal Plk4 is essential for centrosome reconstitution.

Centrosome reconstitution is an essential process for normal animal development. The centrosome becomes inactive after it has undergone complete reduction during spermiogenesis. Immediately after fertilization, this sperm centrosome is restored by recruiting maternal PCM proteins and nucleating astral microtubules, thus gaining full functionality (Simerly et al., 1999). The mechanism by which several centrosomal proteins interact to generate a fully operational centrosome is not known. Gamma-tubulin may be one of the essential players, as it is implicated in nucleating astral microtubules and also maintaining polarity of the microtubule (Simerly et al., 1999). It was previously reported that the maternal mutation spanning the Plk4 gene leads to early miscarriages in humans (Zhang et al., 2017). In addition to this maternal Plk4 has also been implicated in spindle formation in early mouse embryos (Coelho, P.A. et al., 2013). Thus, here we hypothesized that maternal Plk4 might be playing a role in centrosome reconstitution pathway. We used Drosophila as a model organism to elucidate the mechanism of centrosome reconstitution. Our preliminary findings suggest that maternal Plk4 is essential for full restoration of the sperm centrosome post-fertilization. Moreover, trans- autophosphorylation of Plk4 is also necessary for centrosome reconstitution. This auto phosphorylation further activates Plk4, which is essential for this process. These results provide important insights to the contribution of maternal proteins to the process of centrosome reconstitution and in turn to successful fertilization and early development.

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6.2 Future Directions

6.2.1 Development of phosphomimetic in Asl domain 1

We have shown that centrosome reconstitution is impeded when we use a phospho- mutant Asl mothered embryos. This mutation is in the first domain of the Asl protein, where in the two serine and two threonine residues are mutated to alanine. To further characterize the role of phosphorylation of domain 1 of Asl, it will be advantageous to make a phosphomimetic fly, where in we will apply site directed mutagenesis for the

Serine-Threonine residues of the first domain of the Asl and mutate each residue to

Aspartic acid or glutamic acid. We would then analyze these phosphomimetic mothered embryos and check for centrosome reconstitution.

6.2.2 Identification of downstream effectors in centrosome reconstitution pathway

Here, we show that Asl-Plk4-Bld10 interact with each other and post fertilization and centrosome is reconstituted. But, we still do not know the mechanism of this interaction.

It would be interesting to map the exact domains of Bld10 that play a role in centrosome reconstitution. It would be also noteworthy to study whether phosphorylation of Bld10 is also essential for centrosome reconstitution phenomenon. Generation of several phospho- mutant Bld10 flies and analyzing their embryos would answer some of these questions.

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Reference

Ahanger S. H., Shouche Y. S., Mishra R. K., 2013. Functional sub-division of the

Drosophila genome via chromatin looping: the emerging importance of

CP190. Nucleus 4: 115–122.

Archambault V., Glover D. M., 2009. Polo-like kinases: conservation and divergence in their functions and regulation. Nat. Rev. Mol. Cell Biol. 10: 265–275.

Arquint C., Nigg E. A., 2014. STIL microcephaly mutations interfere with APC/C- mediated degradation and cause centriole amplification. Curr. Biol. 24: 351–360.

Avasthi, P., and W. F. Marshall, 2012. Stages of ciliogenesis and regulation of ciliary length. Differentiation 83: S30-42.

Avidor-Reiss, T., and J. Gopalakrishnan, 2013. Building a centriole. Curr Opin Cell Biol

25: 72-77.

Avidor-Reiss, T., J. Gopalakrishnan, S. Blachon and A. Polyanovsky, 2012 Centriole duplication and inheritance in Drosophila melanogaster in The Centrosome: Cell and

Molecular Mechanisms of Functions and Dysfunctions in Disease, edited by H. Schatten.

Humana Press.

130

Avidor-Reiss, T., Khire, A., Fishman, E. L., & Jo, K. H. 2015. Atypical centrioles during sexual reproduction. Frontiers in Cell and Developmental Biology, 3, 21

Basiri M. L., Ha A., Chadha A., Clark N. M., Polyanovsky A., et al. , 2014. A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids. Curr. Biol. 24: 2622–2631.

Basto R., Lau J., Vinogradova T., Gardiol A., Woods C. G., et al. , 2006. Flies without centrioles.Cell 125: 1375–1386.

Basto R., Brunk K., Vinadogrova T., Peel N., Franz A., et al., 2008. Centrosome amplification can initiate tumorigenesis in flies. Cell 133: 1032.

Bettencourt-Dias M., Giet R., Sinka R., Mazumdar A., Lock W. G., et al., 2004.

Genome-wide survey of protein kinases required for cell cycle progression. Nature 432:

980–987.

Bettencourt-Dias M., Rodrigues-Martins A., Carpenter L., Riparbelli M., Lehmann L., et al., 2005. SAK/PLK4 is required for centriole duplication and flagella development. Curr.

Biol. 15: 2199–2207.

Blachon S., Cai X., Roberts K. A., Yang K., Polyanovsky A., et al., 2009. A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication. Genetics 182: 133–144.

Blachon S., Khire A., Avidor-Reiss T., 2014. The origin of the second centriole in the zygote of Drosophila melanogaster. Genetics 197: 199–205.

131

Bonaccorsi S., Giansanti M. G., Gatti M., 1998. Spindle self-organization and cytokinesis during male meiosis in asterless mutants of Drosophila melanogaster. J. Cell Biol. 142:

751–761.

Bernardini, G., R. Stipani and G. Melone, 1986. The ultrastructure of Xenopus spermatozoon. Journal of ultrastructure and molecular structure research 94: 188-194.

Bowman S. K., Neumüller R. A., Novatchkova M., Du Q., Knoblich J. A., 2006.

