anti-DAAMR4 (RB)
wt 8.1 8.1
anti-glycogen phosphorylase (Rb) loading control
anti-FlagM2 (M)
wt wt 8.1 8.1
100 kDa 130 kDa
Identification of the molecular interaction partners of the formin dDAAM
Ede Migh, Rita Gombos, Andor Udvardy , Imre Molnár and József Mihály
Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
The formin proteins are an important and evolutionarily well conserved class of actin binding proteins with essential biological functions, including cell division, cell migration and organelle transport. In these processes the best understood molecular role of formins is to promote the nucleation and elongation of unbranched actin filaments, although some formins have also been implicated in the regulation of microtubules. We have previously shown that the single Drosophila DAAM ortholog, dDAAM, is involved in multiple aspects of trachea development and axonal growth regulation, however the molecular mechanisms underlying these morphogenetic functions remain to be uncovered. To gain a better understanding of the molecular functions of dDAAM, we aim to identify the protein interaction partners of dDAAM with biochemical and genetic methods. The biochemical interaction partners is aimed to be identified by affinity chromatography.
To this end, we created a dDAAM-Flag fusion protein by tagging the dDAAM gene in situ by site specific mutagenesis. To complement the biochemical approach, we also aim to identify interaction partners by a genetic interaction screen using the hypomorphic dDAAMEX1 allele exibiting a moderately strong axonal growth phenotype in the adult brain.
Background
Results
1 In situ Flag tagging of the dDAAM gene with P-element mediated gene conversion
Gal4
dDAAM coding 300 300 Flag 3’UTR Flanking region
I-SceI I-SceI
5’UTR dDAAM coding 300 Gal4 300 Flag 3’UTR Flanking region
I-SceI I-SceI
Gene conversion
5’UTR dDAAM coding 3’UTR Flanking region
P-element remobilization:
double strandbreak induction P
I-SceI digestion and recombination
5’UTR dDAAM coding Flag 3’UTR Flanking region
2 Immunohistochemical analyses of dDAAM
Flag3 Biochemical analysis of dDAAM
Flagstrain 8.1
The dDAAM protein is highly enriched in the neuropile region of the adult brain, including the mushroom body (arrows on a). We found that the hypomorphic dDAAMEx1 mutant allele displays axonal projection defects in the mushroom body (b). The moderately strong penetrance (c) of these defects made it an ideal tool for a dominant genetic interaction assay that aims to identify the suppressor and enhancer mutations of dDAAMEx1.
Identification of dDAAM interaction partners by a genetic approach
Bloomington Df kit
Bloomington Df kit
UAS-mCD8GFP
Df ; OK107-Gal4 +
UAS-mCD8GFP
SM6b ; OK107-Gal4
dDAAM Ex1 ; UAS-mCD8GFP
SM6b ; OK107-Gal4 dDAAM Ex1 /Y ; UAS-mCD8GFP
Df ; OK107-Gal4 +
a-dDAAM staining: dDAAMEx1 adult mushroom body phenotypes:
FasII
a
4
b
c
wild type shorter lobe thinner lobe thicker lobe lack of lobe misprojection
dDAAMEx1/Y
dDAAMEx1/Y dDAAMEx1/dDAAMEx1
dDAAMEx1/dDAAMEx1
a lobes
b lobes
354 mutants crossed and evaluated 79 potential interactor regions
65 enhancers 14 supressors
proportion of defective lobes is decreased ≥ 20%
proportion of defective lobes is
increased ≥ 20%
50 brains were dissected for the interaction screen per mutation
(overall approx. 17700 brains)
Genetic interaction screen with the Bloomington Deficiency kit
5
UAS-mCD8GFP
+ ; OK107-Gal4 +
;
UAS-mCD8GFP
+ ; OK107-Gal4 +
dDAAM Ex1 /Y
controls:
Interaction screen:
For the genetic screen we used a mushroom body specific driver (OK107-Gal4) to express the mCD8GFP protein allowing the analysis of the mutant brains without antibody staining. We identified 79 potential interactor deficiencies (14 supressors and 65 enhancers). Two overlapping enhancer deficiencies (Df(2L)ED334 and (Df(2L)ED385) uncover the chic gene that is a known dDAAM interacting partner, and therefore this observation nicely validates our screen. The mapping of the other interacting regions is still in progress.
