Functional, organizational, and biochemical analysis of actin in hyphal tip cells of Allomyces macrogynus

The actin cytoskeleton in hyphal tip cells of Allomyces macrogynus was detected using an actin monoclonal antibody and analyzed with microscopic and protein electrophoresis techniques. Indirect immunofluorescence microscopy revealed that actin was concentrated in small plaques located in the peripheral cytoplasm within the first 10 to 12 αm of the hyphal tip. In a small number of hyphae, actin was detected in regions of the Spitzenkörper and nuclei. Ultrastructural immunocytochemistry suggested that the fibrillar coating of filasomes represented the ultrastructural equivalent of actin plaques. The role of actin during hyphal growth was addressed by treating cells with micromolar concentrations of cytochalasin A, D, or E. For microscopic and biochemical analysis, cytochalasins were used at concentrations which produced approximately 50% inhibition of hyphal growth. Cytochalasin-treated hyphae exhibited a cessation of polarized growth followed by a swelling of the hyphal apex. The Spitzenkörper persisted for up to 30 min in most hyphae. Removal of cytochalasin resulted in reorganization of the Spitzenkörper and resumption of apical growth. Transmission electron microscopy of cytochalasin-treated hyphae confirmed the presence of a Spitzenkörper. Peripheral electron-dense fibrillar patches were common in the apical cytoplasm. Though reminiscent of filasomes, fibrillar patches were not observed in association with microvesicles in cytochalasin-treated hyphae. Immunoblot analysis of total protein extracts from control and cytochalasin-treated hyphae showed that the actin antibody bound predominantly to a single 45 kilodalton polypeptide band.