No abstract

In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin-3 activity into the hyphal apex, where they reverse direction and move backwards in a dyneindependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus-ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin-1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus-ends. Furthermore, biologically active GFPdynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction.

Blasting Through The fungus that causes rice blast disease, Magnaporthe oryzae , can lead to devastating reductions in rice yields. M. oryzae enters the plant by developing specialized infection structures called appressoria. Appressoria generate enormous internal turgor pressure that somehow creates invasive forces that physically break the plant cuticle. Dagdas et al. (p. 1590 ) found that a toroidal (doughnut-shaped) filamentous actin network forms at the base of the appressorium at the precise point where the penetration peg, which ruptures the rice leaf cuticle, will emerge. This network is scaffolded by means of four septin guanosine triphosphatases, which form a dynamic ring structure that colocalizes with F-actin. The findings reveal how fungi translate extreme pressure into localized physical force.

Filamentous fungi are a large and evolutionarily successful group of organisms of enormous ecological importance (27,114). Fungi also have a considerable impact on our economy because they serve as bio-factories for the industrial production of proteins (90,130) and because many fungi are human and plant pathogens that pose a threat to public health and agriculture (1,105,124).The basic unit of a filamentous fungus is the hypha, which usually consists of a chain of elongated cells that expand at the apex of the tip cell (10,41,115). Hyphal tip growth is characterized by the initial establishment of one growth site, which is followed by its continuous maintenance. This growth mode is different from budding in the yeast Saccharomyces cerevisiae.Here a short period of polarized apical growth is followed by extended isotrophic growth, which allows delivery of cell wall material over the entire bud surface and which leads to the almost spherical daughter cell (28,68). In contrast, hyphal growth results in an elongated tip cell, which raises new requirements. Among these is the long-distance transport between the subapical part and the apex of the tip cell. It is thought that microtubule (MT)-based motors, including kinesin-1 and kinesin-3, deliver vesicles and growth supplies over long distances to the hyphal tip (see below). However, in agreement with the described differences between hyphal growth and budding, these motors are not even encoded by the genome of S. cerevisiae.Hyphal growth is accompanied by the secretion of exoenzymes that participate in lysis of the substrate or are involved in the synthesis of the fungal cell wall (3, 42). The cell wall can be considered as an extracellular matrix consisting mainly of a meshwork of polysaccharides and manno-proteins that shelters the cell and resists the internal turgor pressure to maintain the shape of the hypha (112).It is currently thought that growth of fungal hyphae is mediated by cytoskeleton-based polar exocytosis at the hyphal tip and cytoplasmic expansion forces that push the cytoplasm against the flexible apical wall (summarized in reference 7). This model is in part based on the phenotypic similarities between fungal cells and plant cells that grow at their tips, such as pollen tubes or root hairs (36). However, phylogenetic comparison of rRNA or conserved proteins demonstrates that fungi are more closely related to animals (6, 131), and there is evidence for amoeboid motility of fungal cells (50). In this article, I will summarize recent results from different fields of fungal cell biology that are instrumental for understanding hyphal tip growth. This includes research on the Spitzenkör-per, the polarisome, the role of the cytoskeleton in endo-and exocytosis, the cellular function of sterol-rich plasma membrane domains, and the mechanism that generates the force for extension of the hypha. Finally, I will integrate all of these results from different fungal systems in a model for hyphal tip growth, and I will end the review by providing directions for ...

In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3-based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the longrange motility behavior of the organelles. membrane trafficking | modeling