Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery - PubMed (original) (raw)

Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery

Kyle B Quinney et al. Proc Natl Acad Sci U S A. 2019.

Abstract

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.

Keywords: CRISPR/Cas9; ESCRT microdomain; epidermal growth factor receptor; lattice light sheet microscopy; organelle.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Generation of functionally tagged ESCRT subunits using CRISPR/Cas9-mediated genome editing. (A) Cartoon highlighting the placement of the HaloTag on three ESCRT subunits to track ESCRT-0 (green), ESCRT-I (blue), and Vps4 (purple) dynamics. The sequence of the flexible linker used in each case, which contains the tobacco etch virus protease recognition sequence, is also shown. (B) Representative immunoblot analyses of control and CRISPR/Cas9-modified cell lines (n = 3 each) using antibodies directed against Hrs (Left), Tsg101 (Center), Vps4B (Right), and β-actin (load control). (C) Control and CRISPR/Cas9-modified clonal cell lines were incubated in the presence or absence of 30 ng/mL EGF for 3 h following serum starvation, and extracts were immunoblotted using antibodies directed against EGFR (Top) and β-actin (Bottom, load control). (D) Quantification of the percentage of EGFR remaining after 3 h of EGF treatment in control and CRISPR/Cas9-modified clonal cell lines. Error bars represent mean ± SEM (n = 4 each). No statistically significant difference was found, as calculated using an ANOVA test. (E) Representative thin-section EM images of MVEs from control and CRISPR/Cas9-modified clonal cell lines (more than 35 MVEs and 200 ILVs examined in each). (Scale bar: 200 nm.) The size distribution of MVEs (F) and ILVs (G) in control and CRISPR/Cas9-modified clonal cell lines is shown. No statistically significant differences were found, as calculated using an ANOVA test.

Fig. 2.

Fig. 2.

ESCRT-0 associates more stably with endosomes compared with downstream ESCRT complexes under normal growth conditions. (A) Representative immunoblot analysis (n = 3) of control and CRISPR/Cas9-modified cell lines using antibodies directed against HaloTag (Top) and β-actin (Bottom, load control). (B and C) Representative CRISPR/Cas9-modified cells imaged live using SFC optics following dye labeling with the JF549-HaloTag ligand (more than 15 different cells each, more than three biological replicates each). Maximum intensity projections of z-stacks (B) or individual confocal sections over time (C) are shown. Arrows highlight transient Vps4-positive endosomes. (Scale bars: 10 μm.) (D_–_F) Representative CRISPR/Cas9-modified cells imaged live using LLSM following dye labeling using the JF646-HaloTag ligand (more than 10 cells each, more than three biological replicates each). Projected z-stacks are shown for each time point. Arrows highlight the appearance of ESCRT-positive endosomes, and red circles denote their disappearance. (Scale bars: 5 μm; Insets, 2 μm.) (G) Quantification of the average duration of each HaloTag-ESCRT fusion protein on endosomes. Error bars represent mean ± SEM (more than 600 endosomes analyzed for each cell line).

Fig. 3.

Fig. 3.

