Browsing by Author "Viljanen, Samu"

Autophagy is a eukaryotic cellular process where intracellular material is recycled by transporting it in newly formed vesicles to lysosomes for degradation. In normal conditions autophagy supports cellular homeostasis. Different stress conditions can induce autophagy and then it helps the cell to avoid an unnecessary or uncontrolled cell death. RAB proteins are small GTPases that regulate vesicle traffic and fusion events in endocytic and exocytic pathways. RAB24 has recently been shown to participate in autophagy, but there is very little information about how it works at the molecular level. GOSR1 is a Golgi SNARE protein that regulates membrane fusion events, and it has been observed to interact indirectly with RAB24. The participation of GOSR1 in autophagy has not been studied yet. The aim of the study was to find out if RAB24 and GOSR1 colocalize into the same vesicle structures and if they interact within each other. HeLa cells were used as a model organism, and to induce autophagy amino acid starvation was used. For GOSR1 detection a DNA construct was created where GOSR1 was tagged with green fluorescent protein GFP-sequence. Localization was studied with immunofluorescence staining where in addition to RAB24 and GOSR1 also the autophagosomal marker protein LC3 was labeled. The labeled cells were photographed with a confocal microscope. The pictures were analyzed with ImagePro software. Interaction between the proteins was studied using immunoprecipitation. GOSR1 and RAB24 were not observed to colocalize into same structures in significant amount. Instead it was found that GOSR1 colocalized into LC3-positive autophagic vesicles. In immunoprecipitation studies no interaction between RAB24 and GOSR1 could be shown. In order to ensure the results more immunofluorescence stainings should be done using several time points and GFP-tagged GOSR1. Also GOSR1 silencing with siRNA should be used in order to find out if GOSR1 is necessary for autophagy. The immunoprecipitation protocol should be optimized, and the possible interaction could be studied by using other methods, for example yeast-two hybride technology.