Apical localization of actin patches and vacuolar dynamics in Ashbya gossypii depend on the WASP homolog Wal1p

Abstract

Analysis of the Ashbya gossypii Wiskott-Aldrich syndrome-like gene AgWAL1 indicates that it is required for the maintenance of polarized hyphal growth. Growth and organelle dynamics of the wild type and of wal1 and other mutant strains were monitored by in vivo (fluorescence) time-lapse microscopy. Loss of WAL1 led to slow growth and defects in polarized growth that produced swellings in subapical regions, whereas formation of hyphal tips and dichotomous tip branching occurred as in the wild-type. Few actin cables in Agwal1 cells were found to insert into the hyphal tip, but specific clustering of cortical actin patches was observed in subapical regions of hyphal tips instead of at the hyphal apex. Distribution and movement of vacuoles was observed in vivo using FM4-64. In the wild type and in the slowly growing mutant strains bem2 and cla4, which lack a Rho-GTPase-activating protein and a PAK kinase, respectively, early endosomes appeared in the hyphal tip, whereas very few early endosomes and small vacuoles were found in the wal1 mutant hyphal tips, thus linking the cortical patch defect of wal1 hyphae with the distribution of endosomes. Vivid movement of vacuoles seen in the wild type and in the bem2 mutant in subapical regions was largely reduced in the wal1 and cla4 mutants. The tubular structure of mitochondria (as visualized by DIOC6 in vivo) was similar in the wild type and the wal1 mutant, although wal1 mitochondria appeared to be larger. Interestingly, mitochondria were found to insert into the hyphal tips in both strains. Our results indicate a function for Wal1p in filamentous fungi in coordinating actin patch distribution with polarized hyphal tip growth.

Molecular Determinants of Sporulation in Ashbya gossypii

“…Yet, uptake of the lipophilic dye FM4-64 and its delivery to the vacuoles demonstrated active endocytosis under these conditions. Upon feeding of new nutrients the vacuolar compartments became more motile, changes in shape from round to ovoid occurred, and newly formed hyphal tip compartments were filled with endosomes and small vacuoles, as has previously been documented for growing A. gossypii hyphae (Walther and Wendland 2004). These results indicate that sporangia of either wild-type or mutant strains can reinitiate hyphal growth even after prolonged incubation in sporulation-inducing conditions.…”

“…Actin staining was done with Alexa Fluor 488 phalloidin (filter: excitation: 470/20 nm, emission: 525/25 nm) or rhodamine phalloidin (filter: excitation: 545/15 nm, emission: 620/30 nm). The staining procedure was performed as described previously (Walther and Wendland 2004). All dyes were purchase from Life Technologies (Naerum, Denmark).…”

Yeast-to-Hyphal Transition Triggers Formin-dependent Golgi Localization to the Growing Tip inCandida albicans

“…Cell membranes were stained with the lipophilic dicarbocyanine dye DiOC 6 (Eastman Kodak) at a final concentration of 10 -100 ng/ml, which results primarily in mitochondrial membrane staining (Koning et al, 1993Pringle et al, 1989Weisman et al, 1990, Walther andWendland, 2004). A stock solution of 3,3-dihexyloxacarbocyanine iodide (DiOC 6 ) (10 mg/ml in ethanol) was added directly to cells (10 7 cells/ml in YPAD) and imaged using an FITC filter set.…”

“…After incubation with the primary antibody cells were washed and incubated with Alexa 488-conjugated anti-rabbit or anti-mouse IgG antibody (Invitrogen, Carlsbad, CA) diluted 1:300. After washing extensively with phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin, cells were resuspended in mounting media and applied to slides before imaging with a fluorescein isothiocyanate (FITC) filter.Cell membranes were stained with the lipophilic dicarbocyanine dye DiOC 6 (Eastman Kodak) at a final concentration of 10 -100 ng/ml, which results primarily in mitochondrial membrane staining (Koning et al, 1993Pringle et al, 1989 Weisman et al, 1990, Walther andWendland, 2004). A stock solution of 3,3-dihexyloxacarbocyanine iodide (DiOC 6 ) (10 mg/ml in ethanol) was added directly to cells (10 7 cells/ml in YPAD) and imaged using an FITC filter set.…”

Mechanism of Nuclear Movements in a Multinucleated Cell

“…This condition, which is derived from electron tomography measurements, is referred to as "wild type" and was used for all simulations of this article. In the third set, referred to as "highly crowded," "mitochondria" were unaltered, but the diameter of the "vacuoles" was inflated to 1.4 μm ( Figure 7A), simulating the condition in older hyphae (Walther and Wendland, 2004). A soft excluded-volume interaction is present between all objects (see Materials and Methods).…”