FIG 6.

Altering the amount, distribution, and stability of Mlc1 at the Spitzenkörper. (A) Mlc1 is required for regulating the number of secretory vesicles. Indicated strains mlc1Δ/MLC1 (Control; PY5018) and mlc1Δ/mlc1Δ (mlc1; PY5451), expressing Scarlet-Sec4, were imaged after incubation with FCS at 37°C for 1 h with images showing sum projection of filament tips (the two examples for mlc1 illustrate the variation of Spitzenkörper signals). Vesicle clusters were identified in sum projections by signal intensities 8 standard deviations above the mean. The intensity values were normalized to the mean of the control strain. Means (horizontal lines) and standard deviations (error bars) (n = 57) are shown, with ** indicating a P value of <0.005. rel., relative. (B) Stabilization of Mlc1-Sec4 interaction results in an increase in secretory vesicles at the Spitzenkörper. Indicated strains, control expressing Scarlet-Sec4 (PY5018), Mlc1·GFP prenylated expressing Scarlet-Sec4 (PY5831), control expressing GFP-Sec4 (PY4809), Mlc1·Sec4-stabilized (PY5405 with GFP-Sec4), were grown and imaged as described above for panel A. The images at the top of panel A show examples of sum projection of filament tips (bar, 2.5 μm) (the two examples for Mlc1·Sec4-stabilized strain illustrate the variation of Spitzenkörper signals). Vesicle clusters were identified in sum projections by signal intensities 8 standard deviations above the mean for Scarlet-Sec4 and 13 standard deviations above the mean for GFP-Sec4. Intensity values were normalized to the means of the control strains (mlc1Δ/MLC1 expressing Scarlet-Sec4, PY5018 for the Mlc1·GFP prenylated strain, and wild-type expressing GFP-Sec4, PY4809 for the Mlc1·Sec4-stabilized strain). Values are means (horizontal lines) ± standard deviations (error bars) (n = 54), with *** and **** indicating P values of 0.0004 and <0.0001, respectively.