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(PKD2), Eccentric (XNTA), and Meckelin (MKS3) in the Ciliated Model Organism Paramecium Tetraurelia Megan Smith Valentine University of Vermont

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(PKD2), Eccentric (XNTA), and Meckelin (MKS3) in the Ciliated Model Organism Paramecium Tetraurelia Megan Smith Valentine University of Vermont University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2015 Polycystin-2 (PKD2), Eccentric (XNTA), and Meckelin (MKS3) in the Ciliated Model Organism Paramecium tetraurelia Megan Smith Valentine 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 Valentine, Megan Smith, "Polycystin-2 (PKD2), Eccentric (XNTA), and Meckelin (MKS3) in the Ciliated Model Organism Paramecium tetraurelia" (2015). Graduate College Dissertations and Theses. 419. https://scholarworks.uvm.edu/graddis/419 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]. POLYCYSTIN-2 ( PKD2 ), ECCENTRIC ( XNTA ), AND MECKELIN ( MKS3 ) IN THE CILIATED MODEL ORGANISM PARAMECIUM TETRAURELIA A Dissertation Presented by Megan Smith Valentine 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, 2015 Defense Date: June 4 th , 2015 Dissertation Examination Committee: Judith Van Houten, Ph.D., Advisor Alan Howe, Ph.D., Chairperson Anthony Morielli, Ph.D. Jim O. Vigoreaux, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College Abstract Paramecium tetraurelia is a ciliated single cell used as a model organism for the study of ciliopathies. Ciliopathies are mammalian diseases involving the dysfunction of cilia, including cilia maintenance, construction, and signaling. P. tetraurelia and its cilia provides an excellent non-canonical system for the investigation and elucidation of proteins important for the structure, maintenance and function of cilia and ciliary beating. We utilize features of this cell such as its 1000’s of cilia and highly organized and patterned cell surface to observe changes in swimming behavior or disruptions in the ordered cell surface which are not feasible in mammalian cells. Here, we present research on three proteins in Paramecium , two of which are homologs to human ciliopathy genes. Using combinations of epitope-tagging, RNA interference (RNAi), immunofluorescence, immunoprecipitations, LC-MS/MS analysis and electrophysiology, we have attempted to elucidate the location, function, and potential interacting partners of these three proteins. The first protein, meckelin (MKS3), is a contributing factor in Meckel-Gruber syndrome, among other ciliopathies. Using epitope tagging, we identified the location of the Mks3 protein above each basal body. Depletion of MKS3 using RNAi leads to global loss of cilia, a severe disruption in the surface organization and a mislocalization of basal bodies out of the anterior- posterior axis of the cell. We show that depletion of Mks3 leads to abnormal backward swimming in ionic stimuli and depleted secretion of trichocysts. Based on our data, we propose two functions for Mks3 in P. tetraurelia . The first function is a transition zone component important for proper regulation of ciliary protein content, consistent with MKS3 function in other organisms. The depletion of MKS3 led to global ciliary loss, but also an imbalance in the ciliary ion channels that was different from the loss of cilia due to interference with intraflagellar transport as observed in cells depleted of IFT88 . The second novel role for MKS3 is as a transient connection to the kinetodesmal fiber which is important for basal body guidance when daughter basal bodies migrate away from the mother basal body before cell division. We also examine the contribution of the non-selective cation channel Polycystin-2 (Pkd2) in Paramecium to Mg 2+ permeability and Mg 2+ -induced behavior. When mutated in humans, Pkd2 leads to 15% of the cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). When PKD2 is depleted using RNAi in Paramecium , cells show short backward swimming in Mg 2+ solutions, a resistance to heavy metal paralysis, and depleted membrane permeability to Mg 2+ . The channel-like protein XntA which is unique to Paramecium and Tetrahymena , is also important for these phenotypes. Therefore, we utilized the Paramecium XntA1 mutant in our studies, which lacks Mg 2+ -induced behavior. We demonstrate that both Pkd2 and XntA are present in the cell membrane and in the cilia. Co-IP assays show that the IP of XntA-myc co-IPs the Pkd2-FLAG channel, but not vice versa, possibly because of an occluded FLAG epitope due to protein interactions. To tease apart the contributions of Pkd2 in the cilia and the cell membrane, electrophysiology was used to measure membrane potential of ciliated and deciliated