ESCRT subunit dynamics are regulated by the action of downstream ESCRT complexes. (A, Left) Representative HaloTag-Hrs–expressing cells imaged live using STED microscopy following dye labeling using the SiR-HaloTag ligand (more than 10 cells imaged, more than three biological replicates). Arrows highlight endosomes of two different size classes (blue, less than 25th percentile; red, 25th–75th percentile). (Scale bar: 1 μm.) (A, Right) Fluorescence intensity based on line-scan analysis around the circumference of each is shown, reflecting the distribution of Hrs. (B_–_D) Averaged fluorescence intensity profiles of labeled endosomes classified by compartment size (more than 95% of Vps4B-HaloTag–labeled compartments were diffraction-limited). Only compartments that acquire and lose fluorescence during the imaging series were used (more than 350 endosomes per condition in more than 10 cells each, more than three biological replicates). (E) Individual residency times of Vps4B-HaloTag at endosomes are plotted (more than 140 endosomes, at least 10 different cells in more than three biological replicates). A black line indicates the average. (F_–_H) Representative CRISPR/Cas9-modified cells imaged live using LLSM following dye labeling with the JF646-HaloTag ligand and treatment with siRNA targeting either Tsg101 or both Vps4 isoforms (more than 10 cells each, more than three biological replicates each). Projected z-stacks are shown for each time point. Arrows highlight the appearance of ESCRT-positive endosomes, and red circles denote their disappearance. (Scale bars: 5 μm; Insets, 2 μm.) (I) Quantification of the average duration of HaloTag-Hrs on endosomes in control and Tsg101-depleted cells. Error bars represent mean ± SEM (more than 400 endosomes analyzed for each cell line, more than 10 cells imaged per condition in more than three biological replicates). **P < 0.01, as calculated using a Student’s t test. (J_–_L) Averaged fluorescence intensity profiles of labeled endosomes classified by compartment size under the conditions shown (relative to sizes determined under normal conditions). Only compartments that acquire and lose fluorescence during the imaging series were used (more than 400 endosomes per condition, more than 10 cells imaged per condition in more than three biological replicates). (M) Size distributions of ESCRT-positive endosomes under the conditions indicated (more 400 endosomes per condition, more than 10 different cells each in more than three biological replicates).

Fig. 4.

Fig. 4.

Growth factor stimulation alters ESCRT dynamics at endosomes. (A) Quantification of the average duration of each HaloTag-ESCRT fusion protein on endosomes in the presence or absence of EGF stimulation. Error bars represent mean ± SEM (more than 600 endosomes analyzed for each cell line, more than 10 cells each in more than three biological replicates). ***P < 0.005, as calculated using an ANOVA test. (B_–_D) Averaged fluorescence intensity profiles of labeled endosomes classified by compartment size following treatment with 30 ng/mL EGF. Only compartments that acquire and lose fluorescence during the imaging series were used (more than 350 endosomes per condition, more than 10 cells each in more than three biological replicates). (E) Time differences between Alexa Fluor 555-EGF and JF646-labeled HaloTag-ESCRT achieving maximum fluorescence intensity at endosomes are plotted (more than 400 endosomes analyzed for each cell line, more than 10 cells each in more than three biological replicates). (F) Percentages of ESCRT-labeled endosomes that exhibited EGF accumulation at any point during time-lapse imaging were calculated for each Halo-Tag ESCRT fusion. Error bars represent mean ± SEM (endosomes from more than 10 cells each in more than three biological replicates). (G) Quantification of the average duration of HaloTag-Tsg101 on endosomes, either EGF-positive or EGF-negative, following EGF stimulation. Error bars represent mean ± SEM (more than 500 endosomes analyzed, more than 10 cells each in more than three biological replicates). ***P < 0.005, as calculated using a t test. (H) Representative HaloTag-Hrs–expressing cells imaged live using LLSM following dye labeling using the JF646-HaloTag ligand and incubation with Alexa Fluor 555-EGF for 2 min, followed by washout (more 10 cells each, more than three biological replicates each). Projected z-stacks are shown for each time point. Arrows highlight HaloTag-Hrs–positive endosomes that also show accumulation of EGF. (Scale bar, 5 μm; Insets, 2 μm.) Representative HaloTag-Hrs–expressing cells (I and J) or HaloTag-Tsg101–expressing cells (K) imaged live using STED microscopy following dye labeling using the SiR-HaloTag ligand and incubation with Alexa Fluor 594-EGF (Left; more than 10 cells in more than three biological replicates) are shown. Fluorescence intensity based on line-scan analysis around the circumference of representative endosomes (Right, indicated by arrows on Left) is also shown, reflecting the relative distributions of each ESCRT complex and EGF. (Scale bars: I and J, 500 nm; K, 1 μm.)

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