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Figure 2.
vac17ΔH cells show a delay in the initiation of vacuole inheritance. (A and C) Kymographs of vacuole dynamics in wild-type and vac17ΔH cells. Images were taken on a single z-plane to reduce photobleaching. The Y axis represents time (10 s per frame), and the X axis is the directionality of vacuole movement along the segmented path of 8.5 μm (0.06788 μm/pixel). Vacuoles are labeled with endogenously expressed Vph1-mCherry, which localizes to the vacuole. Dashed lines (yellow) indicate the mother-bud neck; solid lines (red) represent the growing bud tip. Yellow arrows indicate vacuole anterograde events prior to crossing the mother-bud neck. Yellow arrowheads indicate anterograde movements following vacuole inheritance. Only cells in which the bud had not yet received a vacuole at the start of imaging were included in the following quantifications. Scale bar = 2 μm. (B and D) Montage of a time series of cells undergoing vacuole inheritance. The montage corresponds to the blue box regions of the kymographs. Scale bar = 5 μm. (E) The ratio of bud to mother cell diameter when the vacuole first crossed the mother-bud neck (n = 13 and n = 22, representing the total number of cells pooled from three independent experiments for wild type and vac17ΔH, respectively). A two-tailed, an unpaired Student’s t test was used to analyze differences between the two groups. ***P < 0.001; ns = not significant. Data are presented as box plots, with colored areas indicating the population from the first to the third quartile, and the median indicated. The whiskers represent the spread of the data. ***P < 0.001; ns = not significant. (F) Instantaneous velocities of vacuole movement (n = 24 and n = 33 for wild type and vac17ΔH, respectively). (G) Rate of all anterograde vacuole movements until the vacuole reached the bud cortex (n = 20 and n = 51 for wild type and vac17ΔH, respectively). Challenges that impacted quantification included: (1) multiple nondirectional vacuole movements, (2) short distances from the edge of the mother vacuole to the small bud tip (average of 3.4 μm for wild-type cells versus 5.9 μm for the mutant), and (3) growing small buds and the mother cells did not always remain in the same plane of focus.

vac17ΔH cells show a delay in the initiation of vacuole inheritance. (A and C) Kymographs of vacuole dynamics in wild-type and vac17ΔH cells. Images were taken on a single z-plane to reduce photobleaching. The Y axis represents time (10 s per frame), and the X axis is the directionality of vacuole movement along the segmented path of 8.5 μm (0.06788 μm/pixel). Vacuoles are labeled with endogenously expressed Vph1-mCherry, which localizes to the vacuole. Dashed lines (yellow) indicate the mother-bud neck; solid lines (red) represent the growing bud tip. Yellow arrows indicate vacuole anterograde events prior to crossing the mother-bud neck. Yellow arrowheads indicate anterograde movements following vacuole inheritance. Only cells in which the bud had not yet received a vacuole at the start of imaging were included in the following quantifications. Scale bar = 2 μm. (B and D) Montage of a time series of cells undergoing vacuole inheritance. The montage corresponds to the blue box regions of the kymographs. Scale bar = 5 μm. (E) The ratio of bud to mother cell diameter when the vacuole first crossed the mother-bud neck (n = 13 and n = 22, representing the total number of cells pooled from three independent experiments for wild type and vac17ΔH, respectively). A two-tailed, an unpaired Student’s t test was used to analyze differences between the two groups. ***P < 0.001; ns = not significant. Data are presented as box plots, with colored areas indicating the population from the first to the third quartile, and the median indicated. The whiskers represent the spread of the data. ***P < 0.001; ns = not significant. (F) Instantaneous velocities of vacuole movement (n = 24 and n = 33 for wild type and vac17ΔH, respectively). (G) Rate of all anterograde vacuole movements until the vacuole reached the bud cortex (n = 20 and n = 51 for wild type and vac17ΔH, respectively). Challenges that impacted quantification included: (1) multiple nondirectional vacuole movements, (2) short distances from the edge of the mother vacuole to the small bud tip (average of 3.4 μm for wild-type cells versus 5.9 μm for the mutant), and (3) growing small buds and the mother cells did not always remain in the same plane of focus.

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