cells. Depletion of BBS8 eliminates Pkd2 in the cilia, allowing us to examine Pkd2 activity restricted to the cell membrane of ciliated cells. Depletion of Pkd2 or XntA decreases membrane permeability to Mg 2+ . When Pkd2 was restricted to the cell membrane via BBS8 depletion, the membrane permeability to Mg 2+ increased, much like over-expressing the Pkd2 protein. Depletion of Pkd2, especially in the deciliated XntA1 mutant, leads to a dramatic decrease in Mg 2+ membrane permeability. Based on these data, we propose that Pkd2 is the Mg 2+ channel in Paramecium and XntA is not a channel, but is perhaps important for stabilizing Pkd2 in membrane microdomains. We have uncovered novel function roles for the proteins mentioned here, leading to a broader understanding of their function. These studies also highlight to usefulness and importance of the model organism Paramecium tetraurelia to the study of human ciliopathy genes. Citations Material from this dissertation has been published in the following form: *Picariello, T., *Valentine, M.S., Yano, J., and Van Houten, J.. (2014). Reduction of Meckelin Leads to General Loss of Cilia, Ciliary Microtubule Misalignment, and Distorted Cell Surface Organization. Cilia , 3:2. *These authors contributed equally to this work. ii Acknowledgements I cannot begin to express my appreciation and gratitude to my supervisor, advisor, and mentor, Dr. Judith Van Houten, University Distinguished Professor. You have taught me so much and I am forever indebted to you for the opportunities and experiences you have provided me. I have great admiration for you. You have been a role model and an inspiration. I would also like to thank my other committee members, Dr. Alan Howe, Dr. Jim Vigoreaux, and Dr. Tony Morielli. You have been helpful and supportive and your insight and interest in my research have been very encouraging. Even though two of you were on my Master’s committee, you continued to see me through this next stage. I also appreciate Dr. Morielli’s guidance with my latest adventure of learning electrophysiology. I truly appreciate all of you and your insights. I cannot move forward without thanking Dr. Junji Yano. There is not a student whom has graduated or left Dr. Van Houten’s lab that hasn’t learned something from “The Master of Microinjection.” I have learned a great deal from Dr. Yano and never would have been able to complete my work without his patience and help. I would also like to thank other members of the Van Houten lab, past and present. This includes Heather Czapla, Carlie Wilson, Cassandra Love (Jacobs), Mallory Romanovitch, Dilhan Weeraratne, Sukanya Lodh, Meagan Keiser, Anbazhagan Rajendran, Wade Bell, Samsudeen Ponissery Saidu, among others. I would like to thank all of the individuals whom have provided technical assistance, including Dr. Todd Clason for DeltaVision microscopy. Also, Michele Von Turkovich and Jan Schwarz for SEM and TEM assistance and training. I would like to thank Julia Fields, Dr. Wai Lam and Dr. Bin Deng for all their help with sample preparation and LC-MS/MS analysis and support. Thank you to Jeremy Arenos and Dr. Jon Vick for their help with getting me off the ground with starting electrophysiology work. iii My family and friends have been my help behind the scenes. Especially my mom and dad. The pursuit of a Ph.D. was partially my dad’s idea. Without him and his guidance, I would never have made it to where I am now. My mother has also been a great source of strength and continued support. I appreciate all the long runs, talks, support, and love from all my parents and my brother. Although we don’t talk much, my brother is another inspiration in my life. My husband Scott has been supportive throughout this process and I truly appreciate his patience and most of all, his encouragement. My daughter has been a constant source of laughter and light through this whole process, a true source of joy. I also want to thank my Uncle Jim for his support and “tough love.” Also, I want to thank my late grandparents Jack and Kathleen. I wish you were here. Lastly, all my friends - who constantly ask me when I will be done! From my Reach the Beach Van-mates to my college hallmates, they have all helped in some way. I especially want to thank my closest friends, Jess C., Jess G., Heather K., KP, Cindy, Noel, and everyone else along the way whom has offered a run, a hug or a laugh, and a swift kick in the butt when required, thank you. This has been a journey, a challenge, and a test. I have enjoyed the ride. iv Table of Contents Acknowledgements ....................................................................................................................
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