Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkö rper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 m behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position. INTRODUCTIONPolarized cell growth occurs in most eukaryotic phyla, and it includes a plethora of important phenomena, such as neuronal growth cone extension in animals and pollen tube extension in vascular plants. It is particularly important in filamentous fungi where nearly all growth occurs by hyphal tip extension (reviewed by Momany, 2002). Given that some filamentous fungi are important fermentation organisms, the growth of which is of considerable economic importance, whereas others are serious plant, animal, and human pathogens, there is considerable interest in the mechanisms of tip growth in these organisms.A great deal of progress has been made in understanding fungal tip growth (summarized by Harris et al., 2005; Steinberg, 2007a,b;Riquelme et al., 2007), but key questions remain unanswered. There is general agreement that fungal tip growth involves the synthesis of cell wall components in the cell body, the incorporation of these components into vesicles, the transport of these vesicles to the cell tip, the fusion of these vesicles with the plasma membrane in the area of the cell tip (exocytosis) to release their contents, and the cross-linking of the components after release. It is clear that both microtubules and actin microfilaments play important roles in fungal tip growth, but their exact functions are not yet defined. The exact sites of exocytosis and endocytosis also remain to be determined. The positions of the site(s) of exocytosis are particularly important because fungal walls are relatively stiff structures, and once they have formed the shape of the hypha is established. Hyphal shape is thus determined to a very significant extent by where the wall precursors are released from the cytoplasm, i.e., by the positioning of the site(s) of exocyto...

It has previously been shown that two populations of microtubules coexist in a dynamically unstable manner in vitro: those in one population elongate while those in the other shorten and finally disappear. This conclusion was based on changes in the number and length distribution of microtubules after dilution of the microtubule solution. Here, we demonstrate directly that growing and shortening populations coexist in steady-state conditions, by visualization of the dynamic behaviour of individual microtubules in vitro by dark-field microscopy. Real-time video recording reveals that both ends of a microtubule exist in either the growing or the shortening phase and alternate quite frequently between the two phases in a stochastic manner. Moreover, growing and shortening ends can coexist on a single microtubule, one end continuing to grow simultaneously with shortening at the other end. We find no correlation in the phase conversion either among individual microtubules or between the two ends of a single microtubule. The two ends of any given microtubule have remarkably different characteristics; the active end grows faster, alternates in phase more frequently and fluctuates in length to a greater extent than the inactive end. Microtubule-associated proteins (MAPs) suppress the phase conversion and stabilize microtubules in the growing phase.

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.