Limits...
Movement of Bax from the cytosol to mitochondria during apoptosis.

Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ - J. Cell Biol. (1997)

Bottom Line: Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol.The diffuse localization of GFP-Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL.These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Section, Surgical Neurology Branch, Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP-Bcl-2 and GFP-Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP-Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP-Bax is a soluble protein, in contrast to organelle-bound GFP-Bcl-2. The diffuse localization of GFP-Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP-Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP-Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP-Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

Show MeSH

Related in: MedlinePlus

GFP–Bax distribution in Cos-7 cells is not affected by coexpression of  Bcl-2. Cells were transiently  transfected with a 3:1 ratio of  plasmid containing Bcl-2 and  GFP–Bax, and examined 48 h  later by confocal microscopy.  (A) In Cos-7 cells cotransfected with Bcl-2 and GFP– Bax, the GFP–Bax distributes  throughout the cell in a pattern that is indistinguishable  from that of cells expressing  GFP–Bax alone. (B) The coexpression of GFP–Bax and  human Bcl-2 in Cos-7 cells  was monitored by Western  blotting with α human Bax  2D2 and α human Bcl-2 8C8  monoclonal antibodies. Lanes  a are the Cos-7 cell lysates from transfection with the pcDNA 3 vector alone. Lanes b are the cell lysates from the pcDNA 3–Bcl-2 transfection, and lanes c are from the cotransfection of C3–EGFP-Bax and pcDNA 3–Bcl-2. Arrows a, b, and c represent the GFP–Bax, its  NH2-terminal translational variant, and endogenous simian Bax, respectively. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140220&req=5

Figure 7: GFP–Bax distribution in Cos-7 cells is not affected by coexpression of Bcl-2. Cells were transiently transfected with a 3:1 ratio of plasmid containing Bcl-2 and GFP–Bax, and examined 48 h later by confocal microscopy. (A) In Cos-7 cells cotransfected with Bcl-2 and GFP– Bax, the GFP–Bax distributes throughout the cell in a pattern that is indistinguishable from that of cells expressing GFP–Bax alone. (B) The coexpression of GFP–Bax and human Bcl-2 in Cos-7 cells was monitored by Western blotting with α human Bax 2D2 and α human Bcl-2 8C8 monoclonal antibodies. Lanes a are the Cos-7 cell lysates from transfection with the pcDNA 3 vector alone. Lanes b are the cell lysates from the pcDNA 3–Bcl-2 transfection, and lanes c are from the cotransfection of C3–EGFP-Bax and pcDNA 3–Bcl-2. Arrows a, b, and c represent the GFP–Bax, its NH2-terminal translational variant, and endogenous simian Bax, respectively. Bar, 20 μm.

Mentions: L929 murine fibrosarcoma and Cos-7 green monkey renal epithelia cell lines (American Type Culture Collection, Rockville, MD) were grown in Minimum essential medium, Eagle's in Earle's balanced salt solution and DME, respectively, each supplemented with 2 mM glutamine, 1× nonessential amino acids, 2.5 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ ml streptomycin (all from Biofluids Inc., Rockville, MD), 50 μg/ml gentamicin (GIBCO BRL), and 10% heat-treated FCS. Cells were cultured at 37°C in 5% CO2 and then split twice weekly using 0.05% trypsin/0.02% versene (Biofluids Inc.). In preparation for transfection, cells were plated at a density of 2 × 105 cells per well in six-well tissue-culture plates (Becton Dickinson Labware, Bedford, MA). Either one (Cos-7) or two (L929) days later, cells were transiently transfected using the cationic lipid LipofectAmine (GIBCO BRL) as described by the manufacturer, using 2 μg of plasmid DNA and either 8 (L929) or 10 μl (Cos-7) of LipofectAmine per well. After 5 h in serum-free medium, an equal volume of medium containing 2× normal FCS was added. 1 d after transfection, the cells were washed once and placed in normal growth media. Transfected cells were then cultured for an additional 24 h, and visualized by microscopy. For cotransfection experiments (see Fig. 7), the above procedure was followed, except that a total of 4 μg of plasmid DNA was used per well: 1 of C3–EGFP–Bax and 3 of either pcDNA3–Bcl-2 or pcDNA3–Bcl-XL.


