Single-cell image analysis of cellular localization and colocalization of endonuclease G, AIF and AMID in living cells
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| Year of publication | 2006 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | We studied the cellular localization and colocalization of proteins AIF, endonuclease G and AMID, which were previously found to be involved in caspase-independent apoptosis. We designed mammalian expression vectors, which carry genes encoding the proteins of interest fused to the fluorescent proteins to study cellular localization and colocalization of these proteins. We predicted the cellular localization of the proteins of interest using software, which analyses the aminoacid sequence of the protein with various algorithms. We also designed and applied a novel method of single-cell image analysis of the translocation of the fluorescent proteins into the nucleus. Using the listed methods, we managed to confirm by software prediction the localization of AIF and endoG to mitochondria. We confirmed experimentally the colocalization of AIF and endoG in the mitochondria of living cells using cotransfection of U-2 OS cells with mammalian expression vectors coding fusions proteins AIF-HcRed-tandem and endoG-EYFP. We also observed and confirmed their translocation from mitochondria to the nucleus during the staurosporine-induced apoptosis by time-lapse microscopy of living cells. Using same methods for protein AMID indicates, that AMID is not localised exclusively on the outer mitochondrial membrane, neither dissolved in the cytoplasm, as it was observed and published. Overexpression of fusion protein AMID-HcRed-tandem didnt have lethal effect on cells or mitochondria as it was also previously described. According to all our predicted and experimental results, AMID is localised to the cytoplasm, but more likely bound to the lipids of the cellular membranes. We did not observe its role in the staurosporine-induced apoptosis, because AMID was not transferred into the nucleus as was endoG and AIF and therefore it could not directly participate in the chromatin degradation process. The possible role of AMID in apoptosis was not observed and our results contribute to the comprehension of localization and function of AMID, AIF, and endoG proteins. |
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