Transcript Slide 1

Characterization of Apoptosis Inducing Factor (AIF) in
the Drosophila Visual System
Zachary Lemmon and Joseph E. O’Tousa. Biological Sciences, University of Notre Dame, Notre Dame, IN.
Introduction
We are investigating the localization and apparent activity of
Apoptosis Inducing Factor (AIF) in both wild type
photoreceptors and photoreceptors undergoing age dependent
retinal degeneration.
AIF is a phylogenically ancient
flavoprotein that localizes to mitochondria with an amino
terminal mitochondrial localization sequence (MLS) consisting
of the first 162 amino acids of the protein. Mature protein is
formed by proteolytic cleavage of the MLS in mitochondria
(Cande et al. 2002). AIF has putative roles in electron transfer
and apoptotic processes. Electron transport activities of AIF
are conducted by FAD-binding and NADH-binding domains
(aa 184-540). Upon cellular stress, AIF is released from the
mitochondria and relocated to the nucleus, where it promotes
chromatin alteration and cell death through an unknown
mechanism that may involve direct DNA interaction or
recruitment of various factors such as nucleases (Mate et al.
2002). The mechanism is speculated to involve exposed
positively charged residues in the protein and an AIF specific
insertion of 50 amino acids in the carboxy terminal domain (aa
572-622) (Cande et al. 2002). Previous work on AIF focuses
on mammalian cell culture (Cande et al. 2002), biochemical
(Mate et al. 2002), and mouse AIF models (Cheung et al.
2006). Several of these studies have shown AIF can be
redirected to the nucleus and trigger apoptosis by removal of
the amino MLS. To study AIF function in adult Drosophila
photoreceptors, three mRFP-tagged AIF transgenic strains (full
length AIF, AIF amino terminal domain, and AIF carboxy
terminal domain) were generated. As predicted by previous
studies, we show that the amino terminal domain and full
length constructs specify mitochondrial localization in wild type
Drosophila photoreceptors. In wild type photoreceptor cells,
the carboxy terminal AIF construct failed to localize to
mitochondria. We also used a recessive lethal AIF piggyback
insertion allele (CG7263e04281) in a genetic mosaic to study the
effect of AIF loss of function on photoreceptor development
and degeneration.
The Action of Apoptosis Inducing
Factor (AIF)
Ommatidial Whole Mount Fluorescent Microscopy
Figure 3: Localization of cellular
structures in photoreceptors. A
single ommatidium composed of eight
photoreceptors is prepared by manual
dissection of the retina and mounted
on standard microscope slides with
0.5% paraformaldehyde fix and DAPI
mounting
media.
Fluorescent
microscopy is then used to view the
localization of various tagged proteins
in different cellular structures. This
type of preparation allows for
colocalization studies using multiple
proteins with GFP, RFP, or other
fluorescent tags.
Amino Terminal AIF-RFP Sufficient for Mitochondrial
Localization
4a
4b
Retinal Structure Disrupted by
AIF Loss of Function
FRT
CG7263e04281
7a
SM1, Cy
AIF insertion
heterozygotes
have normal eye
structure.
4c
7b
FRT CG7263e04281
GMR-Hid 40A FRT
AIF insertion
homozygotes
(eyFLP retina
mosaic) disrupt
retinal
development.
<mitoGFP>
<AIF N-term-RFP>
DAPI (nuclei)
<mitoGFP>
<AIF N-term-RFP>
Figure 4: Amino terminal AIF specifies mitochondrial localization. The green fluorescent
image shows mitochondria in the photoreceptor cells marked by a GFP tagged protein (panel 4a).
A complementary red fluorescent image shows similar expression of the mRFP tagged AIF
construct (panel 4b). A merged photograph combining the green, red, and blue (DAPI) fluorescent
images shows strong colocalization of the amino terminal AIF construct and mitochondrial GFP
marker (panel 4c). This shows that the amino terminal AIF construct, consisting of the MLS, is
sufficient for mitochondrial localization.
7c
SM1, Cy
GMR-Hid 40A FRT
GMR-Hid, in nonmosaic flies,
eliminates
eye tissues
Full Length AIF-RFP Localizes to Mitochondria
Figure 5: Full length
AIF construct suggests
mitochondrial
localization. The AIF full
length transgene shows
a punctate expression
pattern similar to amino
terminal AIF, suggesting
full length AIF also
localizes to mitochondria.
There are no green
fluorescent images due
to complications with the
cross.
