Morphogenesis of a filamentous fungus : dynamics of the actin cytoskeleton and control of hyphal integrity in Ashbya gossypii

Abstract

PART I - THE DYNAMIC ACTIN CYTOSKELETON OF ASHBYA GOSSYPII: Polarized growth is an intriguing aspect in a continuously elongating organism like A. gossypii. We therefore attempted a detailed study of the live actin cytoskeleton in this model filamentous fungus. We analyse the different components of the actin cytoskeleton tagged with Green Fluorescent Protein (GFP) by means of rapid, multi-dimensional video microscopy, studying their structural and dynamic properties. Cap1p and Cap2p are the subunits making up capping protein, a heterodimer which binds the barbed end of actin filaments. GFP-labelled variants of each were studied. Cap1-GFP and Cap2-GFP colocalize with actin patches in rhodamine-phalloidin stainings. They are highly enriched in the first six micrometers from the tip, mostly cortical, and at sites of septation and branch formation. Cap1p-GFP and Cap2p-GFP patches moved at 224 (+-98) nm/s over distances of 0.8 μm (+/-0.7μm) and generally had a lifetime of 14 seconds ((+/-6.5). Sequential recordings of the entire hypha were analysed, suggesting that these particles undergo a pattern of movement consistent with their role in endocytosis. That is, following an initial non-motile stage, actin patches undergo random movement near their site of formation, often followed by a secondary, linear retrograde movement away from the tip. Co-stainings with the endocytosis marker FM4-64 show partial colocalization, further supporting the notion that actin patches are involved in endocytosis. A second movement type is that of retrograde patches returning to the tip, resulting in a cycling pattern. This suggests maintenance of polarization by endocytic recycling, a mechanism which was corroborated by experiments concerning lateral diffusion in the apical membrane. Application of Latrunculin A results in depolarized, spherical tips. The combination of these results suggests that apart from their role in endocytosis, Cap-GFP patches are charged with the task of maintaining polarization by endocytic recycling. Actin cables and actin rings were made visible by using a GFP tagged variant of Abp140p, an F-actin binding and crosslinking protein. Abp140p-GFP colocalizes fully with actin cables, actin patches and actin rings in rhodamine phalloidin stainings. Abp140p-GFP cables are mostly cortical, often helical, can be as long as 40μm and are highly motile. The different fluorescent intensities indicate existence of actin bundles with different numbers of cables. Elongation of the tip of a cable is 184 (+/-62) nm/s. Fine cables in the apical zone often feature Abp140p-GFP patches moving to the tip, where they desintegrate. This is strongly reminiscent of the short, straight actin cables in S. cerevisiae, which have been shown to transport exocytic vesicles to the site where a new cell wall is formed. We conclude with a model of the hyphal organisation of the actin cytoskeleton in A. gossypii. PART II - FAR11P IS REQUIRED TO PREVENT PREMATURE HYPHAL ABSCISSION IN THE FILAMENTOUS FUNGUS ASHBYA GOSSYPII: AgFar11p belongs to the Far proteins which have diverse functions. In the budding yeast Saccharomyces cerevisiae, the syntenic homolog ScFar11p links pheromone response to the cell cycle. In the filamentous fungus Neurospora crassa, the Far11p homolog (NcHAM-2) is required for hyphal fusion. While this process is important for communication and homeostasis in filamentous fungi, it has not been observed in A.gossypii. We investigated the structure and role of AgFar11p. It is a putative transmembrane protein and bears conserved domains found in the homologs of S.cerevisiae and N.crassa. Deletion of the FAR11 gene in Ashbya gossypii leads to premature hyphal abscission at septa and lysis of hyphal compartments. This chain of events occurs in wild type only at the end of the life cycle, when spores are released from hyphal compartments. We conclude that hyphal abscission in far11Δ strains is premature and suggest that in A.gossypii, Far11p is involved in the timing of sporangium formation.