ASHFORD, ANNE, LOUISE COLE, GEOFF HYDE & DAVID ORLOVICH.
School of Biological Science, UNSW, Sydney 2052, AUSTRALIA.
Large vacuoles in subapical regions of Pisolithus tinctorius are attached to the plasma membrane by bonds that are so strong that vacuoles break rather than separating from the plasma membrane during tissue preparation for electron microscopy. This membrane-association is indicated by a straight side in the vacuole profile and may be seen by both light and electron microscopy. These large vacuoles either remain more or less in the same position or slowly creep along the plasma membrane and they may fuse with one another. They are interconnected over distances of several microns by one or more long tubules, and video sequences show that pulses of fluorochrome pass along the vacuole chains via these tubules. We propose that the large vacuoles act as anchors/storage sites from which tubules extend and make contact with either other tubules or vacuoles. The vacuole system thus has both relatively stable and highly motile components. X-ray microanalysis of anhydrously freeze-substituted hyphae shows that tubules as well as large vacuoles contain relatively high levels of P and K, further supporting our hypothesis that these elements are transported by interconnected tubular systems. Although freeze-substituted vacuoles show a number of inclusions, none of these is similar to typical polyphosphate granules. Furthermore fluorochrome-loaded vacuoles viewed by fluorescence microscopy do not show spherical fluorochrome exclusion zones as would be expected if they contained polyphosphate granules in vivo. The tubular component of the vacuole system disappears in the presence of microtubule depolymerising drugs such as oryzalin and nocodazole, while levels of anti-actin drugs such as latrunculin B that destroy the hyphal tip actin caps have no obvious effect on the motility and form of the tubular vacuole system.