Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2003 Characterization of the Cytosolic Proteins Involved in the Amoeboid Motility of Ascaris Sperm Shawnna Marie Buttery Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES CHARACTERIZATION OF THE CYTOSOLIC PROTEINS INVOLVED IN THE AMOEBOID MOTILITY OF ASCARIS SPERM By SHAWNNA MARIE BUTTERY A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Fall Semester, 2003 The members of the Committee approve the dissertation of Shawnna Buttery defended on August 12, 2003. ____________________________________ Thomas M. Roberts Professor Directing Dissertation ____________________________________ Timothy A. Cross Outside Committee Member ____________________________________ Thomas C. S. Keller III Committee Member ____________________________________ Myra M. Hurt Committee Member ____________________________________ Timothy S. Moerland Committee Member Approved: __________________________________________________ Timothy S. Moerland, Chair, Department of Biological Science The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGEMENTS This work would not have been possible without the help of many individuals throughout the years. I would like to thank Dr. Thomas Roberts for his support, guidance, and above all patience. My committee members have been a great source of useful critique and questions, for which I thank them. The members of the Roberts’ lab, both past and present, have provided a great supply of help and more importantly laughter, for that I thank: Joseph Italiano, Lawrence LeClaire, Toni Roberts, Greg Roberts, Tom Morgan, Orion Vanderlinde, Long Miao, Gail Ekman, Jean Chamoun, and Mikel Hofmann. A special thanks should be given to Margaret Seavy for her advice and assistance, especially through the troubled times. The friends that I have made in these last few years have been a great support for me through the trials of graduate school; in particular I want to thank Rosa Greenbaum for keeping me sane through it all. I certainly would not have begun this journey if it were not for the help and support of my parents; I thank them everyday for all they have done. Finally, to my husband Bryan, thank you for your love and encouragement. iii TABLE OF CONTENTS LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS ix ABSTRACT xi INTRODUCTION 1 The Actin Cytoskeleton in Amoeboid Locomotion 1 Dissection of the Actin-Based Machinery 3 Lessons from Listeria 4 The Generation of Protrusive Force 8 Limitations of Actin-Based Systems 9 The Advantages of Nematode Sperm 9 Characteristics of the MSP Cytoskeleton 10 In Vitro Reconstitution of the MSP Motility Machinery 12 Regulation of MSP Assembly 13 Project Rationale and Goals 15 MATERIALS AND METHODS 17 Collection of Ascaris Sperm 17 Fractionation of Cytosol with Subsequent Reconstitution 18 Antibody Production 19 SDS-PAGE and Western Blotting 19 Immunofluorescence 20 Peptide Sequencing 20 Cloning of Ascaris Cytosolic Proteins 21 Protein Purification 22 In Vitro Assays 22 Immunoprecipitation 23 Affinity Chromatography 23 iv RESULTS 25 Reconstitution of In Vitro Motility with a Fraction of Cytosolic Components 25 Characterization of Putative Cytoskeletal Proteins 32 p43 is a Putative Serine Phosphoprotein Based on Tandem Sequence Repeats 32 p38 and p40 are Similar Proteins, Each with a Tandem Sequence Duplication 36 p34 is a Putative Protein Kinase 41 p16 Contains a Putative MSP Domain 44 In Vitro Motility Assays Show that p38 and p16 have Reciprocal Effects on the Rate of Fiber Assembly 46 p38 and p16 interact with MSP 59 DISCUSSION 65 Novel Proteins Identified as Components of the MSP Motility Machinery 65 Function of p38 and p16 66 Revised Model of MSP Polymerization 67 Functional Conservation in Actin-Based Motility 69 Issues that Warrant Further Investigation 71 REFERENCES 73 BIOGRAPHICAL SKETCH 81 v LIST OF TABLES 1. Comparison of the in vitro reconstitution of cytosol and ammonium sulfate fractions 31 2. Comparison of the mean rate of fiber growth in perfusion assays 58 vi LIST OF FIGURES 1. The three steps of amoeboid motility 2 2. Model of Listeria comet tails formation 5 3. The dendritic nucleation model of amoeboid motility 7 4. MSP-based movement of Ascaris sperm in vivo and in vitro 11 5. Model for the mechanism of membrane-associated MSP polymerization 14 6. An MSP fiber grown in vitro 26 7. Cytosol can be fractionated by differential ammonium sulfate precipitation to yield fractions of differing protein composition 27 8. Cytosol can be separated into less complex fractions that retain competence to assemble fibers 29 9. Western blots of the SP-sepharose bound fraction probed with antibodies generated against selected proteins 33 10. Indirect immunofluorescence with anti-Mr ~ 38 antibody labels the MSP cytoskeleton uniformly in vivo and in vitro 34 11. Summary of the characteristics of the six putative cytoskeletal proteins involved