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Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization.

Shi L, Mai S, Israels S, Browne K, Trapani JA, Greenberg AH - J. Exp. Med. (1997)

Bottom Line: GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min.With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB.We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.

ABSTRACT
Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature (4 degrees C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced transient plasma membrane blebbing and nuclear coarsening but the cells did not become frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

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Detection of intracellular GraB by immunogold staining. YAC-1 cells incubated in GraB (2 μg/ml) for 10 min were washed and fixed, then  thin cryosections incubated with murine anti-GraB antibody or colloidal gold goat anti–mouse IgG. (A) Colloidal gold anti-GraB (arrows) localized to an  area of increased electron density on the external leaf of the plasma membrane. (B) High power magnification of gold particles (arrows) in the cytoplasm  near the nuclear membrane. (C) A lower magnification of B showing the position of the immunogold in the whole cell (arrows). (D) YAC-1 cell treated  with anti-GraB antibody and immunogold in the absence of GraB had no detectable gold particles.
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Figure 5: Detection of intracellular GraB by immunogold staining. YAC-1 cells incubated in GraB (2 μg/ml) for 10 min were washed and fixed, then thin cryosections incubated with murine anti-GraB antibody or colloidal gold goat anti–mouse IgG. (A) Colloidal gold anti-GraB (arrows) localized to an area of increased electron density on the external leaf of the plasma membrane. (B) High power magnification of gold particles (arrows) in the cytoplasm near the nuclear membrane. (C) A lower magnification of B showing the position of the immunogold in the whole cell (arrows). (D) YAC-1 cell treated with anti-GraB antibody and immunogold in the absence of GraB had no detectable gold particles.

Mentions: To confirm the observation that GraB can enter cells in the absence of perforin by an independent method, we next used colloidal gold labeling of GraB using anti-GraB antibody. YAC-1 cells were treated with 2 μg/ml of GraB and then harvested and fixed at increasing time intervals of 10, 20, 45, and 60 min as described in Materials and Methods. Ultrathin cryosections were prepared and then incubated with anti-human GraB antibody or normal IgG control followed by goat anti–mouse IgG colloidal gold (10 nM), and then the cells were then examined by transmission electron microscopy. Anti-GraB antibody colloidal gold staining was found on the external surface of the plasma membrane associated with areas of increased electron density (Fig. 5 A). Gold particles were also identified in the cell cytoplasm of GraB-treated but not control cells treated with primary and secondary antibody but without GraB preincubation (Fig. 5, B–D). Colloidal gold was not seen associated with any particular cellular structure but was distributed throughout the cytoplasm and was sometimes observed near the nuclear envelope as illustrated in Fig. 5, B and C. No gold particles were detected in the nucleus. At later time points the amount of GraB in the cell cytoplasm was either equivalent to the 15-min time point or somewhat decreased. The nucleus of the cells and the overall cell morphology was not significantly altered (Fig. 5 C ).


Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization.

Shi L, Mai S, Israels S, Browne K, Trapani JA, Greenberg AH - J. Exp. Med. (1997)

Detection of intracellular GraB by immunogold staining. YAC-1 cells incubated in GraB (2 μg/ml) for 10 min were washed and fixed, then  thin cryosections incubated with murine anti-GraB antibody or colloidal gold goat anti–mouse IgG. (A) Colloidal gold anti-GraB (arrows) localized to an  area of increased electron density on the external leaf of the plasma membrane. (B) High power magnification of gold particles (arrows) in the cytoplasm  near the nuclear membrane. (C) A lower magnification of B showing the position of the immunogold in the whole cell (arrows). (D) YAC-1 cell treated  with anti-GraB antibody and immunogold in the absence of GraB had no detectable gold particles.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196167&req=5

Figure 5: Detection of intracellular GraB by immunogold staining. YAC-1 cells incubated in GraB (2 μg/ml) for 10 min were washed and fixed, then thin cryosections incubated with murine anti-GraB antibody or colloidal gold goat anti–mouse IgG. (A) Colloidal gold anti-GraB (arrows) localized to an area of increased electron density on the external leaf of the plasma membrane. (B) High power magnification of gold particles (arrows) in the cytoplasm near the nuclear membrane. (C) A lower magnification of B showing the position of the immunogold in the whole cell (arrows). (D) YAC-1 cell treated with anti-GraB antibody and immunogold in the absence of GraB had no detectable gold particles.
Mentions: To confirm the observation that GraB can enter cells in the absence of perforin by an independent method, we next used colloidal gold labeling of GraB using anti-GraB antibody. YAC-1 cells were treated with 2 μg/ml of GraB and then harvested and fixed at increasing time intervals of 10, 20, 45, and 60 min as described in Materials and Methods. Ultrathin cryosections were prepared and then incubated with anti-human GraB antibody or normal IgG control followed by goat anti–mouse IgG colloidal gold (10 nM), and then the cells were then examined by transmission electron microscopy. Anti-GraB antibody colloidal gold staining was found on the external surface of the plasma membrane associated with areas of increased electron density (Fig. 5 A). Gold particles were also identified in the cell cytoplasm of GraB-treated but not control cells treated with primary and secondary antibody but without GraB preincubation (Fig. 5, B–D). Colloidal gold was not seen associated with any particular cellular structure but was distributed throughout the cytoplasm and was sometimes observed near the nuclear envelope as illustrated in Fig. 5, B and C. No gold particles were detected in the nucleus. At later time points the amount of GraB in the cell cytoplasm was either equivalent to the 15-min time point or somewhat decreased. The nucleus of the cells and the overall cell morphology was not significantly altered (Fig. 5 C ).

Bottom Line: GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min.With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB.We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.

ABSTRACT
Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature (4 degrees C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced transient plasma membrane blebbing and nuclear coarsening but the cells did not become frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

Show MeSH
Related in: MedlinePlus