The Drosophila NuMA homolog Mud regulates spindle orientation in asymmetric cell division. Dev. Cell 10: 731–742.

Borges, E., Jr., Rossi-Ferragut, L.M., Pasqualotto, F.F., dos Santos, D.R., Rocha, C.C., and Iaconelli, A., Jr. 2002. Testicular sperm results in elevated miscarriage rates compared to epididymal sperm in azoospermic patients. Sao Paulo Med J 120, 122-126.

Brevini, T. A., G. Pennarossa, A. Vanelli, S. Maffei and F. Gandolfi, 2012.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity. Theriogenology 77: 766-772

Brownlee C. W., Klebba J. E., Buster D. W., Rogers G. C., 2011. The protein phosphatase 2A regulatory subunit twins stabilizes Plk4 to induce centriole amplification. J. Cell Biol. 195: 231–243.

Calarco, P. G., 2000. Centrosome precursors in the acentriolar mouse oocyte. Microsc

Res Tech 49: 428-434.

132

Callaini, G., and M. G. Riparbelli, 1996. Fertilization in Drosophila melanogaster: centrosome inheritance and organization of the first mitotic spindle. Dev Biol 176: 199-

208.

Callaini, G., W. G. Whitfield and M. G. Riparbelli, 1997. Centriole and centrosome dynamics during the embryonic cell cycles that follow the formation of the cellular blastoderm in Drosophila. Exp Cell Res 234: 183-190.

Carvalho-Santos, Z., P. Machado, I. Alvarez-Martins, S. M. Gouveia, S. C. Jana et al.,

2012. BLD10/CEP135 is a microtubule-associated protein that controls the formation of the flagellum central microtubule pair. Dev Cell 23: 412-424.

Chemes, H. E., and V. Y. Rawe, 2010. The making of abnormal spermatozoa: cellular and molecular mechanisms underlying pathological spermiogenesis. Cell Tissue Res 341:

349-357.

Chenoweth, P.J., and Lorton, S. 2014. Animal Andrology: Theories and Applications,

(Cabi).

Cizmecioglu, O., M. Arnold, R. Bahtz, F. Settele, L. Ehret et al., 2010. Cep152 acts as a scaffold for recruitment of Plk4 and CPAP to the centrosome. J Cell Biol 191: 731-739.

Coelho, P. A., L. Bury, B. Sharif, M. G. Riparbelli, J. Fu et al., 2013. Spindle formation in the mouse embryo requires Plk4 in the absence of centrioles. Dev Cell 27: 586-597.

Comizzoli, P., Wildt, D.E., and Pukazhenthi, B.S. 2006. Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection. Biology of reproduction 75, 252-260.

133

Connolly, J. A., and V. I. Kalnins, 1978. Visualization of centrioles and basal bodies by fluorescent staining with nonimmune rabbit sera. J Cell Biol 79: 526-532.

Courtois, A., M. Schuh, J. Ellenberg and T. Hiiragi, 2012. The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development. J Cell Biol

198: 357-370.

Conduit P. T., Raff J. W., 2010. Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts. Curr. Biol. 20: 2187–

2192.

Conduit P. T., Brunk K., Dobbelaere J., Dix C. I., Lucas E. P., et al. , 2010. Centrioles regulate centrosome size by controlling the rate of Cnn incorporation into the PCM. Curr.

Biol. 20: 2178–2186.

Conduit P. T., Feng Z., Richens J. H., Baumbach J., Wainman A., et al.,2014a. The centrosome-specific phosphorylation of Cnn by Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation. Dev. Cell 28: 659–669.

Conduit P. T., Richens J. H., Wainman A., Holder J., Vicente C. C., et al., 2014b. A molecular mechanism of mitotic centrosome assembly in Drosophila. Elife 3: e03399.

Conduit P. T., Wainman A., Novak Z. A., Weil T. T., Raff J. W., 2015a. Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM

FRAP. Elife 4: e08483.

Conduit P. T., Wainman A., Raff J. W., 2015b. Centrosome function and assembly in animal cells. Nat. Rev. Mol. Cell Biol. 16: 611–624.

134

Cottee M. A., Muschalik N., Johnson S., Leveson J., Raff J. W., et al. , 2015. The homo- oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies. Elife 4: e07236.

Crozet, N., M. Dahirel and P. Chesne, 2000. Centrosome inheritance in sheep zygotes: centrioles are contributed by the sperm. Microsc Res Tech 49: 445-450.

Cunha-Ferreira I., Rodrigues-Martins A., Bento I., Riparbelli M., Zhang W., et al. , 2009.

The SCF/Slimb ubiquitin ligase limits centrosome amplification through degradation of

SAK/PLK4.Curr. Biol. 19: 43–49.

Cunha-Ferreira I., Bento I., Pimenta-Marques A., Jana S. C., Lince-Faria M., et al. , 2013.

Regulation of autophosphorylation controls PLK4 self-destruction and centriole number. Curr. Biol. 23: 2245–2254.

Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai et al., 2004.

Centriole assembly requires both centriolar and pericentriolar material proteins. Dev Cell

7: 815-829.

Daniels, E. W., A. C. Longwell, J. M. McNiff and R. W. Wolfgang, 1971. Ultrastructure of spermatozoa from the American oyster Crassostrea virginica. Trans Am Microsc Soc

90: 275-282. de Fried, E. P., P. Ross, G. Zang, A. Divita, K. Cunniff et al., 2008. Human parthenogenetic blastocysts derived from noninseminated cryopreserved human oocytes.

Fertil Steril 89: 943-947.

