Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube

The actin cytoskeleton plays a crucial role in the growth and polarity of the pollen tube. Due to inconsistencies in the conventional preservation methods, we lack a unified view of the organization of actin micro. laments, especially in the apical domain, where tip growth occurs. In an attempt to improve fixation methods, we have developed a rapid freeze-whole mount procedure, in which growing pollen tubes ( primarily lily) are frozen in liquid propane at - 180 degrees C, substituted at - 80 degrees C in acetone containing glutaraldehyde, rehydrated, quenched with sodium borohydride, and probed with antibodies. Confocal microscopy reveals a distinct organization of actin in the apical domain that consists of a dense cortical fringe or collar of micro. laments starting about 1 - 5 mu m behind the extreme apex and extending basally for an additional 5 - 10 mu m. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal. laments that are evenly dispersed throughout the cytoplasm. We have also developed an improved ambient-temperature chemical fixation procedure, modified from a protocol based on simultaneous fixation and phalloidin staining. We removed EGTA, elevated the pH to 9, and augmented the fixative with ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS). Notably, this protocol preserves the actin cytoskeleton in a pattern similar to that produced by cryofixation. These procedures provide a reproducible way to preserve the actin cytoskeleton; employing them, we find that a cortical fringe in the apex and finely dispersed longitudinal. laments in the shank are consistent features of the actin cytoskeleton.