growth factors in drosophila: characterization of two novel families of growth factors, idgfs and...
TRANSCRIPT
Growth factors in Drosophila:
Characterization of two novel families of growth factors,
IDGFs and ADGFs
IDGFs = Imaginal Disc Growth FactorsIDGFs = Imaginal Disc Growth Factors
• secreted glycoproteins
• isolated from conditioned medium, where they are abundantly secreted by various Drosophila cell lines exhibiting macrophage properties (Cl8+, S2, Kc) (Kirkpatrick et al. 1995, Kawamura et al. 1999)
Detection of DS47 secreted by Dm cell lines1 - M3 media + serum2 - supernatant from S2 culture, serum-free3 - supernatant from S2 culture, serum+4 - supernatant from KcO culture, serum-free5 - supernatant from 1006-2 culture, serum+6 - supernatant from 1006-2 M culture, serum+7 - supernatant from Ox736-3 M culture, serum+8 - supernatant from 5697-5 culture, serum+9 - supernatant from 5697-7 culture, serum+
IdgfIdgf genes in genes in Drosophila melanogasterDrosophila melanogaster
~ 55 % seq. similarity at cDNA level~ 50-60 % seq. similarity at aa level
IDGF = Imaginal Disc Growth FactorsIDGF = Imaginal Disc Growth Factors
• IDGF proteins in cooperation with insulin are able to stimulate proliferation, polarization and motility of Cl8+ cells in culture (Kawamura et al.1999 – recombinant IDGF1 and 2)
Cl8+ in CM
Human homolog HC-gp39Human homolog HC-gp39
• stimulate proliferation of fibroblasts and synovial cells in culture (Recklies et al.2002)
• in vivo is connected with differentiation of peripheral macrophages (Kirkpatick et al. 2007)
• is highly expressed in human body during inflammation (autoimmunity deseases like arthritis, final stage of cancer, viral infections)
IDGFs probably act as lectinsIDGFs probably act as lectins
• binding domain is slighly modified
• Do they bind chitin or different oligosaccharide motive?
A – IDGF2 binding domainB – Chitinase A binding domainC – Ym1 binding domain (murine homolog of insect IDGFs)Varela et al. 2002
IDGFs can be specialized to different functionIDGFs can be specialized to different function
Western blot (Bryant and Zurovec)
2 main groups: IDGFs with constitutive and induced expression
IDGFs 1-3 were identified IDGFs 1-3 were identified as a part of as a part of DrosophilaDrosophila immune response immune response
(DeGregorio et al. 2001, 2002, Irving et al. 2001, Levy et al. 2004,…)
• various MA and proteomic analysis identified IDGF 1-3 as a part of Drosophila immune response in both larvae and adults
• IDGF 1 and 3 are induced with septic injury
• IDGF3 expression is affected with Toll and Imd pathway, however IDGF1 expression is independent on both of them.
• IDGF2 expression is upregulated early after Gram-positive bacterial infection as well as after infection with yeasts.
• All IDGFs were found as a part of haemolymphal clots
17650 17660
ADG F-D ADG F-C CG 9599
9460 9465 9470
ADG F-E
ADA CG 18005
P-EP
P-EP
3L;74E2
3R;87F12
2R;51C1
3R;85C7
ADG F-A ADG F-B
96409635
4870 4872
M SI
ADA-RELATED GENES IN DROSOPHILA
The effect of recombinant ADGF-A on the growth and morphology of the Cl.8+ imaginal disc cells
Dying cells Living cells
It is possible to mimic the effect of ADGFAon cell growth by removal of adenosine
The mutant ADGF has no effect
Expression of different ADGFs ADGF-A is expressed in embryo, larva and adult (arrows)
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Gene targeting by homologous recombination
Mutagenesis of the gene ADGF-A17650 17660
3L;74E2
ADGF-A ADGF-B
MSI
17650 17660
ADGF-A ADGF-B
MSI ADGF-B
MSI ADGF-B
MSI
ADGF-A
3L;74E2
2L;
ADGF-A White
WT
The ADGFA mutant
A rescue construct
Stop kódón
Tests for somatic mutagenesis inDrosophila based on a recessive mutation in a marker gene (left), or a mutant tumor suppressor gene (right)
y = yellow, yellow bristles instead of black normal ones;wts = warts, warts-like patches of overgrown tissue
Heterozygous wild type cells at four-chromatide stage
Mutagen Mutagen
Tumor cell Normal cell
Clone with yellow bristles
Tumorous clone
Wild type homozygousclone
Surrounding heterozygous tissue, phenotypically normal