SummaryOne hallmark of the rapid expansion of the polar surface of fungal hyphae is the spatial separation of regions of exocytosis and endocytosis at hyphal tips, as recently shown for Ashbya gossypii and Aspergillus nidulans. To determine where cortex-associated eisosomes form with respect to these two regions, we monitored fluorescently marked eisosomes in A. gossypii. Each minute, 1.6±0.5 eisosomes form within the first 30 m of each hypha and are exclusively subapical of the endocytosis region. This spatial separation of the processes of eisosome formation and endocytosis, and the much lower frequency of eisosome formation compared with that of endocytic vesicle production do not support a recently proposed role for eisosomes in endocytosis. Levels of mRNA encoding eisosome components are tenfold higher in spores than in hyphae, explaining the observed higher eisosome density at the cortex of germ bubbles. As in Saccharomyces cerevisiae, eisosomes in A. gossypii are very stable. In contrast to S. cerevisiae, however, the A. gossypii homologue of Pil1, one of the main eisosome subunits, is very important for polar growth, whereas the homologue of Nce102, which colocalizes with eisosomes, is not needed for eisosome stability. By testing partial deletions of the A. gossypii homologue of Ymr086w, another component of the eisosome, we identified a novel protein domain essential for eisosome stability. We also compare our results with recent findings about eisosomes in A. nidulans.

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.Migration of nuclei in multinucleated hyphae of filamentous fungi is a fascinating but also a very complex dynamic process which is far from being understood. The efficient polar growth of hyphae continuously generates new cytoplasmic space. Hyphae maintain a relatively even distribution of nuclei by adjusting the migration and the division of nuclei to the hyphal growth speed. Important components of this control system were first identified as mutants exhibiting nuclear distribution defects and were termed nud in Aspergillus nidulans and ropy in Neurospora crassa. Most of the affected genes coded for components of the microtubule motor dynein and its activating complex, dynactin (27,30,42,43). Dynein and dynactin components were found to form comet-like structures at the plus end (ϩend) of cytoplasmic microtubules (cMT) in N. crassa and A. nidulans, thereby indicating that cMTs and very likely the dynamics of cMTs are important for nuclear migration (10,27,45).Dynein was also shown to be essential for functional distribution of nuclei in Nectria haematococca, Ashbya gossypii, and the dimorphic fungus Ustilago maydis (1, 13, 37). However, surprising phenotypic differences were observed, questioning the existence of a single mechanism controlling nuclear distribution in fungi. In the absence of dynein, nuclei remain in the germ bubble of A. nidulans, where they form clusters, whereas in A. gossypii, all nuclei move out of the germ bubble and form clusters at hyphal tips (1, 42). Hyphae lacking dynein generate more stable or longer cMTs in A. nidulans and A. gossypii, whereas fewer and shorter cMTs form in N. haema...