in MSP motility 35 12. Alignment of Ascaris p43 with C. elegans T28H11.5, ssq-2 37 13. p43 is serine phosphorylated 38 14. Alignment of Ascaris p38 and p40 with their C. elegans homologs 39 15. Alignment of four Ascaris EST homologs of p38 and p40 42 16. Alignment of Ascaris p34 predicted protein sequence with C. elegans Y38H8A.3 43 vii 17. Ascaris p16α and β show homology to Ascaris MSP domain protein 1 (MDP1) and C. elegans predicted gene product C35D10.11 45 18. p38 is required for in vitro assembly 47 19. p16 is a negative regulator of fiber growth rate 48 20. Purification of p38 and p16 for in vitro assays 49 21. The effects of p38 on fiber growth rate at varying dilutions of S100 compared to that of dilution with KPM buffer alone 51 22. The effects of p16 on fiber growth rate at varying dilutions of S100 compared to that of dilution with KPM buffer alone 52 23. P38 alters fiber growth rate in a concentration-dependent manner 53 24. The p16 triplet alters fiber growth rates in a concentration-dependent manner 54 25. The effects of p38 are antagonistic to the effects of p16; addition of p38 and inhibition of p16 do not show a synergistic effect 56 26. Perfusion of fibers with cytosol, purified proteins and ATP shows that p38 increases fiber growth rate and p16 decreases the growth rate of individual fibers 57 27. Immunoprecipitations with anti-p38 show that p38 co-precipitates with MSP under assembly conditions 60 28. Immunoprecipitations with anti-p16 antibody 61 29. Immunoprecipitations with anti-MSP show that MSP co-precipitates with p38 and p40 under assembly conditions 62 30. Affinity chromatography with MSP as the ligand shows that MSP and the p38 family bind under assembly conditions 64 31. Revised model for the mechanism of membrane-associated MSP polymerization 68 viii LIST OF ABBREVIATIONS Å Angstrom A600 Absorbance at 600 nm ADF/cofilin actin depolymerizing factor/cofilin ADP adenosine diphosphate Arp2/3 actin related protein 2/3 complex atm atmosphere ATP adenosine triphosphate BDM butanedione monoxime BSA bovine serum albumin C Celsius cDNA complimentary deoxyribonucleic acid Da Dalton Endo-Lys-C Endoproteinase Lys-C EST Expressed Sequence Tag g gravity HKB 50 mM HEPES, 65 mM KCl, 10 mM NaHCO3 (pH 7.0) HPLC high performance liquid chromatography HRP horseradish peroxidase IgG immuno-gammaglobulin IP immunoprecipitation kDa kilo-Dalton KP 8 mM KH2PO4, 2 mM K2HPO4 (pH 6.8) KPM 8 mM KH2PO4, 2 mM K2HPO4, 0.5 M MgCl2 (pH 6.8) L Liter MALDI/TOF Matrix Assisted Laser Desorption Ionization/Time of Flight mg milligram min minute ml milliliter MPOP MSP polymerization and organizing protein Mr relative mobility mRNA messenger ribonucleic acid MSP major sperm protein MSP* polymerization-competent MSP MWCO molecular weight cut-off NaP 10 mM sodium phosphate (pH 6) PBS 136 mM NaCl, 2.7 mM KCl, 1.7 mM KH2PO4, 10 mM Na2HPO4, (pH 7.4) PCR polymerase chain reaction ix PEG polyethylene glycol S10 10,000 × g supernatant S100 100,000 × g supernatant SDS-PAGE sodium dodecyl sulfate-polyacrylimide gel electrophoresis SF soluble factor TBS-T 20 mM Tris, 137 mM NaCl, 0.5% Tween-20, (pH 7.6) µm micron µg microgram µl microliter VASP vasodilator-stimulated phosphoprotein WASp Wiscott Aldrich syndrome protein YOP tyrosine phosphatase, purified from Yersenia entercolitica x ABSTRACT The amoeboid sperm of Ascaris crawl through a cycle of protrusion, adhesion, and retraction, similar to that seen in conventional actin-based cells. However, instead of actin, these cells power their movement through modulation of the major sperm protein (MSP) cytoskeleton. MSP forms dense filament meshworks that pack the sperm lamellipod. Protrusion is associated with the assembly of MSP filaments at the leading edge of the lamellipod, and retraction is connected with the disassembly of the MSP network at the base of the lamellipod. The motility of Ascaris sperm can be reconstituted in cell-free extracts. In vitro, plasma membrane vesicles are pushed forward by the elongation of fibers constructed from a columnar meshwork of MSP filaments. This in vitro motility requires components from both the cytosol and the vesicle. LeClaire et al. (2003) recently identified the 48 kDa membrane protein required to orchestrate MSP cytoskeletal assembly at the leading edge of the lamellipod. In this study, I describe the first cytosolic proteins that are components of the MSP locomotory machinery. I fractionated cytosol with a range of biochemical techniques and reconstituted fiber assembly with a limited subset of cytosolic components. Thus, this fraction contains all the cytosolic accessory proteins required to build fibers. Several of the components in this active fraction were used to generate antibodies, which labeled the cytoskeleton in Ascaris sperm and in fibers grown in vitro and thus, identified six proteins, p43, p42, p40, p38, p34, and p16, as part of the MSP cytoskeleton.