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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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(A) Intracellular fluorescence of cells treated with FITCGraB in the presence and absence of perforin. HeLa cells are shown 30  min after incubation with FITC-GraB (1 μg/ml). The fluorescence is distributed diffusely throughout the cytoplasm and a shadow indicating  lower levels is seen over the nucleus. After treatment with FITC-GraB  and perforin for 30 min both HeLa cells (B) or YAC-1 cells (C) showed a  dramatic redistribution with the FITC-GraB now seen in the nucleus  with some cytoplasmic staining. In HeLa cells (B) the fluorescence was  distributed around the nuclear membrane and in localized areas in the nucleus while in YAC-1 cells (C) the fluorescence was detected in areas of  condensed chromatin or in apoptotic bodies. YAC-1 cells were treated  with GraB and perforin in the presence of rhodamine-conjugated transferrin (D). Transferrin was not detected in the nucleus in cells undergoing  apoptosis (arrow). Cells from GraB and perforin treatment in the presence  of rhodamine-conjugated transferrin shown in D were stained with Hoechst  dye to demonstrate the condensed chromatin in the apoptotic nucleus  (E). The experiments described above were repeated at least three times  with each target cell.
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Figure 2: (A) Intracellular fluorescence of cells treated with FITCGraB in the presence and absence of perforin. HeLa cells are shown 30 min after incubation with FITC-GraB (1 μg/ml). The fluorescence is distributed diffusely throughout the cytoplasm and a shadow indicating lower levels is seen over the nucleus. After treatment with FITC-GraB and perforin for 30 min both HeLa cells (B) or YAC-1 cells (C) showed a dramatic redistribution with the FITC-GraB now seen in the nucleus with some cytoplasmic staining. In HeLa cells (B) the fluorescence was distributed around the nuclear membrane and in localized areas in the nucleus while in YAC-1 cells (C) the fluorescence was detected in areas of condensed chromatin or in apoptotic bodies. YAC-1 cells were treated with GraB and perforin in the presence of rhodamine-conjugated transferrin (D). Transferrin was not detected in the nucleus in cells undergoing apoptosis (arrow). Cells from GraB and perforin treatment in the presence of rhodamine-conjugated transferrin shown in D were stained with Hoechst dye to demonstrate the condensed chromatin in the apoptotic nucleus (E). The experiments described above were repeated at least three times with each target cell.

Mentions: Examination of the distribution of fluorescence in FITCGraB–treated cells, showed the most intense fluorescence in the cytoplasm with a shadow evident over the nucleus suggesting that the granzyme had not crossed the nuclear membrane (Fig. 2, A and B). However, in perforin- and GraB-treated cells the fluorescence pattern had shifted dramatically. Here we observed that the nucleus of cells that were undergoing apoptosis now stained strongly for FITCGraB (Fig. 2, B and C ). In HeLa cells, after 90 min of FITCGraB and perforin treatment, staining was evident around the nuclear membrane with localized areas of fluorescence in the nucleus (Fig. 2 B). In YAC-1 cells treated in the same way, intense staining was evident in areas of chromatin margination and in apoptotic bodies with fainter residual staining in the cytoplasm (Fig. 2 C ). To determine if proteins other than FITC-GraB cross the nuclear membrane as a result of apoptosis induced damage, we examined the fate of rhodamine-conjugated transferrin administered to cells with GraB and perforin. Under these conditions, transferrin was excluded from the nucleus (Fig. 2 D). Cells were also analyzed after treatment with different doses of FITC-GraB ranging from 62.5 to 1,000 ng/ml in the presence or absence of perforin and showed the identical staining pattern but with proportionately lower levels of fluorescence (not shown).


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)

(A) Intracellular fluorescence of cells treated with FITCGraB in the presence and absence of perforin. HeLa cells are shown 30  min after incubation with FITC-GraB (1 μg/ml). The fluorescence is distributed diffusely throughout the cytoplasm and a shadow indicating  lower levels is seen over the nucleus. After treatment with FITC-GraB  and perforin for 30 min both HeLa cells (B) or YAC-1 cells (C) showed a  dramatic redistribution with the FITC-GraB now seen in the nucleus  with some cytoplasmic staining. In HeLa cells (B) the fluorescence was  distributed around the nuclear membrane and in localized areas in the nucleus while in YAC-1 cells (C) the fluorescence was detected in areas of  condensed chromatin or in apoptotic bodies. YAC-1 cells were treated  with GraB and perforin in the presence of rhodamine-conjugated transferrin (D). Transferrin was not detected in the nucleus in cells undergoing  apoptosis (arrow). Cells from GraB and perforin treatment in the presence  of rhodamine-conjugated transferrin shown in D were stained with Hoechst  dye to demonstrate the condensed chromatin in the apoptotic nucleus  (E). The experiments described above were repeated at least three times  with each target cell.
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Figure 2: (A) Intracellular fluorescence of cells treated with FITCGraB in the presence and absence of perforin. HeLa cells are shown 30 min after incubation with FITC-GraB (1 μg/ml). The fluorescence is distributed diffusely throughout the cytoplasm and a shadow indicating lower levels is seen over the nucleus. After treatment with FITC-GraB and perforin for 30 min both HeLa cells (B) or YAC-1 cells (C) showed a dramatic redistribution with the FITC-GraB now seen in the nucleus with some cytoplasmic staining. In HeLa cells (B) the fluorescence was distributed around the nuclear membrane and in localized areas in the nucleus while in YAC-1 cells (C) the fluorescence was detected in areas of condensed chromatin or in apoptotic bodies. YAC-1 cells were treated with GraB and perforin in the presence of rhodamine-conjugated transferrin (D). Transferrin was not detected in the nucleus in cells undergoing apoptosis (arrow). Cells from GraB and perforin treatment in the presence of rhodamine-conjugated transferrin shown in D were stained with Hoechst dye to demonstrate the condensed chromatin in the apoptotic nucleus (E). The experiments described above were repeated at least three times with each target cell.
Mentions: Examination of the distribution of fluorescence in FITCGraB–treated cells, showed the most intense fluorescence in the cytoplasm with a shadow evident over the nucleus suggesting that the granzyme had not crossed the nuclear membrane (Fig. 2, A and B). However, in perforin- and GraB-treated cells the fluorescence pattern had shifted dramatically. Here we observed that the nucleus of cells that were undergoing apoptosis now stained strongly for FITCGraB (Fig. 2, B and C ). In HeLa cells, after 90 min of FITCGraB and perforin treatment, staining was evident around the nuclear membrane with localized areas of fluorescence in the nucleus (Fig. 2 B). In YAC-1 cells treated in the same way, intense staining was evident in areas of chromatin margination and in apoptotic bodies with fainter residual staining in the cytoplasm (Fig. 2 C ). To determine if proteins other than FITC-GraB cross the nuclear membrane as a result of apoptosis induced damage, we examined the fate of rhodamine-conjugated transferrin administered to cells with GraB and perforin. Under these conditions, transferrin was excluded from the nucleus (Fig. 2 D). Cells were also analyzed after treatment with different doses of FITC-GraB ranging from 62.5 to 1,000 ng/ml in the presence or absence of perforin and showed the identical staining pattern but with proportionately lower levels of fluorescence (not shown).

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