Creating and maintaining cell polarity are complex processes that are not fully understood. Fungal hyphal tip growth is a highly polarized and dynamic process involving both F-actin and microtubules (MTs), but the behaviour and roles of the latter are unclear. To address this issue, MT dynamics and subunit distribution were analysed in a strain of Aspergillus nidulans expressing GFP-a-tubulin. Apical MTs are the most dynamic, the bulk of which move tipwards from multiple subapical spindle pole bodies, the only clear region of microtubule nucleation detected. MTs populate the apex predominantly by elongation at rates about three times faster than tip extension. This polymerization was facilitated by the tipward migration of MT subunits, which generated a tip-high gradient. Subapical regions of apical cells showed variable tubulin subunit distributions, without tipward flow, while subapical cells showed even tubulin subunit distribution and low MT dynamics. Short MTs, of a similar size to those reported in axons, also occasionally slid into the apex. During mitosis in apical cells, MT populations at the tip varied. Cells with less distance between the tip and the first nucleus were more likely to loose normal MT populations and dynamics. Reduced MTs in the tip, during mitosis or after exposure to the MT inhibitor carbendazim (MBC), generally correlated with reduced, but continuing growth and near-normal tip morphology. In contrast, the actin-disrupting agent latrunculin B reduced growth rates much more severely and dramatically distorted tip morphology. These results suggest substantial independence between MTs and hyphal tip growth and a more essential role for F-actin. Among MT-dependent processes possibly contributing to tip growth is the transportation of vesicles. However, preliminary ultrastructural data indicated a lack of direct MT-organelle interactions. It is suggested that the population of dynamic apical MTs enhance migration of the 'cytomatrix', thus ensuring that organelles and proteins maintain proximity to the constantly elongating tip.

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Hyphal tip growth has been previously reported as pulsatile, defined as regularly alternating fast and slow rates of extension. The growth of pollen tubes, and hyphae of Neurospora crassa and Saprolegnia ferax were analysed using high spatial and temporal resolution. By using long (100-500 s) records of growth rate, sampled every second, it was possible to apply rigorous statistical analysis of the time series. As previously demonstrated, pollen tubes can show pulsatile growth, detectable with this system. In contrast, hyphal growth rates do not show any evidence of pulsatile growth; instead, growth rates appear to fluctuate randomly. It is concluded that pulsatile growth is not a common feature of hyphal tip growth.

We found previously that in living cells of Oedogonium cardiacum and O. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (< 2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore of Oedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.

Mitotic spindles of the diatom Pinnularia viritiformis stained with the fluorescent actin-labelling reagent bodipyphallacidin revealed actin among the chromosomes and extending along the spindle to the poles. This is the first report of actin's presence within spindles of this ecologically important group of organisms. Since diatom mitoses have a number of marked differences compared to that of many other eukaryotes (Int Rev Cytol 128 (1991) 63; Cell 14 (1978) 455), the present observations substantially extend the diversity of mitotic spindle types in which there is evidence of spindle actin.