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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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GraB crosses the plasma membrane by an energy-dependent  mechanism. (A) HeLa cells were treated with NaF (1 mM) an inhibitor of  glycolysis and DNP (100 μM) which uncouples oxidative phosphorylation at 4°C or cytochalasin B (Cytoch B) (2 μg/ml) and then incubated  with FITC-GraB (GraB) or GraB and perforin (Perf) for 30 min then analyzed as described in Fig. 2. (B) Cells treated with DNP/NaF at 4°C then  FITC GraB showed fluorescence localized to the plasma membrane in  discreet patches or clumps.
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Figure 4: GraB crosses the plasma membrane by an energy-dependent mechanism. (A) HeLa cells were treated with NaF (1 mM) an inhibitor of glycolysis and DNP (100 μM) which uncouples oxidative phosphorylation at 4°C or cytochalasin B (Cytoch B) (2 μg/ml) and then incubated with FITC-GraB (GraB) or GraB and perforin (Perf) for 30 min then analyzed as described in Fig. 2. (B) Cells treated with DNP/NaF at 4°C then FITC GraB showed fluorescence localized to the plasma membrane in discreet patches or clumps.

Mentions: Next we determined whether GraB transit of the plasma membrane was an active energy-dependent process. FITCGraB was incubated with cells that had been pretreated either with NaF an inhibitor of glycolysis and DNP, which uncouples oxidative phosphorylation, or incubated at 4°C. Pretreatment with NaF and DNP in combination or incubation at 4°C reduced fluorescence by 30–50% in either YAC-1 or HeLa cells (not shown). When cells were treated with both inhibitors at 4°C then fluorescence was reduced by over 80% (Fig. 4 A). Cells examined under these conditions revealed GraB in a patchy plasma membrane distribution, indicating that it had bound to but had not crossed the cell membrane (Fig. 4 B). In earlier studies we had shown that pretreatment of target cells with cytochalasin B, an inhibitor of actin polymerization and microfilament formation (29), blocked apoptosis induced by GraB and perforin (7). When cytochalasin B was incubated with cells, we found that the amount of FITC-GraB that entered the cell was also profoundly suppressed (Fig. 4 A).


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)

GraB crosses the plasma membrane by an energy-dependent  mechanism. (A) HeLa cells were treated with NaF (1 mM) an inhibitor of  glycolysis and DNP (100 μM) which uncouples oxidative phosphorylation at 4°C or cytochalasin B (Cytoch B) (2 μg/ml) and then incubated  with FITC-GraB (GraB) or GraB and perforin (Perf) for 30 min then analyzed as described in Fig. 2. (B) Cells treated with DNP/NaF at 4°C then  FITC GraB showed fluorescence localized to the plasma membrane in  discreet patches or clumps.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196167&req=5

Figure 4: GraB crosses the plasma membrane by an energy-dependent mechanism. (A) HeLa cells were treated with NaF (1 mM) an inhibitor of glycolysis and DNP (100 μM) which uncouples oxidative phosphorylation at 4°C or cytochalasin B (Cytoch B) (2 μg/ml) and then incubated with FITC-GraB (GraB) or GraB and perforin (Perf) for 30 min then analyzed as described in Fig. 2. (B) Cells treated with DNP/NaF at 4°C then FITC GraB showed fluorescence localized to the plasma membrane in discreet patches or clumps.
Mentions: Next we determined whether GraB transit of the plasma membrane was an active energy-dependent process. FITCGraB was incubated with cells that had been pretreated either with NaF an inhibitor of glycolysis and DNP, which uncouples oxidative phosphorylation, or incubated at 4°C. Pretreatment with NaF and DNP in combination or incubation at 4°C reduced fluorescence by 30–50% in either YAC-1 or HeLa cells (not shown). When cells were treated with both inhibitors at 4°C then fluorescence was reduced by over 80% (Fig. 4 A). Cells examined under these conditions revealed GraB in a patchy plasma membrane distribution, indicating that it had bound to but had not crossed the cell membrane (Fig. 4 B). In earlier studies we had shown that pretreatment of target cells with cytochalasin B, an inhibitor of actin polymerization and microfilament formation (29), blocked apoptosis induced by GraB and perforin (7). When cytochalasin B was incubated with cells, we found that the amount of FITC-GraB that entered the cell was also profoundly suppressed (Fig. 4 A).

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