135

Debec A., Marcaillou C., Bobinnec Y., Borot C., 1999. The centrosome cycle in syncytial Drosophila embryos analyzed by energy filtering transmission electron microscopy. Biol. Cell 91: 379–391.

Debec, A., and C. Abbadie, 1989. The acentriolar state of the Drosophila cell lines 1182.

Biol Cell 67: 307-311.

Delattre, M., and P. Gonczy, 2004. The arithmetic of centrosome biogenesis. J Cell Sci

117: 1619-1630.

Dias, G., J. Lino-Neto and R. Dallai, 2014. The sperm ultrastructure of Stictoleptura cordigera (Fussli, 1775) (Insecta, Coleoptera, Cerambycidae). Tissue Cell.

Dix C. I., Raff J. W., 2007. Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication. Curr. Biol. 17: 1759–1764.

Dobbelaere J., Josué F., Suijkerbuijk S., Baum B., Tapon N., et al., 2008. A genome-wide

RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biol. 6: e224.

Dzhindzhev, N.S., Yu, Q.D., Weiskopf, K., Tzolovsky, G., Cunha-Ferreira, I., Riparbelli,

M., Rodrigues-Martins, A., Bettencourt-Dias, M., Callaini, G., and Glover, D.M. 2010.

Asterless is a scaffold for the onset of centriole assembly. Nature 467, 714-718.

Dzhindzhev N. S., Tzolovsky G., Lipinszki Z., Schneider S., Lattao R., et al. , 2014.

Plk4 phosphorylates ana2 to trigger SAS6 recruitment and procentriole formation. Curr.

Biol. 24: 2526–2532.

136

Fabian L., Brill J. A., 2012. Drosophila spermiogenesis: big things come from little packages.Spermatogenesis 2: 197–212.

Fawcett, D. W. 1965. The anatomy of the mammalian spermatozoon with particular reference to the guinea pig. Z Zellforsch Mikrosk Anat 67: 279-296.

Fawcett, D. W., and D. M. Phillips, 1969. The fine structure and development of the neck region of the mammalian spermatozoon. Anat Rec 165: 153-164.

Fechter, J., A. Schoneberg and G. Schatten, 1996. Excision and disassembly of sperm tail microtubules during sea urchin fertilization: requirements for microtubule dynamics. Cell

Motil Cytoskeleton 35: 281-288.

Felix, M. A., C. Antony, M. Wright and B. Maro, 1994. Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation. J Cell Biol 124:

19-31.

Fong, C. S., M. Kim, T. T. Yang, J. C. Liao and M. F. Tsou, 2014. SAS-6 assembly templated by the lumen of cartwheel-less centrioles precedes centriole duplication. Dev

Cell 30: 238-245.

Fourrage, C., Chevalier, S., and Houliston, E. 2010. A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies. PLoS One 5, e13994.

Friedlander, M., 1980. The sperm centriole persists during early egg cleavage in the insect Chrysopa carnea (Neuroptera, Chrysopidae). J Cell Sci 42: 221-226.

137

Freeman M., Glover D. M., 1987. The gnu mutation of Drosophila causes inappropriate

DNA synthesis in unfertilized and fertilized eggs. Genes Dev. 1: 924–930.

Freeman M., Nüsslein-Volhard C., Glover D. M., 1986. The dissociation of nuclear and centrosomal division in gnu, a mutation causing giant nuclei in Drosophila. Cell 46: 457–

468.

Fu, J., and D. M. Glover, 2012. Structured illumination of the interface between centriole and peri-centriolar material. Open Biol 2: 120104.

Fu, J., Lipinszki, Z., Rangone, H., Min, M., Mykura, C., Chao-Chu, J., Schneider, S.,

Dzhindzhev, N.S., Gottardo, M., Riparbelli, M.G., et al. 2016. Conserved molecular interactions in centriole-to-centrosome conversion. Nat Cell Biol 18, 87-99.

Fuller, M. T., 1993. Spermatogenesis, The Development of Drosophila melanogaster, edited by M. Bate and A. Martinez-Arias. Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, New York. pp. 71-174 i

Galletta B. J., Guillen R. X., Fagerstrom C. J., Brownlee C. W., Lerit D. A., et al. , 2014.

Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies. Mol. Biol. Cell 25:

2682–2694.

Galletta B. J., Jacobs K. C., Fagerstrom C. J., Rusan N. M., 2016a. Asterless is required for centriole length control and sperm development. J. Cell Biol. 213: 435–450.

138

Galletta B. J., Fagerstrom C. J., Schoborg T. A., McLamarrah T. A., Ryniawec J. M., et al., 2016b. A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4. Nat. Commun. 7: 12476.

Gatti M., Baker B. S., 1989. Genes controlling essential cell-cycle functions in

Drosophila melanogaster.Genes Dev. 3: 438–453.

Ganem, N. J., S. A. Godinho and D. Pellman, 2009. A mechanism linking extra centrosomes to chromosomal instability. Nature 460: 278-282.

Giansanti M. G., Bucciarelli E., Bonaccorsi S., Gatti M., 2008. Drosophila SPD-2 is an essential centriole component required for PCM recruitment and astral-microtubule nucleation. Curr. Biol. 18: 303–309.

Glover D.M., 1989. Mitosis in Drosophila. J. Cell Sci. 92(Pt. 2): 137–146.

Glover D.M., 2005. Polo kinase and progression through M phase in Drosophila: a perspective from the spindle poles. Oncogene 24: 230–237.

Godinho, S. A., R. Picone, M. Burute, R. Dagher, Y. Su et al., 2014. Oncogene-like induction of cellular invasion from centrosome amplification. Nature 510: 167-171.

Goetz, S. C., and K. V. Anderson, 2010. The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 11: 331-344.

