Figure 7
Figure 7. Silencing of adaptin γ causes retention of perforin in transport vesicles and blocks cytotoxicity. (A) YTS cells, transduced with CTRL or adaptin γ RNAi, were fixed, stained with anti–CI-MPR Ab followed by AlexaFluor 568–conjugated anti-mouse Ab (red), and then stained with Alexa Fluor 488–conjugated anti-perforin D48 Ab (green). The area of colocalization between the 2 fluorophores is shown as a heat map image. Scale bars represent 5 μm. Inserts show DIC images. (B) The percentage of colocalization between perforin (D48-reactive) and CI-MPR. The data were determined by analysis of 20 (CTRL RNAi) or 39 (adaptin γ RNAi) cells and are represented as mean values + SD from 2 experiments. The numbers below the graph show Manders overlap coefficients ± SD. ***P < .001; Mann-Whitney U test. (C) Cytotoxic activity of YTS cells, mock-transduced or transduced with CTRL, adaptin γ, or LAMP1 RNAi. The graph shows the percentage of 721.221 target-cell lysis at different E:T ratios and illustrates mean values + SD from 3 independent experiments. The image (right) shows the result of immunoblotting with anti–adaptin γ or anti-actin (loading control) Ab’s in the indicated cells. (D) A model of LAMP1 function. The proteins destined for the lysosomes, such as perforin, leave the TGN in the outgoing transport vesicles and reach their destination due to the action of AP sorting complexes. In normal conditions (1), the AP-1 sorting complex recognizes and binds LAMP1 on the surface of the outgoing vesicles, allowing for transport of the LAMP1-positive, perforin-containing vesicles to the late endosomal/lysosomal (LE/Lys) compartment. Therefore, disrupting the interaction between LAMP1 and AP-1 would negatively affect perforin trafficking to the lysosomes. Silencing of LAMP1 (2), for example, would remove the AP-1 binding partner from the surface of the transport vesicles, preventing binding of AP-1 and causing retention of perforin in those vesicles. Consequently, less perforin would reach the lysosomes/lytic granules. Similarly, silencing of adaptin γ (and subsequent disruption of AP-1 expression) (3) would inhibit vesicle sorting, leading to the accumulation of the perforin-containing transport vesicles and decreased level of perforin in the secretory lysosomes.

Silencing of adaptin γ causes retention of perforin in transport vesicles and blocks cytotoxicity. (A) YTS cells, transduced with CTRL or adaptin γ RNAi, were fixed, stained with anti–CI-MPR Ab followed by AlexaFluor 568–conjugated anti-mouse Ab (red), and then stained with Alexa Fluor 488–conjugated anti-perforin D48 Ab (green). The area of colocalization between the 2 fluorophores is shown as a heat map image. Scale bars represent 5 μm. Inserts show DIC images. (B) The percentage of colocalization between perforin (D48-reactive) and CI-MPR. The data were determined by analysis of 20 (CTRL RNAi) or 39 (adaptin γ RNAi) cells and are represented as mean values + SD from 2 experiments. The numbers below the graph show Manders overlap coefficients ± SD. ***P < .001; Mann-Whitney U test. (C) Cytotoxic activity of YTS cells, mock-transduced or transduced with CTRL, adaptin γ, or LAMP1 RNAi. The graph shows the percentage of 721.221 target-cell lysis at different E:T ratios and illustrates mean values + SD from 3 independent experiments. The image (right) shows the result of immunoblotting with anti–adaptin γ or anti-actin (loading control) Ab’s in the indicated cells. (D) A model of LAMP1 function. The proteins destined for the lysosomes, such as perforin, leave the TGN in the outgoing transport vesicles and reach their destination due to the action of AP sorting complexes. In normal conditions (1), the AP-1 sorting complex recognizes and binds LAMP1 on the surface of the outgoing vesicles, allowing for transport of the LAMP1-positive, perforin-containing vesicles to the late endosomal/lysosomal (LE/Lys) compartment. Therefore, disrupting the interaction between LAMP1 and AP-1 would negatively affect perforin trafficking to the lysosomes. Silencing of LAMP1 (2), for example, would remove the AP-1 binding partner from the surface of the transport vesicles, preventing binding of AP-1 and causing retention of perforin in those vesicles. Consequently, less perforin would reach the lysosomes/lytic granules. Similarly, silencing of adaptin γ (and subsequent disruption of AP-1 expression) (3) would inhibit vesicle sorting, leading to the accumulation of the perforin-containing transport vesicles and decreased level of perforin in the secretory lysosomes.

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