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