Gonczy, P., 2012. Towards a molecular architecture of centriole assembly. Nat Rev Mol

Cell Biol 13: 425-435.

139

Gonzalez C., 2007. Spindle orientation, asymmetric division and tumour suppression in Drosophila stem cells. Nat. Rev. Genet. 8: 462–472.

González C., Tavosanis G., Mollinari C., 1998. Centrosomes and microtubule organisation during Drosophila development. J. Cell Sci. 111: 2697–2706.

Gopalakrishnan, J., V. Mennella, S. Blachon, B. Zhai, A. H. Smith et al., 2011. Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome. Nat

Commun 2: 359.

Goshima, G., R. Wollman, S. S. Goodwin, N. Zhang, J. M. Scholey et al., 2007. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417-421.

Goto, M., D. A. O'Brien and E. M. Eddy, 2010. Speriolin is a novel human and mouse sperm centrosome protein. Hum Reprod 25: 1884-1894.

Gottardo M., Pollarolo G., Llamazares S., Reina J., Riparbelli M. G., et al., 2015a. Loss of centrobin enables daughter centrioles to form sensory cilia in Drosophila. Curr.

Biol. 25: 2319–2324.

Gottardo M., Callaini G., Riparbelli M. G., 2015b. The Drosophila centriole - conversion of doublets into triplets within the stem cell niche. J. Cell Sci. 128: 2437–2442.

Gottardo M., Callaini G., Riparbelli M. G., 2015c. Structural characterization of procentrioles in Drosophila spermatids. Cytoskeleton 72: 576–584.

Gottardo, M., G. Callaini and M. G. Riparbelli, 2013. The cilium-like region of the

Drosophila spermatocyte: an emerging flagellum? J Cell Sci 126: 5441-5452.

140

Gueth-Hallonet, C., C. Antony, J. Aghion, A. Santa-Maria, I. Lajoie-Mazenc et al., 1993. gamma-Tubulin is present in acentriolar MTOCs during early mouse development. J Cell

Sci 105 ( Pt 1): 157-166.

Guichard, P., D. Chretien, S. Marco and A. M. Tassin, 2010. Procentriole assembly revealed by cryo-electron tomography. EMBO J 29: 1565-1572.

Gur, Y., and Breitbart, H. 2006. Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes. Genes Dev 20, 411-416.

Habedanck, R., Stierhof, Y.D., Wilkinson, C.J., and Nigg, E.A. 2005. The Polo kinase

Plk4 functions in centriole duplication. Nat Cell Biol 7, 1140-1146.

Hames, R.S., Hames, R., Prosser, S.L., Euteneuer, U., Lopes, C.A., Moore, W.,

Woodland, H.R., and Fry, A.M. 2008. Pix1 and Pix2 are novel WD40 microtubule- associated proteins that colocalize with mitochondria in Xenopus germ plasm and centrosomes in human cells. Exp Cell Res 314, 574-589.

Hamilton, G. 2015. The mitochondria mystery. Nature, 525(7570), 444-446.

Hatch, E. M., A. Kulukian, A. J. Holland, D. W. Cleveland and T. Stearns, 2010. Cep152 interacts with Plk4 and is required for centriole duplication. J Cell Biol 191: 721-729.

Heidemann, S. R., and M. W. Kirschner, 1975. Aster formation in eggs of Xenopus laevis.

Induction by isolated basal bodies. J Cell Biol 67: 105-117.

Hinchcliffe, E. H., F. J. Miller, M. Cham, A. Khodjakov and G. Sluder, 2001.

Requirement of a centrosomal activity for cell cycle progression through G1 into S phase.

Science 291: 1547-1550.

141

Hilbert M., Erat M. C., Hachet V., Guichard P., Blank I. D.et al., 2013. Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry. Proc. Natl. Acad. Sci. USA 110: 11373–11378.

Hiraki, M., Y. Nakazawa, R. Kamiya and M. Hirono, 2007. Bld10p Constitutes the

Cartwheel-Spoke Tip and Stabilizes the 9-Fold Symmetry of the Centriole. Curr Biol.

Holland, A. J., W. Lan and D. W. Cleveland, 2010. Centriole duplication: A lesson in self-control. Cell Cycle 9: 2731-2736.

Howe, K., and G. Fitzharris, 2013. A non-canonical mode of microtubule organization operates throughout pre-implantation development in mouse. Cell Cycle 12.

Hsu W.-B., Hung L.-Y., Tang C.-J. C., Su C.-L., Chang Y., et al. , 2008. Functional characterization of the microtubule-binding and -destabilizing domains of CPAP and d-

SAS-4. Exp. Cell Res. 314: 2591–2602.

Jana, S. C., G. Marteil and M. Bettencourt-Dias, 2014. Mapping molecules to structure: unveiling secrets of centriole and cilia assembly with near-atomic resolution. Curr Opin

Cell Biol 26: 96-106.

Jin, Y. X., X. S. Cui, X. F. Yu, S. H. Lee, Q. L. Wang et al., 2012. Cat fertilization by mouse sperm injection. Zygote 20: 371-378.

Khire, A., Vizuet, A. A., Davila, E., & Avidor-Reiss, T. 2015. Asterless reduction during spermiogenesis is regulated by Plk4 and is essential for zygote development in

Drosophila. Current Biology, 25(22), 2956-2963.

142

Khire, A., Jo, K. H., Kong, D., Akhshi, T., Blachon, S., Cekic, A. R., Avidor-Reiss, T.

2016. Centriole Remodeling During Spermiogenesis in Drosophila. Current Biology:

CB, 26(23), 3183–3189

Kim, D. Y., and R. Roy, 2006. Cell cycle regulators control centrosome elimination during oogenesis in Caenorhabditis elegans. J Cell Biol 174: 751-757.

