Radial F-actin arrays precede new hypha formation in Saprolegnia: implications for establishing polar growth and regulating tip morphogenesis

Abstract

The roles of cortical F-actin in initiating and regulating polarized cell expansion in the form of hyphal tip morphogenesis were investigated by analyzing long term effects of F-actin disruption by latrunculin B in the oomycete Saprolegnia ferax, and detecting localized changes in the cortical F-actin organization preceding hyphal formation. Tubular hyphal morphology was dependent on proper F-actin organization, since latrunculin induced dose-dependent actin disruption and corresponding changes in hyphal morphology and wall deposition. With long incubation times (1 to 3 hours), abundant subapical expansion occurred, the polar form of which was increasingly lost with increasing actin disruption, culminating in diffuse subapical expansion. These extreme effects were accompanied by disorganized cytoplasm, and novel reorganization of microtubules, characterized by star-burst asters. Upon removing latrunculin, hyperbranching produced abundant polar branches with normal F-actin organization throughout the colony. The results are consistent with F-actin regulating polar vesicle delivery and controlling vesicle fusion at the plasma membrane, and suggest that F-actin participates in establishing polar growth. To test this idea further, we utilized the hyperbranching growth form of Saprolegnia. Early during the recovery time, prior to multiple branch formation, radial arrays of filamentous F-actin were observed in regions with no detectable surface protrusion. Their locations were consistent with those of the numerous branches that formed with longer recovery times. Similar radial arrays preceded germ tube formation in asexual spores. The arrays were important for initiating polar growth since the spores lost their ability to polarize when the F-actin was disrupted with latrunculin, and increased isometrically in size rather than producing germ tubes. Therefore, F-actin participates in initiating tip formation in addition to its previously demonstrated participation in maintenance of hyphal tip growth. The cortical location and radial organization of the arrays suggest that they recruit and stabilize membrane-bound and cytosolic factors required to build a new tip.