Microinjected fluorescent phalloidin in vivo reveals F-actin dynamics in isolated egg cells of wheat (Triticum aestivum, L.) developed in situ and fertilised in vitro

Capitalising on a novel immobilisation method, the microinjection of fluorescent phalloidin into wheat egg cells made it possible to follow the in vivo dynamics of the F-actin cytoskeleton. The fluorophore bound to the actin microfilaments within minutes following microinjection. Altogether, 280 isolated egg cells were analysed. The localisation of F-actin was investigated in egg cells of wheat developed in situ and fertilised in vitro at consecutive time intervals using a low-light CCD camera connected to an image-processing system. The images of filamentous actin revealed the rapid, dynamic reorganisation of the actin filaments upon sperm-egg cell fusion. Whereas unfertilised egg cells showed the polar F-actin localisation, which was most pronounced in receptive egg cells, cortical actin filaments were found to be distributed uniformly in in vitro fertilised egg cells after a characteristic accumulation of actin patch at the site of sperm entry disassembled. Throughout mitosis and cytokinesis, rearrangement of the interphase actin cytoskeleton resulted in transverse cortical F-actin becoming concentrated in a widening band predicting the future division plane. Observations made on the egg cell apparatus and two-celled proembryos hint at the possibility that synergids may play a crucial role in developmental axis fixation presumably by actin accumulation. When the whole egg cell apparatus was isolated after in vivo fertilisation, the strongest fluorescent signal was detected in synergids attached to the fertilised egg cells, although microinjection was performed only in the egg cell. Therefore, it appears likely that the synergids are involved in the actin turnover of the wheat egg cell and may impart spatial information to the egg cell concerning the micropylar-chalazal axis of the embryo sac. The same actin pattern was observed in two-celled proembryos, However, when the synergids were detached, an actin crescent formed at both sides of the common cell wall. Neither the electropolarisation brought about by the AC-field alignment of the cells to be fused, nor the DC-pulses applied to the gamete pairs during electrofusion were found to account for the reorganisation of actin filaments. In cells fixed chemically using glutar-aldehyde no F-actin could be detected.