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Characterization of the Cytosolic Proteins Involved in the Amoeboid Motility of Ascaris Sperm

Title: Characterization of the Cytosolic Proteins Involved in the Amoeboid Motility of Ascaris Sperm.
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Name(s): Buttery, Shawnna Marie, author
Roberts, Thomas M., professor directing dissertation
Cross, Timothy A., outside committee member
Keller, Thomas C. S., III, committee member
Hurt, Myra M., committee member
Moerland, Timothy S., committee member
Department of Biological Science, degree granting department
Florida State University, degree granting institution
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2003
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: 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. Sequence analysis showed that each protein is novel to nematode sperm and has a homolog of unknown function in C. elegans that exhibits sperm-enriched expression (Reinke et al., 2000; Hill et al., 2001). The predicted protein sequence of p43 is based on a tandem array of repeats with serine phosphorylation sites. P38, p40, and p42 appear to be a family of closely related polypeptides. The sequences of p38 and p40 contain a domain duplication as well as proline-rich repeats. P34 shows homology to serine/threonine kinases. The p16 triplet is a family of closely related polypeptides. Two of the proteins had reciprocal effects on fiber growth: p38 increased fiber growth rate and p16 decreased fiber growth rate. The effects of both p38 and p16 were concentration-dependent and antagonistic. Since the rate-enhancement by p38 was not potentiated by MSP, another cytosolic protein is involved in up-regulation of the rate of MSP elongation. Additionally, p16 altered the number of filaments polymerized at the vesicle surface and thus may regulate MSP nucleation. Immunoprecipitations and affinity chromatography established that these two proteins bind to MSP under conditions that promote in vitro assembly. Based on these data, I present a revised model for the mechanism of membrane-associated MSP polymerization, which proposes that p38 influences the rate of MSP elongation by recruiting MSP to the vesicle surface and that p16 incorporates into MSP filaments and blocks further assembly by acting as a capping protein. My results imply that although the specific components differ, actin-based and MSP-based systems both rely on the interplay of positive and negative regulators of cytoskeletal assembly to maintain the pace of locomotion.
Identifier: FSU_migr_etd-2403 (IID)
Submitted Note: 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.
Date of Defense: August 12, 2003.
Keywords: Motility, Sperm, Ascaris, Protein, Amoeboid
Bibliography Note: Includes bibliographical references.
Advisory Committee: 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.
Subject(s): Biology
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_migr_etd-2403
Owner Institution: FSU

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Buttery, S. M. (2003). Characterization of the Cytosolic Proteins Involved in the Amoeboid Motility of Ascaris Sperm. Retrieved from http://purl.flvc.org/fsu/fd/FSU_migr_etd-2403