The OK107-Gal4 (mushroom- body specific) driver activates mCD8GFP (green) expression in the Kenyon cells.
Df(2L)ED334 and Df(2L)ED385 overlap in
the 26B2-26D7 region, that uncovers chic encoding Drosophila
profilin
Conclusions
The dDAAM chromosomal region
The structure of the donor construct
The dDAAMFlag mutant
A’
dDAAM
A”
Actin dDAAM
A
Actin
ventral nerve cord
Actin
B
dDAAM
B’
C C’ C”
Flag dDAAM Flag dDAAM
Figure 2. Confocal images of stage 16 dDAAMFlag embryos. The dDAAM protein can be detected in the prominent dDAAM expression domains, such as the heart tube (arrow in A’ and A”), the dorsal trunk (arrowhead in A’ and A”) and the ventral nerve cord (B and B’) demonstrating that the Flag tagged protein is expressed very similarly to wild type dDAAM. C-C” shows a double staining with anti-Flag (in red) and anti-dDAAM (in green), note that the two stainings overlap almost completely.
Figure 1. A P-element insertion, that is located in dDAAM flanking region, is remobilizied by the ∆2-3 protein. This process induces DNA double stranded breaks, that facilitates the integration of the donor construct into the fly genome in the vicinity of the break by activating the DNA repair mechanisms. The integration can be detected with the help of the Gal-4 marker gene. Subsequently, I-SceI can be used to induce double stranded breaks again to promote the recombination of two homologous (300 nucleotides long) sequences of the donor construct leading to Gal-4 excision and C-term Flag taging of dDAAM.
a
b
c
1h 2h 4h 8h 12h 16h anti-FlagM2 (M)
8.1 8.1 8.1 8.1 8.1 8.1
130 kDa
100 kDa anti-glycogen phosphorylase (Rb)
loading control 100 kDa
130 kDa
head head LP EP L3 L2 L1 anti-FlagM2 (M)
wt 8.1 8.1 8.1 8.1 8.1 8.1 EP: early pupa LP: late pupa
L3: 3. stadium of larva
embryo
Figure 3. Immunoblot experiments of dDAAMFlag (a) We compared the dDAAM protein level in wild type and dDAAMFlag 8.1 mutant Drosophila brains. We revealed no difference in the expression level as compared to wild type type. (b) Comparison of dDAAMFlag expression in different tissues and developmental stages. The most abundant protein level is found in head and 4, 8, 12 hours embryos. (c) Subcellular fractionation with sucrose gradient of the plasmamembrane (HSP), nuclear (LSP) and cytoplasmic (HSS) cpmpartments by centrifugation from protein extracts prepared from Drosophila heads. The highest level of dDAAM protein is found in the HSP fraction. These results suggest that most part of the dDAAM protein is membrane associated.
1. We generated a Drosophila strain (dDAAMFlag) in which we inserted a Flag tag at the C- terminus of the protein in situ.
2. Localization of the dDAAM-Flag protein is identical to that of the wild type protein because it is strongly expressed in the trachea, heart tube and ventral nerve cord.
3. Western-blot experiments demonstrate that the dDAAM-Flag protein is expressed at wild type levels.
4. The largest amount of dDAAM-Flag can be found in the head, and it apperas to be membrane associated. We will aim to purify the dDAAM containing protein complex from head extraxts in near future.
5. We established a genetic interaction assay suitable for the identification of dDAAM interaction partners by using the adult Drosophila brain.
6. In this screen we identified 79 potential interactor deficiencies whose mapping is in progress
TÁMOP-4.2.2/B-10/1-2010-0012 projekt
high-speed pellet (HSP)
O T O T O T O T
O: detergent free
T: 0,5% Triton X-100 130 kDa
160 kDa
high-speed supernatant (HSS)
2.000x g, 15 min
„nuclear fraction”
(LSP)
post-nuclear supernatant
fractions lysate
100.000x g, 1h
100.000x g, 16h
fractions (1-21)
Total extract Pellet
130 kDa
160 kDa suspended HSP with
sucrose gradient
