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BCL-2 Family Proteins


1. Bcl-2 Tail Anchor Protein

Bcl-2 is targeted to membranes by a carboxyl-terminal tail-anchor sequence.

Unlike other proteins with carboxyl-terminal tail-anchor sequences Bcl-2 is targeted to both the endoplasmic reticulum and mitochondria. Although the amino acid sequence of Bax suggests that it has a tail-anchor sequence Bax is found in the cytoplasm or loosely associated with intracellular membranes prior to the onset of apoptosis.

To determine the importance of specific targeting of Bcl-2 we generated versions of Bcl-2 targeted exclusively to either the endoplasmic reticulum or mitochondria and then assayed them for prevention of apoptosis due to either ceramide or etoposide.

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2. BCL-CB5 Endoplasmic Reticulum

Bcl-cb5 is targeted to the endoplasmic reticulum. 
Since they are contiguous the protein is also targeted to the outer nuclear membrane.  Bcl-cb5 is a fusion protein containing the cytoplasmic domain of Bcl-2 (blue) and the insertion sequence from the endoplasmic reticulum specific isoform of cytochrome b5 (orange).

Bcl-cb5 prevents ceramide induced apoptosis but not apoptosis induced by etoposide.

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3. BCL-ACTA Mitochondria

Bcl-acta is targeted to mitochondria. 
Bcl-acta is a fusion protein containing the cytoplasmic domain of Bcl-2 (blue) and the insertion sequence from the mitochondria specific insertion sequence from the ActA protein from Listeria (green).




Bcl-acta prevents etoposide induced apoptosis. During etoposide induced apoptosis activation and translocation of Bax to mitochondria results in  loss of cytochrome c from mitochondria.  BCL-ACTA Immunofluorescence Data.

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4. Bcl-2 Prevents Apoptosis due to either Ceramide or Etoposide

Bcl-2 prevents apoptosis due to either ceramide or etoposide.  The experiments outlined on the next few pages show how we discovered that ceramide targets the endoplasmic reticulum while etoposide targets an apoptosis pathway at mitochondria.



To dissect these pathways we used Bcl-2 mutants that were targeted to either the endoplasmic reticulum or mitochondria

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5. Etoposoide

Etoposoide induced Apoptosis.  Etoposide leads to translocation of Bax to mitochondria, activation of Bax and release of cytochrome c from mitochondria into the cytoplasm




Bcl-2 must be located at mitochondria to prevent apoptosis due to etoposide.  Bcl-acta is as effective as wild-type Bcl-2 in preventing etoposide induced apoptosis.  However, Bcl-cb5 is completely ineffective.  Immunofluorescence micrographs demonstrate translocation and activation of Bax.

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6. Ceramide Induced Apoptosis

Ceramide induced Apoptosis.  Ceramide leads to loss of mitochondrial transmembrane potential (DYm). Translocation of Bax to mitochondria, activation of Bax and release of cytochrome c are all late events in this form of apoptosis.  Loss of DYm is illustrated as the thin black lines surrounding the mitochondria. 


Bcl-cb5 is as effective as wild-type Bcl-2 in preventing ceramide induced apoptosis.  Therefore, Bcl-2 located at endoplasmic reticulum prevents loss of DYm due to ceramide.

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7. Etoposide Treated Rat-1 Myc Cells

Etoposide leads to activation of Bax and cytochrome c release from mitochondria prior to loss of mitochondrial transmembrane potential (DYm).

The upper pair of images shows the same two cells stained for either cytochrome c or activated Bax.  The cell on the left stains with for activated Bax but cytochrome c staining is dim because cytochrome c has been released from mitochondria.  The cell with no active Bax (on the right in both pictures) retains cytochrome c.  

In the lower pair of images a cell containing activated Bax retains mitochondrial transmembrane potential and therefore stains with mitotracker and with an antibody specific for activated Bax.  Bcl-acta (Bcl-2 located at mitochondria) prevents etoposide induced apoptosis and blocks release of cytochrome c from mitochondria.  Bcl-cb5 (located at the endoplasmic reticulum) cannot prevent etoposide induced apoptosis.

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8. The Role of Endoplasmic Reticulum in Apoptosis

Ceramide

Ceramide induced Apoptosis. 

Bcl-cb5 prevents loss of mitochondrial transmembrane potential (DYm). Therefore, there must be a signal that comes from the endoplasmic reticulum to the mitochondria that is blocked by Bcl-cb5. We support that the signal is calcium




Mitochondrial transmembrane potential was visualized in cells using mitotracker.  Click here to see some of the data.

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9. Bcl-cb5 Presents Loss of DYm in Response to Ceramide

Ceramide Apoptosis

Ceramide induced Apoptosis.  Bcl-cb5 prevents loss of DYm. 
Neo cells are controls transfected with the empty vector.  Hsp60 is a marker for mitochondria.  Mito is mitotracker.  The intensity of this dye is proportional to transmembrane potential.




Micrographs of Hsp60 show the location of mitochondria.  Mitochondrial transmembrane potential was visualized in the same cells using mitotracker.  The dark cells in the upper right hand corner (arrowheads) indicate loss of DYm.  Bcl-cb5 efficiently prevents loss of DYm therefore only one cell is not mitotracker positive (arrowhead) in the field shown.  Unlike cell-death due to ceramide, exposure to etoposide does not lead to loss of DYm.  Instead etoposide causes activation of Bax and loss of cytochrome c from mitochondria.

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10. Bax Translocates to Mitochondria In Presence of Myc

Bax

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Structure of Bcl - XL & Bax

Structural comparison of anti apoptotic Bcl-XL & pro apoptotic Bax

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12. E-Cadherin

Cadhesion

Trafficking of E-cadherin.

In epithelia, intercellular adhesion is maintained by adherens junctions and desmosomes.  We are studying adhesion via adherens junctions.  The primary molecule responsible for intercellular adhesion is E-cadherin (blue ellipses).  This picture outlines trafficking of E-cadherin to and from the cell surface. 



After E-cadherin is synthesized at the endoplasmic reticulum it binds to either b- or g- catenin (also called plakoglobin) and then transports via the secretory pathway to the cell surface (plasma membrane).  At some point p120 catenin is also added to the complex.  When E-cadherins from adjacent cells bind to each other (binding is calcium dependent) then an adherens junction is established.  During apoptosis trafficking of E-cadherin to the cell surface is lost.  Therefore, as E-cadherin is turned-over cells detach from one another. 

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