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 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 microfilaments starting about 1-5 microm behind the extreme apex and extending basally for an additional 5-10 microm. 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 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 filaments in the shank are consistent features of the actin cytoskeleton.
Lovy-Wheeler
Alenka
A
Department of Biology and Plant Biology Graduate Program, Morrill Science Center III, University of Massachusetts, 611 North Pleasant St., Amherst, MA 01003-9297, USA.
Wilsen
Kathleen L
KL
Baskin
Tobias I
TI
Hepler
Peter K
PK
D000199:Actins / Q000648:ultrastructure*; D015925:Cryopreservation; D027762:Lilium / Q000166:cytology* / Q000502:physiology*; D011058:Pollen / Q000648:ultrastructure*; D016707:Tissue Fixation
D000199:Actins