Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition
Fig 6
Intracellular trafficking and localization of the β-(1,3)-glucan synthase, Fks1, are impaired in the mar1Δ mutant.
(A) Uptake of the lipophilic dye, FM4-64, is irregular in mar1Δ cells. Cells were incubated overnight in YPD (30°C) or TC (37°) and stained with FM4-64 for 30 minutes with shaking, followed by pelleting and refreshing in the indicated media for an additional 30 minutes with shaking. Stained cells were imaged by fluorescent microscopy with the Texas Red filter and were taken with the same exposure time. Bar, 10 μM. Arrows indicate endocytic vesicles (B, C) Acid phosphatase secretion is intact in the mar1Δ strain; alkaline phosphatase activity is decreased. WT and mar1Δ cultures were pre-incubated for 16–18 hours in phosphate replete minimal medium. The cells were diluted to an OD of 0.9 in either phosphate replete (non-inducing) or phosphate deficient (inducing) minimal medium and incubated for 3 hours at 30°C with shaking. (B) Acidic or (C) alkaline para-Nitrophenylphosphate (pNPP) substrate solution was added to each well and plates were incubated for an additional 2.5 hours at 37°C with shaking. Phosphatase activity was measured as absorbance at 410 nm over cell density at 600 nm. Data represent the mean of 3 replicates per strain per condition. (D) Fks1 is mislocalized in TC medium in mar1Δ cells. The Fks1-GFP fusion protein was expressed in the WT and mar1Δ mutant strains. Cells were incubated for 16–18 hours in TC medium at 37°C. Live cells were imaged using DeltaVision deconvolution fluorescent microscopy with the GFP filter. Images were deconvolved using softWoRx software. Bar, 10 μM.