Kim, H. K., J. G. Kang, S. Yumura, C. J. Walsh, J. W. Cho et al., 2005. De novo formation of basal bodies in Naegleria gruberi: regulation by phosphorylation. J Cell Biol

169: 719-724.

Kim, T.-S., Park, J.-E., Shukla, A., Choi, S., Murugan, R. N., Lee, J. H., … Lee, K. S.

2013. Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152. Proceedings of the National Academy of

Sciences of the United States of America, 110(50), E4849–E4857

Kimura, A., and S. Onami, 2005. Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration. Dev Cell 8: 765-775.

Kirkham, M., T. Muller-Reichert, K. Oegema, S. Grill and A. A. Hyman, 2003. SAS-4 is a C. elegans centriolar protein that controls centrosome size. Cell 112: 575-587.

Klebba, J.E., Galletta, B.J., Nye, J., Plevock, K.M., Buster, D.W., Hollingsworth, N.A.,

Slep, K.C., Rusan, N.M., and Rogers, G.C. 2015. Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability. J Cell Biol 208,

401-414.

143

Ko, M.A., Rosario, C.O., Hudson, J.W., Kulkarni, S., Pollett, A., Dennis, J.W., and

Swallow, C.J. 2005. Plk4 haploinsufficiency causes mitotic infidelity and carcinogenesis.

Nat Genet 37, 883-888.

Krioutchkova, M. M., and G. E. Onishchenko, 1999. Structural and functional characteristics of the centrosome in gametogenesis and early embryogenesis of animals.

Int Rev Cytol 185: 107-156.

La Terra, S., C. N. English, P. Hergert, B. F. McEwen, G. Sluder et al., 2005. The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation. J Cell Biol 168: 713-722.

Langreth, S. G., 1969. Spermiogenesis in Cancer crabs. J Cell Biol 43: 575-603.

Lau, L., Y. L. Lee, S. J. Sahl, T. Stearns and W. E. Moerner, 2012. STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein.

Biophys J 102: 2926-2935.

Lee, J., S. Kang, Y. S. Choi, H. Kim, C. Yeo et al., 2014. Identification of a Cell Cycle-

Dependent Duplicating Complex that Assembles Basal Bodies de novo in Naegleria.

Protist 166: 1-13.

Leidel, S., and P. Gonczy, 2003. SAS-4 is essential for centrosome duplication in C elegans and is recruited to daughter centrioles once per cell cycle. Dev Cell 4: 431-439.

Li, K., E. Y. Xu, J. K. Cecil, F. R. Turner, T. L. Megraw et al., 1998. Drosophila centrosomin protein is required for male meiosis and assembly of the flagellar axoneme. J

Cell Biol 141: 455-467.

144

Longo, F. J., and E. Anderson, 1968. The fine structure of pronuclear development and fusion in the sea urchin, Arbacia punctulata. J Cell Biol 39: 339-368.

Longo, F. J., and E. Anderson, 1969. Sperm differentiation in the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus. J Ultrastruct Res 27: 486-509.

Luksza, M., I. Queguigner, M. H. Verlhac and S. Brunet, 2013. Rebuilding MTOCs upon centriole loss during mouse oogenesis. Dev Biol 382: 48-56.

Mahjoub, M. R., and T. Stearns, 2012. Supernumerary centrosomes nucleate extra cilia and compromise primary cilium signaling. Curr Biol 22: 1628-1634.

Malicki, J., and T. Avidor-Reiss, 2014. From the cytoplasm into the cilium: bon voyage.

Organogenesis 10: 138-157.

Manandhar, G., and G. Schatten, 2000. Centrosome reduction during Rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration. Mol Reprod Dev

56: 502-511.

Manandhar, G., H. Schatten and P. Sutovsky, 2005. Centrosome reduction during gametogenesis and its significance. Biol Reprod 72: 2-13.

Manandhar, G., C. Simerly and G. Schatten, 2000. Centrosome reduction during mammalian spermiogenesis. Curr Top Dev Biol 49: 343-363.

Manandhar, G., P. Sutovsky, H. C. Joshi, T. Stearns and G. Schatten, 1998. Centrosome reduction during mouse spermiogenesis. Dev Biol 203: 424-434.

145

Marescalchi, O., C. Zauli and V. Scali, 2002. Centrosome dynamics and inheritance in related sexual and parthenogenetic Bacillus (Insecta Phasmatodea). Mol Reprod Dev 63:

89-95.

Maro, B., M. H. Johnson, S. J. Pickering and G. Flach, 1984. Changes in actin distribution during fertilization of the mouse egg. J Embryol Exp Morphol 81: 211-237.

Martinez-Campos, M., R. Basto, J. Baker, M. Kernan and J. W. Raff, 2004. The

Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis. J Cell Biol 165: 673-683.

Matsuura, K., P. A. Lefebvre, R. Kamiya and M. Hirono, 2004. Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly. J Cell Biol 165: 663-671.

McGill, M., and B. R. Brinkley, 1975. Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin. J Cell Biol 67:

189-199.

McNally, K. L., A. S. Fabritius, M. L. Ellefson, J. R. Flynn, J. A. Milan et al., 2012.

Kinesin-1 prevents capture of the oocyte meiotic spindle by the sperm aster. Dev Cell 22:

788-798.

Mennella, V., B. Keszthelyi, K. L. McDonald, B. Chhun, F. Kan et al., 2012.

Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nat Cell Biol 14: 1159-1168.

146

Mikeladze-Dvali, T., L. von Tobel, P. Strnad, G. Knott, H. Leonhardt et al., 2012.