<AIF Full Length-RFP>
Carboxy Terminal AIF-RFP does not
localize to the mitochondria
Figure 1: AIF Structure and Function. The 162 amino acid N-terminal
mitochondrial localization sequence directs AIF to mitochondria. Cleavage
of the N-terminal sequence results in the active form of AIF responsible for
electron transport (FAD/NADH-Binding Domains) and apoptotic (Cterminus) functions.
Upon apoptotic stress, permeabilization of the
mitochondrial membrane occurs and causes AIF release and relocalization
to the nucleus, where it takes part in an unknown apoptotic mechanism.
Investigation of AIF in Drosophila
Photoreceptor Cells
6a
Figure 6: Localization of the
Carboxy Terminal AIF-RFP
construct.
with
a
mitochondrial GFP marker.
Localization of the carboxy
terminal AIF construct was
expected to be nuclear and
cytosolic.
Fluorescent
pictures of the ommatidial
bundles show the expected
punctate green fluorescence
(panel
6a)
for
the
mitochondrial GFP marker.
Red fluorescent pictures of
the carboxy terminal AIF
construct show a weak signal
(panel 6b) that could be
indicative
of
cytosolic
localization, but there is no
indication
of
nuclear
localization. A merged image
(panel 6c) of the green, red,
and blue (DAPI) signal clearly
shows carboxy AIF does not
localize to the mitochondria.
<mitoGFP>
6b
<AIF C-term-RFP>
6c
Figure 2: AIF transgene constructs. Three AIF constructs were produced
using standard PCR cloning methods and the Drosophila Gateway Cloning
System. The constructs consisted of a UAS promoter, AIF specific
sequence, and carboxy terminal mammalian RFP tag. A mitochondrial GFP
tagged protein, mitoGFP (A. Pilling and B. Saxton), was used for the
purpose of colocalization and was also under control of the UAS promoter.
Expression of RFP and GFP tagged proteins was specifically driven in adult
photoreceptor cells with GAL4 under control of the rhodopsin promoter
(Rh1).
<AIF C-term-RFP>
<mitoGFP>
DAPI (nuclei)
Figure 7: Eye phenotype observed in AIF mutant. CG7263e04281
is a PBac element insertion in the second intron of AIF. This creates
a loss of function homozygous lethal mutation. For this reason,
FLP/FRT site somatic recombination was used to study the effect of
AIF loss of function in the adult retina. Genetic mosaic flies (panel
7b) showed reduced eye size and a rough eye phenotype in
comparison to an FRT CG7263e04281 control (panel 7a). A GMR-Hid
eye (panel 7c) is shown for comparison.
Conclusions
 The amino terminal domain of AIF is
sufficient
to
target
the
protein
into
mitochondria.
 The AIF full length-RFP gene construct also
is correctly targeted to mitochondria. This
reagent now puts us in position to assess the
movement of AIF during the retinal disease
process.
 Carboxy terminal AIF does not localize to
mitochondria, nor predominately to the nucleus
as predicted by mammalian cell culture work.
Currently we are investigating its role as a
trigger of retinal degeneration.
 Altered eye morphology in AIF genetic
mosaics indicates a cellular defect, likely
impacting mitochondrial function, due to AIF
mutation.
Works Cited
•Cande, Celine, F. Cecconi, P. Dessen, and G. Kroemer. 2002. Apoptosis-inducing
factor (AIF): key to the conserved caspase-independent pathways of cell death?
Journal of Cell Science, 115: 4727-4734.
•Cande, Celine. I. Cohen, E. Daugas, L. Ravagnan, N. Larochette, N. Zamzami,
and G. Kroemer. 2002. Apoptosis-inducing factor (AIF): a novel caspaseindependent death effector released from mitochondria. Biochimie, 84: 215-222.
•Mate, Maria. M. Ortiz-Lombardia, B. Boitel, A. Haouz, D. Tello, S. Susin, J.
Penninger, G. Kroemer, and P. Alzari. 2002. The crystal structure of the mouse
apoptosis-inducing factor AIF. Nature Structural Biology, 9 (6): 442-446.
•Cheung, Eric. N. Joza, N. Steenaart, K. McClellan, M. Neuspiel, S. McNamara, J.
MacLaurin, P. Rippstein, D. Park, G. Shore, H. McBride, J. Penninger, and R.
Slack. 2006. Dissociating the dual roles of apoptosis-inducing factor in maintaining
mitochondrial structure and apoptosis. The EMBO Journal, 25 (17): 4061-4073.