ACTIN ASSOCIATED WITH PLASMODESMATA

We have used several methods to localise actin associated with plasmod esmata. In meristematic plant material fixed in 0.1% glutaraldehyde/1% paraformaldehyde and embedded in LR White resin, actin was localised (in TEM using 5 nm gold-labelled secondary antibody to C-4 anti-actin primary antibody) in the neck region by the plasma membrane and endopl asmic reticulum, and also down the length of the plasmodesma, deep in the cell wall. When the chemical fixation was replaced by rapid freezi ng in liquid propane (without cryoprotectants) and substitution in ace tone, the plasmodesmata were labelled in similar positions, but with l ess background label on sections. While only 8-20% of plasmodesmata we re labelled, the label was 10 to 100 fold denser over plasmodesmata th an over the surrounding wall indicating specific association with plas modesmata. We presume the apparent extracellular location of some labe l was due to the size of the antibodies between the site of attachment and the observed position of the gold particle. Gold label was found in similar locations in material fixed in 3% paraformaldehyde, infiltr ated with sucrose, frozen, sectioned (10-12 mu m thick), then labelled with antibodies before resin embedding. Furthermore, cell walls in ep idermal peels stained with rhodamine-phalloidin showed localised patch es of fluorescence, presumably at the site of plasmodesmata (or primar y pit-fields), which were connected on either side to fluorescent stra nds of actin in the cytoplasm. Suspension cultured cells of Nicotiana plumbaginifolia similarly stained showed very faint, narrow fluorescen t strands crossing the walls of sister cells, which may indicate actin associated with individual plasmodesmata, shown in TEM to be sparsely distributed in these walls. In addition, the neck regions of cytochal asin-treated plasmodesmata were greatly enlarged and lacked the normal extracellular ring of particles. We propose that actin associated wit h plasmodesmata stabilizes the neck region and possibly also the cytop lasmic sleeve, and may be actively involved in regulating cell-to-cell transport.