Analysis of centriole elimination during C. elegans oogenesis. Development 139: 1670-

1679.

Morito, Y., Y. Terada, S. Nakamura, J. Morita, T. Yoshimoto et al., 2005. Dynamics of microtubules and positioning of female pronucleus during bovine parthenogenesis. Biol

Reprod 73: 935-941.

Moritz, M., Braunfeld, M. B., Guénebaut, V., Heuser, J., & Agard, D. A., 2000. Structure of the γ-tubulin ring complex: a template for microtubule nucleation. Nature cell biology, 2(6), 365-370.

Morgan, T. H., & Bridges, C. B., 1916. Sex-linked inheritance in Drosophila (No. 237).

Carnegie institution of Washington.

Mottier-Pavie, V., and T. L. Megraw, 2009. Drosophila bld10 is a centriolar protein that regulates centriole, basal body, and motile cilium assembly. Mol Biol Cell 20: 2605-2614.

Nakashima, S., and K. H. Kato, 2001. Centriole behavior during meiosis in oocytes of the sea urchin Hemicentrotus pulcherrimus. Dev Growth Differ 43: 437-445.

Nakazawa, Y., M. Hiraki, R. Kamiya and M. Hirono, 2007. SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole. Curr Biol 17: 2169-2174.

Navara, C. S., N. L. First and G. Schatten, 1994. Microtubule organization in the cow during fertilization, polyspermy, parthenogenesis, and nuclear transfer: the role of the sperm aster. Dev Biol 162: 29-40.

147

Nigg, E. A., and J. W. Raff, 2009. Centrioles, centrosomes, and cilia in health and disease.

Cell 139: 663-678.

Nigg, E. A., and T. Stearns, 2011. The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries. Nat Cell Biol 13: 1154-1160.

Noguchi, T., and K. G. Miller, 2003. A role for actin dynamics in individualization during spermatogenesis in Drosophila melanogaster. Development 130: 1805-1816.

O'Connell, K. F., C. Caron, K. R. Kopish, D. D. Hurd, K. J. Kemphues et al., 2001. The

C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo. Cell 105: 547-558.

O'Donnell, L., P. K. Nicholls, M. K. O'Bryan, R. I. McLachlan and P. G. Stanton, 2011.

Spermiation: The process of sperm release. Spermatogenesis 1: 14-35.

O'Toole, E. T., and S. K. Dutcher, 2014. Site-specific basal body duplication in

Chlamydomonas. Cytoskeleton (Hoboken) 71: 108-118.

Paffoni, A., T. A. Brevini, E. Somigliana, L. Restelli, F. Gandolfi et al., 2007. In vitro development of human oocytes after parthenogenetic activation or intracytoplasmic sperm injection. Fertil Steril 87: 77-82.

Palazzo, R. E., J. M. Vogel, B. J. Schnackenberg, D. R. Hull and X. Wu, 2000.

Centrosome maturation. Curr Top Dev Biol 49: 449-470.

Pampliega, O., I. Orhon, B. Patel, S. Sridhar, A. Diaz-Carretero et al., 2013. Functional interaction between autophagy and ciliogenesis. Nature 502: 194-200.

148

Paweletz, N., D. Mazia and E. M. Finze, 1987. Fine structural studies of the bipolarization of the mitotic apparatus in the fertilized sea urchin egg. I. The structure and behavior of centrosomes before fusion of the pronuclei. Eur J Cell Biol 44: 195-204.

Pelletier, L., E. O'Toole, A. Schwager, A. A. Hyman and T. Muller-Reichert, 2006.

Centriole assembly in Caenorhabditis elegans. Nature 444: 619-623.

Peters, N., D. E. Perez, M. H. Song, Y. Liu, T. Muller-Reichert et al., 2010. Control of mitotic and meiotic centriole duplication by the Plk4-related kinase ZYG-1. J Cell Sci

123: 795-805.

Phillips, D. M., 1970. Insect sperm: their structure and morphogenesis. J Cell Biol 44:

243-277.

Rattner, J. B., 1972. Observations of centriole formation in male meiosis. J Cell Biol 54:

20-29.

Rawe, V. Y., E. S. Diaz, R. Abdelmassih, C. Wojcik, P. Morales et al., 2008. The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans. Hum Reprod 23: 573-580.

Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., & Bier, E., 2001. A Systematic

Analysis of Human Disease-Associated Gene Sequences In Drosophila melanogaster. Genome Research, 11(6), 1114–1125.

Riparbelli, M. G., and G. Callaini, 2010. Detachment of the basal body from the sperm tail is not required to organize functional centrosomes during Drosophila embryogenesis.

Cytoskeleton (Hoboken) 67: 251-258.

149

Riparbelli, M. G., G. Callaini and D. M. Glover, 2000. Failure of pronuclear migration and repeated divisions of polar body nuclei associated with MTOC defects in polo eggs of Drosophila. J Cell Sci 113 ( Pt 18): 3341-3350.

Riparbelli, M. G., G. Callaini and T. L. Megraw, 2012. Assembly and persistence of primary cilia in dividing Drosophila spermatocytes. Dev Cell 23: 425-432.

Riparbelli, M. G., R. Dallai and G. Callaini, 2010. The insect centriole: A land of discovery. Tissue Cell 42: 69-80.

Riparbelli, M. G., W. G. Whitfield, R. Dallai and G. Callaini, 1997. Assembly of the zygotic centrosome in the fertilized Drosophila egg. Mech Dev 65: 135-144.

Robert D. Marshall, and Peter Luykx, 1973. Oobservations on the centrioles of the sea urchin spermatozoon. Development Growth & Differentiation 14: 311-324.

