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Journal of Cell Science, Vol 101, Issue 4 847-857, Copyright © 1992 by Company of Biologists
JOURNAL ARTICLES |
KL King, M Stewart, TM Roberts and M Seavy
Department of Biological Science, Florida State University, Tallahassee 32306.
Ascaris sperm are amoeboid cells that crawl by extending pseudopods. Although amoeboid motility is generally mediated through an actin-based cytoskeleton, Ascaris sperm lack this system. Instead, their major sperm protein (MSP) forms an extensive filament system that appears to fulfil this function. Because their motility appears to be essentially the same as that of their actin-rich counterparts, Ascaris sperm offer a simple alternative system for investigation of the molecular mechanism of amoeboid movement. To examine the structure and composition of the cytoskeleton, we stabilized the extremely labile native MSP filaments by detergent lysis of sperm in the presence of either glutaraldehyde or polyethylene glycol (PEG). Biochemical analysis showed that the cytoskeleton contained two isoforms of MSP, designated alpha- and beta-, that we purified and sequenced. Both contain 126 amino acids and have an acetylated N-terminal alanine, but differ at four residues so that alpha-MSP is 142 Da larger and 0.6 pH unit more basic than beta-MSP. Neither isoform shares sequence homology with other cytoskeletal proteins. In ethanol, 2-methyl-2,4-pentanediol (MPD), and other water-miscible alcohols each isoform assembled into filaments 10 nm wide with a characteristic substructure repeating axially at 9 nm. These filaments were indistinguishable from native fibers isolated from detergent-lysed sperm. Pelleting assays indicated a critical concentration for assembly of 0.2 mM for both isoforms in 30% ethanol, but alpha-MSP formed filaments at lower solvent concentration than beta-MSP. When incubated in polyethylene glycol, both isoforms formed thin, needle-shaped crystals that appeared to be constructed from helical fibers, with a 9 nm axial repeat that matched that seen in isolated filaments. These crystals probably contained a parallel array of helical filaments, and may enable both the structure of MSP molecules and their mode of assembly into higher aggregates to be investigated to high resolution.
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This article has been cited by other articles:
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  K. Yi, X. Wang, M. R. Emmett, A. G. Marshall, M. Stewart, and T. M. Roberts Dephosphorylation of Major Sperm Protein (MSP) Fiber Protein 3 by Protein Phosphatase 2A during Cell Body Retraction in the MSP-based Amoeboid Motility of Ascaris Sperm Mol. Biol. Cell, July 15, 2009; 20(14): 3200 - 3208. [Abstract] [Full Text] [PDF]
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 L. Miao, O. Vanderlinde, J. Liu, R. P. Grant, A. Wouterse, K. Shimabukuro, A. Philipse, M. Stewart, and T. M. Roberts The role of filament-packing dynamics in powering amoeboid cell motility PNAS, April 8, 2008; 105(14): 5390 - 5395. [Abstract] [Full Text] [PDF]
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S. M. Buttery, G. C. Ekman, M. Seavy, M. Stewart, and T. M. Roberts Dissection of the Ascaris Sperm Motility Machinery Identifies Key Proteins Involved in Major Sperm Protein-based Amoeboid Locomotion Mol. Biol. Cell, December 1, 2003; 14(12): 5082 - 5088. [Abstract] [Full Text] [PDF]
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 D. Bottino, A. Mogilner, T. Roberts, M. Stewart, and G. Oster How nematode sperm crawl J. Cell Sci., January 15, 2002; 115(2): 367 - 384. [Abstract] [Full Text] [PDF]
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 T. M. Roberts and M. Stewart Acting like Actin: The Dynamics of the Nematode Major Sperm Protein (Msp) Cytoskeleton Indicate a Push-Pull Mechanism for Amoeboid Cell Motility J. Cell Biol., April 3, 2000; 149(1): 7 - 12. [Full Text] [PDF]
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K. King, M Stewart, and T. Roberts Supramolecular assemblies of the Ascaris suum major sperm protein (MSP) associated with amoeboid cell motility J. Cell Sci., January 10, 1994; 107(10): 2941 - 2949. [Abstract] [PDF]
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