Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.Abbreviation DMSO dimethylsulfoxide