Fig 1.
USMB treatment rapidly reduces cell surface TfR levels.
RPE (A, B) or MDA-MB-231 (C-D) cells grown on glass coverslips were treated with microbubbles and/or ultrasound, as indicated. 5 min following USMB treatment, cells were placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in (A, C) are representative epifluorescence micrographs of cell surface TfR levels and in (B, D) the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiments, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05 relative to the control condition.
Fig 2.
USMB treatment enhances the rate of Transferrin uptake.
RPE cells grown on glass coverslips were treated with microbubbles and ultrasound (USMB), or left untreated (control), as indicated. Following treatment, cells were incubated with A555-Tfn for 7.5 min, and then immediately placed on ice, washed and fixed, and subjected to staining to detect EEA1. (A) Shown are representative epifluorescence micrographs depicting EEA1 and internalized A555-Tfn. (B) Shown are mean ± SEM of internalized A555-Tfn intensity in each condition (C) Shown are the mean colocalization index between A555-Tfn and EEA1 (determined by Pearson’s coefficient). For B, C: n = 3 independent experiments, each experiment >20 cells per condition. Scale = 20 μm, Magnified image scale 10 μm. *, p < 0.05 relative to the control condition.
Fig 3.
USMB treatment alters the properties of clathrin-coated pits.
RPE cells stably expressing clathrin light chain fused to green fluorescent protein (RPE GFP-CLC) cells grown on glass coverslips were treated with microbubbles and ultrasound, or left untreated (control), as indicated. Cells were then incubated with A555-Tfn for 3 min to allow labeling of internalizing TfR, and then immediately subjected to fixation and processing for imaging by total internal reflection fluorescence microscopy (TIRF-M). (A) Shown are representative fluorescence micrographs obtained by TIRF-M. Scale = 5 μm. Images are higher magnification insets of larger images shown in S1 Fig. (B-C) Images obtained by TIRF-M were subjected to automated detection and analysis of clathrin-coated pits (CCPs), as described in Material and Methods. The mean GFP-CLC (B) and A555-Tfn (C) intensity within each detected object (CCP) in each cell are shown. Each diamond symbol represents the mean fluorescence of all objects within a single cell; also shown are the mean of the cellular fluorescence values and interquartile range (red bars). The number of CCPs analyzed (n) and cells (k) from 3 independent experiments for each condition are control: n = 37,762, k = 89; USMB n = 29,897 k = 80.
Fig 4.
USMB treatment results in a delayed increase in fluid-phase internalization.
RPE cells were treated with microbubbles and ultrasound, or left untreated (control), as indicated. Following treatment, HRP uptake was measured as described in Materials and Methods. Shown are the mean ± SE of the HRP uptake values at different times following commencement of the assay, which also corresponds to the time following USMB treatment. n = 5, *, p < 0.05 relative to the corresponding timepoint of the control condition.
Fig 5.
USMB treatment increases the cell surface abundance of the lysosomal marker LAMP1.
RPE cells grown on glass coverslips were treated with 5.0 μM vacuolin-1 for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. Following treatment, cells were immediately placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface LAMP1 levels. Shown in (A) are representative epifluorescence micrographs of cell surface LAMP1 levels and in (B) the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiment, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05 relative to the control, vehicle-treated condition.
Fig 6.
Vacuolin-1 treatment impairs the reduction in cell surface TfR levels by USMB treatment.
RPE cells grown on glass coverslips were treated with 5.0 μM vacuolin-1 for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. 5 min after USMB treatment, cells were placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in (A) are representative epifluorescence micrographs of cell surface TfR levels and in (B) the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiments, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05.
Fig 7.
Vacuolin-1 treatment does not affect the regulation of fluid-phase endocytosis by USMB treatment.
RPE cells were treated with 5.0 μM vacuolin-1 for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. Following treatment, HRP uptake was measured as described in Materials and Methods. Shown are the mean ± SE of the HRP uptake values at different times following commencement of the assay, which also corresponds to the time following USMB treatment. n = 3. *, p < 0.05 relative to the corresponding timepoint of the control condition.
Fig 8.
Desipramine treatment impairs the reduction in cell surface TfR levels by USMB treatment.
RPE cells grown on glass coverslips were treated with 50 μM desipramine for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. 5 min following USMB treatment, cells were immediately placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in (A) are representative epifluorescence micrographs of cell surface TfR levels and in (B) the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiment, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05.
Fig 9.
Desipramine enhances the rate of fluid-phase uptake in USMB-treated cells.
RPE cells or MDA-MB-231 cells were treated with 50 μM desipramine for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. (A) Following treatment, HRP uptake was measured in RPE cells as described in Materials and Methods. Shown are the mean ± SE of the HRP uptake values at different times following commencement of assay, which also corresponds to the time following USMB treatment. n = 4, Following treatment, FITC-dextran was measured in RPE cells (B-C) or MDA-MB-231 cells (D-E) as described in Materials and Methods. Representative fluorescence micrographs of FITC-dextran uptake for 20 min are shown in (B) and (D), scale 20 μm. The mean ± SE of the FITC-dextran values at different times following commencement of the assay, which also corresponds to the time following USMB treatment is shown in (C) and (E) (see S2 Fig). n = 3. * p < 0.01 (relative to vehicle control cells not treated with USMB), ** p < 0.001 (relative to vehicle control cells treated with USMB).