Rodrigues-Martins, A., M. Bettencourt-Dias, M. Riparbelli, C. Ferreira, I. Ferreira et al.,

2007a. DSAS-6 Organizes a Tube-like Centriole Precursor, and Its Absence Suggests

Modularity in Centriole Assembly. Curr Biol 17: 1465-1472.

Rodrigues-Martins, A., M. Riparbelli, G. Callaini, D. M. Glover and M. Bettencourt-Dias,

2007b. Revisiting the role of the mother centriole in centriole biogenesis. Science 316:

1046-1050.

Sathananthan, A. H., S. S. Ratnam, S. C. Ng, J. J. Tarin, L. Gianaroli et al., 1996. The sperm centriole: its inheritance, replication and perpetuation in early human embryos.

Hum Reprod 11: 345-356.

150

Sathananthan, A. H., K. Selvaraj, M. L. Girijashankar, V. Ganesh, P. Selvaraj et al., 2006.

From oogonia to mature oocytes: inactivation of the maternal centrosome in humans.

Microsc Res Tech 69: 396-407.

Schatten, G., 1994. The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev Biol 165:

299-335.

Schatten, G., C. Simerly and H. Schatten, 1985. Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. Proc Natl Acad Sci U S A

82: 4152-4156.

Schatten, H., G. Schatten, D. Mazia, R. Balczon and C. Simerly, 1986. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs.

Proc Natl Acad Sci U S A 83: 105-109.

Schatten, H., and Q. Y. Sun, 2009. The role of centrosomes in mammalian fertilization and its significance for ICSI. Mol Hum Reprod.

Schatten, H., and Q. Y. Sun, 2011. New insights into the role of centrosomes in mammalian fertilization and implications for ART. Reproduction 142: 793-801.

Schatten, G., & Stearns, T. 2015. Sperm Centrosomes: Kiss Your Asterless Goodbye, for

Fertility’s Sake. Current Biology, 25(24), R1178-R1181.

Scheer, U., 2014. Historical roots of centrosome research: discovery of Boveri's microscope slides in Wurzburg. Philos Trans R Soc Lond B Biol Sci 369.

151

Shirato, Y., M. Tamura, M. Yoneda and S. Nemoto, 2006. Centrosome destined to decay in starfish oocytes. Development 133: 343-350.

Simerly, C., G. J. Wu, S. Zoran, T. Ord, R. Rawlins et al., 1995. The paternal inheritance of the centrosome, the cell's microtubule-organizing center, in humans, and the implications for infertility. Nat Med 1: 47-52.

Simerly, C., Zoran, S. S., Payne, C., Dominko, T., Sutovsky, P., Navara, C. S., ... &

Schatten, G., 1999. Biparental inheritance of γ-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm. Molecular Biology of the Cell, 10(9),

2955-2969.

Sluder, G., 2014. One to only two: a short history of the centrosome and its duplication.

Philos Trans R Soc Lond B Biol Sci 369.

Sluder, G., and A. Khodjakov, 2010. Centriole duplication: analogue control in a digital age. Cell Biol Int 34: 1239-1245.

Sluder, G., F. J. Miller and C. L. Rieder, 1989. Reproductive capacity of sea urchin centrosomes without centrioles. Cell Motil Cytoskeleton 13: 264-273.

Sluder, G., and C. L. Rieder, 1985a. Centriole number and the reproductive capacity of spindle poles. J Cell Biol 100: 887-896.

Sluder, G., and C. L. Rieder, 1985b. Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes. J

Cell Biol 100: 897-903.

152

Sonnen, K. F., Gabryjonczyk, A. M., Anselm, E., Stierhof, Y. D., & Nigg, E. A. 2013.

Human Cep192 and Cep152 cooperate in Plk4 recruitment and centriole duplication. J

Cell Sci, 126(14), 3223-3233

Stearns, T., and M. Kirschner, 1994. In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell 76: 623-637.

Stevens, N. R., J. Dobbelaere, K. Brunk, A. Franz and J. W. Raff, 2010. Drosophila Ana2 is a conserved centriole duplication factor. J Cell Biol 188: 313-323.

Stevens, N. R., A. A. Raposo, R. Basto, D. St Johnston and J. W. Raff, 2007. From stem cell to embryo without centrioles. Curr Biol 17: 1498-1503.

Stevermann, L., and D. Liakopoulos, 2012. Molecular mechanisms in spindle positioning: structures and new concepts. Curr Opin Cell Biol 24: 816-824.

Sun, Q. Y., and H. Schatten, 2007. Centrosome inheritance after fertilization and nuclear transfer in mammals. Adv Exp Med Biol 591: 58-71.

Sutovsky, P., and G. Schatten, 2000. Paternal contributions to the mammalian zygote: fertilization after sperm-egg fusion. Int Rev Cytol 195: 1-65.

Szollosi, A., 1975. Electron microscope study of spermiogenesis in Locusta migratoria

(Insect Orthoptera). J Ultrastruct Res 50: 322-346.

Szollosi, D., P. Calarco and R. P. Donahue, 1972. Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J Cell Sci 11: 521-541.

153

Tachibana, M., Terada, Y., Ogonuki, N., Ugajin, T., Ogura, A., Murakami, T., Yaegashi,

N., and Okamura, K., 2009. Functional assessment of centrosomes of spermatozoa and spermatids microinjected into rabbit oocytes. Molecular reproduction and development

76, 270-277.

Tang, Z., M. G. Lin, T. R. Stowe, S. Chen, M. Zhu et al., 2013. Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites. Nature 502: 254-257.

Tates, A. D., 1971. Cytodifferentiation during Spermatogenesis in Drosophila melanogaster: An Electron Microscope Study. Rijksuniversiteit de Leiden. Leiden,

Netherlands.

