University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2018 Multiple Functions Of The trS iated Rootlet Proteins Of The aP ramecium Basal Body Md Ashikun Nabi University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biology Commons, and the Cell Biology Commons Recommended Citation Nabi, Md Ashikun, "Multiple Functions Of The trS iated Rootlet Proteins Of The aP ramecium Basal Body" (2018). Graduate College Dissertations and Theses. 951. https://scholarworks.uvm.edu/graddis/951 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. MULTIPLE FUNCTIONS OF THE STRIATED ROOTLET PROTEINS OF THE PARAMECIUM BASAL BODY A Dissertation Presented by Md Ashikun Nabi to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Specializing in Biology October, 2018 Defense Date: August 9, 2018 Dissertation Examination Committee: Judith L. Van Houten Ph.D., Advisor Alan K. Howe Ph.D., Chairperson Bryan A. Ballif, Ph.D. Alicia M. Ebert, Ph.D. Cynthia J. Forehand Ph.D., Dean of the Graduate College 1 ABSTRACT Paramecium ciliary basal bodies align in straight rows from posterior to anterior. Each basal body is connected to three rootlets ((Post Ciliary Rootlet (PCR), Transverse Rootlet (TR) and Striated Rootlet (SR)). The SR, the longest, projects from the basal body toward the anterior past several more anterior basal bodies. The depletion of Meckelin (MKS3) misaligns SRs, disorganizes basal body rows and makes the SRs appear ragged and serpentine. In this study we clarify the composition of the Paramecium ciliary basal body’s SR and demonstrate that the SR plays a critical role in creating the orderly array of basal bodies in rows that run from pole to pole of the cell, likely through the interactions with centrins and other cytoskeletal elements underlying the cell surface. Here in this study we first report the reciprocal relationship between the SR and centrin related infraciliary lattice (ICL) protein that can dictate the cell surface morphology. The SR of Chlamydomonas is the best studied. Using the single SR Chlamydomonas gene SF-assemblin to search in Paramecium DB, we found thirty Paramecium genes in thirteen Paralog Groups. Proteins from 13 paralog groups were confirmed to be in the SR structure using immunofluorescence. LC-MS/MS analyses of density fractions from SRs isolation show all thirty SR members are within the same density fraction. We further categorized all 30 SR genes in five Structural Groups based on their ability to form coiled coil domain and evaluate the function of all five Structural Group using RNA interference (RNAi). Silencing the transcripts of the any of the Structural Group showed misaligned basal body rows and the disordered organization of the SRs with abnormal appearance of SRs all over the cell surface. Silencing of Paralog Group showed normal phenotype except for the two Paralog Group (Paralog Group 1 or Paralog Group 7) which themselves constitute Structural Group individually. Isolated SRs from the control or Paralog Group depleted cells show a characteristic striation pattern that includes characteristic major and minor striations. Isolated SRs from any of the Structural Group depleted cells demonstrate abnormal shapes and striation periodicity. There is a correlation between the SR Structural Group RNAi surface misalignment phenotype and the isolated SR Structural Group RNAi phenotype for shape and periodicity of the SR. Strikingly our study of SR clearly demonstrates the role of SRs in shaping the other cytoskeleton structures of the cell cortex e.g., ICL, epiplasm territory and cortical unit territory. In another follow up study of MKS3 (Picariello et al., 2014), we depleted the transcripts of MKS5 gene in Paramecium tetraurelia. Depletion of MKS5 transcripts in Paramecium causes cilia loss all over the cell surface. Unlike MKS3 depletion, MKS5 depletion does not affect the straight basal body rows and the ordered organization of SRs. Moreover, data presented in this study clearly demonstrates depletion of MKS5 transcripts somehow affect the localization of another transition zone protein, B9D2. It appears when lacking any of the SR Structural Group, the rest fail to interact properly with each other to maintain the SRs structure and directionality toward the anterior. As a result, abnormal SRs appear to lose the interaction with other cytoskeleton structures such as ICL network complex, which eventually results in misaligned basal body rows and altered swimming behavior. From the data presented in this study it is reasonable to postulate ICL1e subfamily and SRs are in a reciprocal relationship to maintain the straight basal body rows and the highly ordered organization of the SRs all over the cell surface. i ACKNOWLEDGEMENTS I would first like to thank my advisor Dr. Judith Van Houten for providing me with the opportunity to work in her lab. Her constant encouragement and dedication helped make this possible. I would also like to thank Dr. Bryan Ballif, Dr. Alicia Ebert and Dr. Alan Howe for agreeing to be part of my dissertation committee. Your insight and critiques about this work were invaluable. I would like to thank Dr. Junji Yano and Megan Valentine for their amazing technical skills, suggestions and in depth discussion about this work. Thanks to all past members of the Van Houten lab, it was a pleasure to work with you. Thanks to Dr. Ying Wai Lam, Dr. Bin Deng and Julia Fields for their expert technical assistance with the mass spectrometry analysis. Thanks to Todd Clason for his patience and his technical assistance with my immunofluorescence imaging. Thanks to Dr. Janine Beisson for the gift of the anti-SR antibody and the opportunity to use it in this project. Continued thanks to Anne Aubusson-Fleury for the anti-CTS32 antibody for epiplasm staining and Dr. Mark Winey for his gift of the anti-centrin antibody. Without either of these the project would not have been possible. Finally, a special thank you to my parents who have supported me through my entire life. I want to extend my thanks to my wife, Nahin. Without all of you none of this would be possible. ii TABLE OF CONTENTS Abstract ................................................................................................................................ i Acknowledgements ............................................................................................................. ii List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii Chapter 1: Comprehensive Literature Review .................................................................... 1 I. General overview ............................................................................................................ 1 Motility and behavior of the Paramecium tetraurelia ....................................................... 2 Cilia and basal bodies ........................................................................................................ 3 Basal body duplication in Paramecium .............................................................................. 5 Cortex of the Paramecium and the associated structure ..................................................... 7 Intraflagellar transport (IFT) ............................................................................................ 10 Ciliopathies ....................................................................................................................... 12 II. Experimental approaches ............................................................................................. 15 RNA interference .............................................................................................................. 15 Expression of exogenous protein in Paramecium ............................................................ 17 iii Isolation of SRs ................................................................................................................. 18 Experimental Approach for the SR project ....................................................................... 19 Chapter 2: Multiple functions of the Striated Rootlet (SR) proteins of the Paramecium basal body .................................................................................................... 30 Introduction ....................................................................................................................... 30 Materials and Methods ...................................................................................................... 34 Results ............................................................................................................................... 47 Discussion ......................................................................................................................... 66 Chapter 3: Depletion of MKS5 causes loss of cilia but does not affect the basal body rows straight alignment .........................................................................................