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 microfilaments, 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°C, substituted at –80°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 microfilaments starting about 1–5 m behind the extreme apex and extending basally for an additional 5–10 m. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal filaments 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 bissulfosuccinimidylsuccinate] (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 filaments in the shank are consistent features of the actin cytoskeleton.