Filamentous networks in crane-fly spermatocytes: osmotic lysis of cell monolayers attached to poly-l-lysine-derivatized coverslips

Spermatocytes of the crane fly, Nephrotoma suturalis, were prepared as monolayers by centrifuging cell suspensions onto glass coverslips to which poly-l-lysine had been bound covalently using a water-soluble crosslinking reagent. Cells attached to coverslips were osmotically lysed under conditions that preserve the integrity of actin filaments. Lysed spermatocyte remnants then were examined using scanning electron microscopy. While the morphology of the remnants was variable, all were observed to contain filamentous networks that appeared similar to actin-containing cortical networks observed in other types of nonmuscle cells. The organization of networks also resembled that of critical point dried F-actin. SDS-polyacrylamide gel electrophoresis used in conjunction with DNase-I affinity chromatography showed a twofold enrichment of actin in remnants in comparison to whole cells. These methods may be applicable to combined structural and biochemical studies on other types of cells that do not attach naturally to glass surfaces.