A dissertation entitled Centriole Inheritance during Fertilization of Drosophila melanogaster by 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 Copyright 2017, Atul D. Khire 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 by 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. iv To my wife, my sister and my parents. This would not have been possible without your unwavering support. v 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. vi 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