Investigation Function Of Cytochrome C During Apoptosis Of Dictyostelium Discoideum

Dictyostelium discoideum which we usually called"social amoebae" is a simple eukaryote and phagocytosis soil bacteria for food. Under favorable condition, it multiplies as a unicellular organism which propagation by binary fission. Upon starvation, a multi-cellular structure was formed by aggregation of thousands of single cells which differentiate gradually into two types of cells called prestalk cells and prespore cells. The pathway involving aggregation, mound, slug, culmination stages induces the formation of a fruiting body consisting of a head of spores supported on a stalk of vacuolated cells. Spores await dispersal and germinate in favourable conditions for amoeboid growth. However, the formation of stalk cells is a process of cell death.So it is a combined course of differentiation and apoptosis.Current evidences suggest that cytochrome c not only take part in energy producing for the cell, but also play a crucial role in cell differentiation and cell apoptosis. It induce apoptosis by activates apaf-1(apoptotic protease activating factor 1) and caspase-9 which stimulate the caspase-3 and the following chemical and morphological appearance of apoptosis. However it's fairly know and barely reported the role of cytochrome c in apoptosis of prestalk cells in Dictyostelium.KAx-3 cells (wild type) and AK127 cells (mutant type) were taken as experimental materials and the mitochondria were got at different developmental stages. Appearance of aggregation, mound, slug, culmination stages was observed in KAx-3 cells. However, the mutant-type strain AK127 undergoes normal chemotaxis, but is arrested at the loose aggregate stage. AK127 cells eventually dissociate into single cell. In addition, cell-type differentiation is blocked in these mutant cells.The methods of western blot and Semi-quantitative evaluation RT-PCR were used to detect the changes of quantity and expression level of cytochrome c between prestalk cells and prespore cells at 16h,18h,20h of multi-cellular development , in order to discuss the role of cytochrome c in apoptosis of prestalk cells. The results showed that: 1,the amounts of mitochondrial cytochrome c presents a increasing tendency while the amounts of cytoplasmic cytochrome c presents inversely a decreasing tendency in prestalk cells during developmental 16h-20h.The two related events indicate that cytochrome c which may cause subsequently apoptosis was released gradually from mitochondria into cytoplasmic.But the phenomenon of lots of cytochrome c still stayed in mitochondria showed that the apoptotic prestalk cells still alive and need energy produced by Respiratory Chain participated by cytochrome c.2, the total amounts of cytochrome c (include mitochondria cytochrome c and cytoplasmic cytochrome c) in prestalk cells which shows a decreasing tendency in later 18h,20h was higher than in prespore cells.This result suggested some mechanism has improved to make up the deficiency of electron-carrier cytochrome c which reduced by releasing,and relieve of the efficiency of this mechanism was helping cells to goto death.3,The expression level of cytochrome c in prestalk cells is higher than in prespore cells in each developmental stage.This results shows the requirements of cytochrome c in transctiption level of apoptosis and vertifies the hypothesis proposed previously.As we know, when Dictyostelium .whose developmental cycle is 24h, develops 20h , most organelle was disrupted and the requirements of cytochrome c was low, so the transcription of cytochrome c was to decrease and dcrease the total amount in late apoptosis. As a result, the regulation of the transcription of cytochrome c was closely related with apoptosis.4, It's a gradual process in apoptosis of prestalk.Results of our laboratory showed cytochrome c was taking part in the apoptosis of prestalk of Dictyostelium KAx-3 cells.