Localization Of Apoptosis-related Proteins And Ultrastructure Of Mitochondria In SL-1 Cells Undergoing Apoptosis Induced By AfMNPV

Apoptosis has been one of the research hotspots. Since the twentieth century nineties, apoptosis pathways have been gradually clear, especially in model organisms such as C. elegans, mammalian etc. However, The mechanisms of insect apoptosis induced by baculovirus are still unclear.Our previous studies showed that Lepidoptera (Spodoptera litura) SL-1 cells can be induced to apoptosis by Anagrapha (Syngrapha) falifera multiple nuclear polyhedrosis virus(AfMNPV). We found mitochondrial membrane potential decline,and detected cytochrome c release from mitochondria into the cell matrix during apoptosis. The apoptosis signaling pathway in Sl-1 cells may be similar with the mammalian mitochondrial apoptosis pathway. In this study, we investigate the localization of apoptosis-related proteins and mitochondrial structural changes to further elucidate the mechanism of SL-1 apoptosis induced by AfMNPV.1.Ultrastructure observation of mitochondria in SL-1 cells undergoing apoptosis induced by AfMNPV.Mitochondria ultrastructure of Spodoptera litura cells undergoing apoptosis induced by Anagrapha (Syngrapha) falifera multiple nuclear polyhedrosis virus were studied with transmission electron microscope. It was observed that mitochondria of SL-1 cells began to appear apoptotic features at 4h post infection:the number of mitochondria significantly increased; 8 hours later,the mitochondrias began to swell, turned round and vacuolizate and the cristae reduces,16 hours later, the mitochondrias pyknosised shrinkage,the electron density increased and the volume decreased by several times, and that the other organelles, such as nucleus and cytoplasm also undergone significant changes.2. Localization of apoptosis-related proteinsCytochrome C and Bax are very important proteins in mammalian mitochondrial pathway.In normal cells, Bax protein locates in the cytoplasmic matrix and cytochrome C locates in the mitochondrial membrane.After Cells stimulated by apoptotic signals, Bax transfers from cytoplasm to mitochondrial membrane.This translocation makes mitochondrial membrane permeability increased significantly,leading to the release of cytochrome C from mitochondria into cytoplasm matrix. In this study, we observe the localization of cytochrome C and Bax-like protein by immunofluorescence staining.2.1 Localization of cytochrome cTwo group SL-1 cells are incubated at 28℃for 4h and 8h after infected with AfMNPV, then stained with Mitochondria and cytochrome c by MitoTracker Red CMXRos and FITC-antibody respectively.The localization of cytochrome C was observe by confocal microscope. Microscoyic observation showed that mitochondria and cytochrome c were co-localize in normal SL-1 cells.Cells were infected with AfMNPV 4h later, cytochrome c no longer co-localized with mitochondria, suggesting the releases of cytochrome from the mitochondria into cytosol.2.2 Localization of Bax-like ProteinSL-1 cells were each incubated at 28℃for 3h and 6h after infected with AfMNPV, then stained with Mitochondria and Bax-like Protein respectively. Under confocal microscope t, Bax-like protein was disperse in the cell matrix in control cells, and did not co-localized with mitochondria. After cells are infected by AfMNPV for 3h, mitochondrias and Bax-like protein were turn to co-localization.This indicate that Bax-like protein translocate from the cytoplasm to the mitochondria during apoptosis.