SUMMARYFilamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established.

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3 +. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3 + gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3 + gene product had no gross effect on mitotic spindle morphology, overexpression of mal3 + compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.

We identified a truncated allele of dam1 as a multicopy suppressor of the sensitivity of cdc13-117 (cyclin B) and mal3-1 (EB-1) cells to thiabendazole, a microtubule poison. We find that Dam1 binds to the plus end of spindle microtubules and kinetochores as cells enter mitosis and this is dependent on other components of the fission yeast DASH complex, including Ask1, Duo1, Spc34 and Dad1. By contrast, Dad1 remains bound to kinetochores throughout the cell cycle and its association is dependent on the Mis6 and Mal2, but not Mis12, Nuf2 or Cnp1, kinetochore proteins. In cells lacking Dam1, or other components of the DASH complex, anaphase is delayed due to activation of the spindle assembly checkpoint and lagging sister chromatids are frequently observed and occasionally sister chromatid pairs segregate to the same spindle pole. We find that the mitotic centromere-associated Klp5/Klp6 kinesin complex is essential in cells lacking components of the DASH complex. Cells lacking both Dam1 and Klp5 undergo a first cell cycle arrest in mitosis due to a failure to establish bipolar chromosome attachment.