Terada, Y., H. Hasegawa, T. Ugajin, T. Murakami, N. Yaegashi et al., 2009. Microtubule organization during human parthenogenesis. Fertil Steril 91: 1271-1272.

Terada, Y., G. Schatten, H. Hasegawa and N. Yaegashi, 2010. Essential roles of the sperm centrosome in human fertilization: developing the therapy for fertilization failure due to sperm centrosomal dysfunction. Tohoku J Exp Med 220: 247-258.

Tokuyasu, K. T., 1975. Dynamics of spermiogenesis in Drosophila melanogaster. V.

Head-tail alignment. J Ultrastruct Res 50: 117-129.

Uetake, Y., K. H. Kato, S. Washitani-Nemoto and S. Nemoto Si, 2002. Nonequivalence of maternal centrosomes/centrioles in starfish oocytes: selective casting-off of reproductive centrioles into polar bodies. Dev Biol 247: 149-164. van Breugel, M., M. Hirono, A. Andreeva, H. A. Yanagisawa, S. Yamaguchi et al., 2011.

Structures of SAS-6 suggest its organization in centrioles. Science 331: 1196-1199.

154

Varmark, H., S. Llamazares, E. Rebollo, B. Lange, J. Reina et al., 2007. Asterless is a centriolar protein required for centrosome function and embryo development in

Drosophila. Curr Biol 17: 1735-1745.

Venoux, M., Tait, X., Hames, R.S., Straatman, K.R., Woodland, H.R., and Fry, A.M.

2013. Poc1A and Poc1B act together in human cells to ensure centriole integrity. J Cell

Sci 126, 163-175.

Wasbrough, E. R., S. Dorus, S. Hester, J. Howard-Murkin, K. Lilley et al., 2010. The

Drosophila melanogaster sperm proteome-II (DmSP-II). J Proteomics 73: 2171-2185.

Washitani-Nemoto, S., C. Saitoh and S. Nemoto, 1994. Artificial parthenogenesis in starfish eggs: behavior of nuclei and chromosomes resulting in tetraploidy of parthenogenotes produced by the suppression of polar body extrusion. Dev Biol 163:

293-301.

Wakimoto, B.T., Lindsley, D.L., and Herrera, C. 2004. Toward a comprehensive genetic analysis of male fertility in Drosophila melanogaster. Genetics 167, 207-216.

Wang, Y., Hu, X.J., Zou, X.D., Wu, X.H., Ye, Z.Q., and Wu, Y.D. 2015. WDSPdb: a database for WD40-repeat proteins. Nucleic acids research 43, D339-344.

Wheatley, D. N., 1982. The centriole, a central enigma of cell biology. Elsevier

Biomedical Press.

White-Cooper, H. 2012. Tissue, cell type and stage-specific ectopic gene expression and

RNAi induction in the Drosophila testis. Spermatogenesis 2, 11-22

155

Wilson, P. G., Y. Zheng, C. E. Oakley, B. R. Oakley, G. G. Borisy et al., 1997.

Differential expression of two gamma-tubulin isoforms during gametogenesis and development in Drosophila. Dev Biol 184: 207-221.

Winey, M., and E. O'Toole, 2014. Centriole structure. Philos Trans R Soc Lond B Biol

Sci 369.

Woolley, D. M., and D. W. Fawcett, 1973. The degeneration and disappearance of the centrioles during the development of the rat spermatozoon. Anat Rec 177: 289-301.

Xiao, X., and W. X. Yang, 2007. Actin-based dynamics during spermatogenesis and its significance. J Zhejiang Univ Sci B 8: 498-506.

Yabe, T., Ge, X., and Pelegri, F. 2007. The zebrafish maternal-effect gene cellular atoll encodes the centriolar component sas-6 and defects in its paternal function promote whole genome duplication. Dev Biol 312, 44-60

Yang, J., M. Adamian and T. Li, 2006. Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells. Mol Biol Cell 17:

1033-1040.

Yasuno, Y., and M. T. Yamamoto, 2014. Electron microscopic observation of the sagittal structure of Drosophila mature sperm. Microsc Res Tech.

Zamboni, L., J. Chakraborty and D. M. Smith, 1972. First cleavage division of the mouse zygot. An ultrastructural study. Biol Reprod 7: 170-193.

Zamboni, L., and M. Stefanini, 1971. The fine structure of the neck of mammalian spermatozoa. Anat Rec 169: 155-172.

156

Zhang, Q., Li, G., Zhang, L., Sun, X., Zhang, D., Lu, J., ... & Chen, Z. J., 2017. Maternal common variant rs2305957 spanning PLK4 is associated with blastocyst formation and early recurrent miscarriage. Fertility and Sterility, 107(4), 1034-1040.

157

Appendix A

List of Publications

Blachon S., Khire A., Avidor-Reiss T., 2014. The origin of the second centriole in the zygote of Drosophila melanogaster. Genetics 197: 199–205.

Avidor-Reiss, T., Khire, A., Fishman, E. L., & Jo, K. H. 2015. Atypical centrioles during sexual reproduction. Frontiers in Cell and Developmental Biology, 3, 21

Khire, A., Vizuet, A. A., Davila, E., & Avidor-Reiss, T. 2015. Asterless reduction during spermiogenesis is regulated by Plk4 and is essential for zygote development in

Drosophila. Current Biology, 25(22), 2956-2963.

Khire, A., Jo, K. H., Kong, D., Akhshi, T., Blachon, S., Cekic, A. R., Avidor-Reiss, T.

2016. Centriole Remodeling During Spermiogenesis in Drosophila. Current Biology:

CB, 26(23), 3183–3189

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