Movement of Bax from the cytosol to mitochondria during apoptosis.

Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ - J. Cell Biol. (1997)

GFP–Bax distribution in Cos-7 cells is not affected by coexpression of  Bcl-2. Cells were transiently  transfected with a 3:1 ratio of  plasmid containing Bcl-2 and  GFP–Bax, and examined 48 h  later by confocal microscopy.  (A) In Cos-7 cells cotransfected with Bcl-2 and GFP– Bax, the GFP–Bax distributes  throughout the cell in a pattern that is indistinguishable  from that of cells expressing  GFP–Bax alone. (B) The coexpression of GFP–Bax and  human Bcl-2 in Cos-7 cells  was monitored by Western  blotting with α human Bax  2D2 and α human Bcl-2 8C8  monoclonal antibodies. Lanes  a are the Cos-7 cell lysates from transfection with the pcDNA 3 vector alone. Lanes b are the cell lysates from the pcDNA 3–Bcl-2 transfection, and lanes c are from the cotransfection of C3–EGFP-Bax and pcDNA 3–Bcl-2. Arrows a, b, and c represent the GFP–Bax, its  NH2-terminal translational variant, and endogenous simian Bax, respectively. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140220&req=5

Figure 7: GFP–Bax distribution in Cos-7 cells is not affected by coexpression of Bcl-2. Cells were transiently transfected with a 3:1 ratio of plasmid containing Bcl-2 and GFP–Bax, and examined 48 h later by confocal microscopy. (A) In Cos-7 cells cotransfected with Bcl-2 and GFP– Bax, the GFP–Bax distributes throughout the cell in a pattern that is indistinguishable from that of cells expressing GFP–Bax alone. (B) The coexpression of GFP–Bax and human Bcl-2 in Cos-7 cells was monitored by Western blotting with α human Bax 2D2 and α human Bcl-2 8C8 monoclonal antibodies. Lanes a are the Cos-7 cell lysates from transfection with the pcDNA 3 vector alone. Lanes b are the cell lysates from the pcDNA 3–Bcl-2 transfection, and lanes c are from the cotransfection of C3–EGFP-Bax and pcDNA 3–Bcl-2. Arrows a, b, and c represent the GFP–Bax, its NH2-terminal translational variant, and endogenous simian Bax, respectively. Bar, 20 μm.
Mentions: L929 murine fibrosarcoma and Cos-7 green monkey renal epithelia cell lines (American Type Culture Collection, Rockville, MD) were grown in Minimum essential medium, Eagle's in Earle's balanced salt solution and DME, respectively, each supplemented with 2 mM glutamine, 1× nonessential amino acids, 2.5 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ ml streptomycin (all from Biofluids Inc., Rockville, MD), 50 μg/ml gentamicin (GIBCO BRL), and 10% heat-treated FCS. Cells were cultured at 37°C in 5% CO2 and then split twice weekly using 0.05% trypsin/0.02% versene (Biofluids Inc.). In preparation for transfection, cells were plated at a density of 2 × 105 cells per well in six-well tissue-culture plates (Becton Dickinson Labware, Bedford, MA). Either one (Cos-7) or two (L929) days later, cells were transiently transfected using the cationic lipid LipofectAmine (GIBCO BRL) as described by the manufacturer, using 2 μg of plasmid DNA and either 8 (L929) or 10 μl (Cos-7) of LipofectAmine per well. After 5 h in serum-free medium, an equal volume of medium containing 2× normal FCS was added. 1 d after transfection, the cells were washed once and placed in normal growth media. Transfected cells were then cultured for an additional 24 h, and visualized by microscopy. For cotransfection experiments (see Fig. 7), the above procedure was followed, except that a total of 4 μg of plasmid DNA was used per well: 1 of C3–EGFP–Bax and 3 of either pcDNA3–Bcl-2 or pcDNA3–Bcl-XL.

Bottom Line: Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol.The diffuse localization of GFP-Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL.These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Section, Surgical Neurology Branch, Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP-Bcl-2 and GFP-Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP-Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP-Bax is a soluble protein, in contrast to organelle-bound GFP-Bcl-2. The diffuse localization of GFP-Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP-Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP-Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP-Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

Show MeSH
Related in: MedlinePlus