Cryo-Electron Microscopy, Tomography, and Single-Particle Analysis of MSP Filaments Derived from the Amoeboid Sperm of Ascaris Suum

Abstract

Locomotion of nematode sperm is remarkably similar to that of most other crawling cells but is powered by a system of filaments composed of major sperm protein (MSP) instead of the actin-myosin machinery typically associated with amoeboid motility. The MSP motility apparatus has been reconstituted in vitro, and individual MSP filaments can be generated by the addition of ATP to detergent-treated Ascaris sperm cytosol. Filaments formed in this way have been successfully vitrified and examined using cryo-electron microscopy, and this has allowed for the structural analysis of filaments formed in the presence of MSP accessory proteins. Single particle reconstruction techniques have been applied to individual filaments sampled along their lengths, and whole filament meshworks have been reconstructed using electron cryo-tomography. These resulting physiological models were compared to earlier models derived using purified MSP, including a helical reconstruction of filaments polymerized in ethanol (King et al. 1992. JCS 101:847) and an x-ray crystal model of MSP subfilaments (Bullock et al. 1998. NSB 5:184). Comparisons suggest important differences between filaments formed under physiological and nonphysiological conditions. There are currently six Ascaris sperm proteins known to modulate MSP filament dynamics in sperm; these same proteins are absent from filaments prepared using purified MSP. Comparing MSP filament models with and without these accessory proteins allows us to map the binding sites of these accessory proteins and provides a greater understanding of how they effect MSP filament dynamics and influence motility. Supported by NIH Grant R37 GM29994 and by the American Heart Assoc.