Fig. 4: T9 proteins inhibit vacuole generation by ClC-3 overexpression in a CID-dependent manner. | Nature Communications

Fig. 4: T9 proteins inhibit vacuole generation by ClC-3 overexpression in a CID-dependent manner.

From: Endosomal chloride/proton exchangers need inhibitory TMEM9 β-subunits for regulation and prevention of disease-causing overactivity

Fig. 4

a Large lamp2-positive vacuoles 48 h after HeLa cell transfection with ClC-3, but not ClC−4 or ClC−5 (green). b Live cell imaging for Venus fluorescence (green) and acidic pH (blue, using Lysosensor) of cells 48 h after transfection with Venus-ClC-3 (top), Venus-ClC-3unc (center), or Venus-ClC-3td (bottom). Variable Lysosensor staining of vacuoles and inhibition of vacuole formation by unc and td mutants. c Flow cytometry (FC) analysis of Lysosensor reveals cell population with acidified vacuoles in ClC-3 transfected cells, which is reduced with Venus-ClC-3unc and absent with Venus-ClC-3td. Same reference for ClC-3 + CD4 in both panels. d Co-transfection of T9A or T9B suppresses vacuolization as shown by FC (left) and IF (right). Same reference for ClC-3 + CD4 in both panels. e The ΔCID mutation in either T9A or T9B suppressed inhibitory effect on vacuolization. Data values in insets of panels c-e represent percentage of cells exhibiting high Lysosensor intensity from n = 4 biological replicates. Data given as mean ± SD. *, p < 0.05 using non-parametric two-tailed Mann-Whitney test. Numerical data and exact p values are given in Source Data provided with this paper. Same set of control data (ClC-3 + CD4, blue) was used in upper and lower panels in (c). Similarly, same set of control data was used in upper and lower panels of (d) (blue). Same data set for ClC-3 + T9B (red) was used in (d) (lower panel) and (e). f T9A is detected on ClC-3-positive puncta (green) in ClC-3/HA-T9A transfected cells by anti-HA (magenta), but not with the T9AC2 (red) antibody (arrowheads). g Both anti-HA and T9AC2 antibodies detect T9A on large vacuoles in cells co-expressing HA-T9AΔCID with ClC-3. h Both anti-HA and T9AC2 antibodies detect T9A on large vacuoles in cells expressing HA-T9A with ClC-3JK_C2 (416RRRKST to QVMRKQ). Representative images in a, b-h, from technical duplicates and n≥3 biological replicates. Individual channels are depicted from the zoomed area in (a, d, and e). All scale bars, 10 μm.

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