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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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Microinjection of GraB into the cytoplasm of B16 melanoma. After GraB (2 μg/ml) (A) or medium (B) injection into B16 cells  (injected cells indicated by arrows), the B16 cells were examined by DIC  microscopy using image analysis. After 6 min the cell nucleoli became  prominent and by 12 min plasma membrane blebbing was noted (arrowheads). No further changes were observed over the next 60 min. Plasma  membrane and nuclear changes were transient. (C) Hoechst dye staining  of GraB microinjected B16 melanoma cells. A group of cells were injected with GraB (1 μg/ml) and after 45 min stained with Hoechst dye.  Similar to the DIC microscopy (Fig. 3) the only nuclear changes observed  was the increasing prominence of the nucleoli (arrows). Microinjection  experiments were repeated at different doses and times in at least five separate experiments. (D) Cells incubated in GraB (1 μg/ml) and perforin  (60 ng/ml) for 45 min display the typical chromatin condensation pattern  of apoptotic cells (arrows). (E) Control cells incubated in medium.
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Figure 6: Microinjection of GraB into the cytoplasm of B16 melanoma. After GraB (2 μg/ml) (A) or medium (B) injection into B16 cells (injected cells indicated by arrows), the B16 cells were examined by DIC microscopy using image analysis. After 6 min the cell nucleoli became prominent and by 12 min plasma membrane blebbing was noted (arrowheads). No further changes were observed over the next 60 min. Plasma membrane and nuclear changes were transient. (C) Hoechst dye staining of GraB microinjected B16 melanoma cells. A group of cells were injected with GraB (1 μg/ml) and after 45 min stained with Hoechst dye. Similar to the DIC microscopy (Fig. 3) the only nuclear changes observed was the increasing prominence of the nucleoli (arrows). Microinjection experiments were repeated at different doses and times in at least five separate experiments. (D) Cells incubated in GraB (1 μg/ml) and perforin (60 ng/ml) for 45 min display the typical chromatin condensation pattern of apoptotic cells (arrows). (E) Control cells incubated in medium.

Mentions: It was evident from the above experiments that GraB crossed the cell membrane and was found within the cytoplasm in the absence of perforin but produced no obvious damage to the cells. If GraB can enter the target cell but fails to initiate apoptosis then it is possible that it is unable to enter the cell in sufficient quantity on its own to cleave and activate its substrates. Another possibility is that GraB is in some way segregated from the substrates that it uses to initiate apoptosis. To test these hypotheses, we directly microinjected GraB into the cell cytoplasm. B16 melanoma cells, which are highly susceptible to GraB and perforin-induced apoptosis within 2 h, were injected with either GraB (2 μg/ml) or buffer control and observed by DIC microscopy using an Image 1 time lapse image analysis system over a 2–3-h period. We observed changes to both the nucleus and plasma membrane within a few minutes of placing the protease in the cytoplasm. In 4–6 min the nuclear membrane and nucleolus appeared coarse and more prominent compared to control injections (Fig. 6, A and B). The plasma membrane began blebbing starting ∼5–7 min after injection which became pronounced by 8–12 min. However, in no instance did we observe progression to obvious membrane or nuclear disruption. Membrane blebs were usually transient in that they appeared and disappeared quickly. Nuclear changes also did not progress over the observation period. The rate at which the blebbing and nuclear changes occurred was dependent on the concentration of the GraB in the injection buffer. At high concentrations the changes were evident by 4–12 min, while at lower concentrations the effects were delayed to 20 min or longer. The experiment was repeated using the Rat-1 cell, microinjecting GraB at a 10-fold high concentration (20 μg/ml) using an automated injection protocol in which 1.2 × 10−12 ml was injected per cell containing 0.2–0.4 femtograms. Cells were again followed for 2–3 h and no apoptosis was observed.


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)

Microinjection of GraB into the cytoplasm of B16 melanoma. After GraB (2 μg/ml) (A) or medium (B) injection into B16 cells  (injected cells indicated by arrows), the B16 cells were examined by DIC  microscopy using image analysis. After 6 min the cell nucleoli became  prominent and by 12 min plasma membrane blebbing was noted (arrowheads). No further changes were observed over the next 60 min. Plasma  membrane and nuclear changes were transient. (C) Hoechst dye staining  of GraB microinjected B16 melanoma cells. A group of cells were injected with GraB (1 μg/ml) and after 45 min stained with Hoechst dye.  Similar to the DIC microscopy (Fig. 3) the only nuclear changes observed  was the increasing prominence of the nucleoli (arrows). Microinjection  experiments were repeated at different doses and times in at least five separate experiments. (D) Cells incubated in GraB (1 μg/ml) and perforin  (60 ng/ml) for 45 min display the typical chromatin condensation pattern  of apoptotic cells (arrows). (E) Control cells incubated in medium.
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Figure 6: Microinjection of GraB into the cytoplasm of B16 melanoma. After GraB (2 μg/ml) (A) or medium (B) injection into B16 cells (injected cells indicated by arrows), the B16 cells were examined by DIC microscopy using image analysis. After 6 min the cell nucleoli became prominent and by 12 min plasma membrane blebbing was noted (arrowheads). No further changes were observed over the next 60 min. Plasma membrane and nuclear changes were transient. (C) Hoechst dye staining of GraB microinjected B16 melanoma cells. A group of cells were injected with GraB (1 μg/ml) and after 45 min stained with Hoechst dye. Similar to the DIC microscopy (Fig. 3) the only nuclear changes observed was the increasing prominence of the nucleoli (arrows). Microinjection experiments were repeated at different doses and times in at least five separate experiments. (D) Cells incubated in GraB (1 μg/ml) and perforin (60 ng/ml) for 45 min display the typical chromatin condensation pattern of apoptotic cells (arrows). (E) Control cells incubated in medium.
Mentions: It was evident from the above experiments that GraB crossed the cell membrane and was found within the cytoplasm in the absence of perforin but produced no obvious damage to the cells. If GraB can enter the target cell but fails to initiate apoptosis then it is possible that it is unable to enter the cell in sufficient quantity on its own to cleave and activate its substrates. Another possibility is that GraB is in some way segregated from the substrates that it uses to initiate apoptosis. To test these hypotheses, we directly microinjected GraB into the cell cytoplasm. B16 melanoma cells, which are highly susceptible to GraB and perforin-induced apoptosis within 2 h, were injected with either GraB (2 μg/ml) or buffer control and observed by DIC microscopy using an Image 1 time lapse image analysis system over a 2–3-h period. We observed changes to both the nucleus and plasma membrane within a few minutes of placing the protease in the cytoplasm. In 4–6 min the nuclear membrane and nucleolus appeared coarse and more prominent compared to control injections (Fig. 6, A and B). The plasma membrane began blebbing starting ∼5–7 min after injection which became pronounced by 8–12 min. However, in no instance did we observe progression to obvious membrane or nuclear disruption. Membrane blebs were usually transient in that they appeared and disappeared quickly. Nuclear changes also did not progress over the observation period. The rate at which the blebbing and nuclear changes occurred was dependent on the concentration of the GraB in the injection buffer. At high concentrations the changes were evident by 4–12 min, while at lower concentrations the effects were delayed to 20 min or longer. The experiment was repeated using the Rat-1 cell, microinjecting GraB at a 10-fold high concentration (20 μg/ml) using an automated injection protocol in which 1.2 × 10−12 ml was injected per cell containing 0.2–0.4 femtograms. Cells were again followed for 2–3 h and no apoptosis was observed.

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