Figure 4. Mechanotransduction regulates... | Rockefeller University Press

Figure 4.

$Mechanotransduction regulates the localization of ATAT1 and controls lysosome positioning at the IS. (A) Representative images of fixed B cells seeded on stiff or soft substrates containing BCR+ ligands for different time points. ATAT1 is shown in green, the nucleus in red (Hoechst), and the cell border (white dashed outline) was defined by F-actin phalloidin staining. Scale bar: 10 μm. (B) Quantification of nuclear and cytoplasmic ATAT1 accumulation from the cells in A. The distribution index (y axis) represents the fraction of ATAT1 fluorescence in the nucleus (N, blue) or the cytoplasm (C, excluding the nucleus, gold) relative to the total cell area. (C) Representative images of control (siCTL) or ATAT1-silenced (siATAT1) B cells seeded on glass coverslips containing BCR+ ligands for different time points. F-actin is shown in red, Ac-Tub is shown in green, and Ac-Tub density was calculated based on MFI at the IS for the images in C. (D) Western blot showing Ac-Tub and GAPDH from control or siATAT1 cells under activated (30 min) and resting conditions. Quantification is shown in the graph below. (E and F) Representative images of control (siCTL) or ATAT1-silenced (siATAT1) activated as in C. LAMP1 is shown in green, F- actin in red (phalloidin), and pericentrin (F) in cyan. Insets highlight the central pool of lysosomes, shown in grayscale. Graphs below show the quantification of cell spreading areas and LAMP1+ lysosome accumulation at the IS center of B cells activated during 30 min, from cells represented in E. Z-scan profiles are shown from cells analyzed in F. (G and H) Representative images of B cells preincubated with vehicle (EtOH) or LMB and activated for 30 min on glass slides. In G ATAT1 is shown in green, the nucleus in red (Hoechst), and the cell border (white dashed outline), defined by actin staining. Graph shows ATAT1 density in the cytoplasm and nucleus, calculated based on MFI. (H) Cells were stained for LAMP1 and actin. Graph shows LAMP1+ lysosome accumulation at the IS center of B cells activated during 30 min. Data consider n ≥ 30 cells pooled from N = 3 independent experiments. Scale bars are 10 μm. Inset scale bars are 2 μm. (B, C, and E) (Right), G: Two-way ANOVA with Sidak’s multiple comparison test. (E) (Left), H: t test. P values illustrated with asterisks are * <0.05, ** <0.01, *** <0.001, and **** <0.0001. Error bars are mean ± SEM. Source data are available for this figure: SourceData F4. Refer to the image caption for details.$

Mechanotransduction regulates the localization of ATAT1 and controls lysosome positioning at the IS. (A) Representative images of fixed B cells seeded on stiff or soft substrates containing BCR⁺ ligands for different time points. ATAT1 is shown in green, the nucleus in red (Hoechst), and the cell border (white dashed outline) was defined by F-actin phalloidin staining. Scale bar: 10 μm. (B) Quantification of nuclear and cytoplasmic ATAT1 accumulation from the cells in A. The distribution index (y axis) represents the fraction of ATAT1 fluorescence in the nucleus (N, blue) or the cytoplasm (C, excluding the nucleus, gold) relative to the total cell area. (C) Representative images of control (siCTL) or ATAT1-silenced (siATAT1) B cells seeded on glass coverslips containing BCR⁺ ligands for different time points. F-actin is shown in red, Ac-Tub is shown in green, and Ac-Tub density was calculated based on MFI at the IS for the images in C. (D) Western blot showing Ac-Tub and GAPDH from control or siATAT1 cells under activated (30 min) and resting conditions. Quantification is shown in the graph below. (E and F) Representative images of control (siCTL) or ATAT1-silenced (siATAT1) activated as in C. LAMP1 is shown in green, F- actin in red (phalloidin), and pericentrin (F) in cyan. Insets highlight the central pool of lysosomes, shown in grayscale. Graphs below show the quantification of cell spreading areas and LAMP1⁺ lysosome accumulation at the IS center of B cells activated during 30 min, from cells represented in E. Z-scan profiles are shown from cells analyzed in F. (G and H) Representative images of B cells preincubated with vehicle (EtOH) or LMB and activated for 30 min on glass slides. In G ATAT1 is shown in green, the nucleus in red (Hoechst), and the cell border (white dashed outline), defined by actin staining. Graph shows ATAT1 density in the cytoplasm and nucleus, calculated based on MFI. (H) Cells were stained for LAMP1 and actin. Graph shows LAMP1⁺ lysosome accumulation at the IS center of B cells activated during 30 min. Data consider n ≥ 30 cells pooled from N = 3 independent experiments. Scale bars are 10 μm. Inset scale bars are 2 μm. (B, C, and E) (Right), G: Two-way ANOVA with Sidak’s multiple comparison test. (E) (Left), H: t test. P values illustrated with asterisks are * <0.05, ** <0.01, *** <0.001, and **** <0.0001. Error bars are mean ± SEM. Source data are available for this figure: SourceData F4.