Team:Kyoto/CiC/Results

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Results & Discussion

Result of Subgoal A

Confirmation of function of TAT-liposome

To confirm the function of the constructed TAT-anchor peptide, we prepare the liposome containing NBD-PE with or without TAT-anchor which was synthesized or translated in vitro. Since the cells incubated with liposome did not emit any fluorescent signal, no liposome was uptaken by cells nonspecifically. In contrast, the TAT-liposome since if the liposome was uptaken by the cells, the fluorescent

(A)Fluorescent images of liposome with or without TAT-anchor. (B)Microscopic images of the cells incubated with liposome. The green arrowheads indicated the positions of the fluorescent signals.

Sliced cell treated with TAT-liposome. A merged image bright field and NBD-PE (left). The red dashed-lines indicate outline of cross-section of sliced cell. Red and green arrowheads are described in a text.

Result of Subgoal B

Result of Subgoal C

sig-GFP expression in HeLa

To confirm the function of the signal sequence for importing protein into mitochondria, we compared the expressioin pattern of sig-GFP or GFP with mitotracker signal. The GFP signal was detected throughout the cell except for the black granules in the cytoplasm or the nuclear, while sig-GFP signal showed the string-like pattern in the cytoplasm. The black granules in the cytoplasm observed in the GFP-expressing cell were stained by mitotracker, and the result indicated that the GFP was normally eliminated from mitochondria (Fig. NUMBER, GFP and mitotracker merged). In case of sig-GFP, mitochondria stained by a mitotracker and the GFP signal showed almost the same pattern. the yellow color in the merge images (Fig. Number, sig-GFP and mitotracker merged) suggested that the sig-GFP and mitochondria were colocalized in the HeLa cell. We, consequently, our constructed signal sequence has the function of importing protein into mitochondria as expected.

Figure Caption: Conforcal microscopic images of GFP or sig-GFP transfected cells. The image lines titled "mitotracker(+)" indicated the samples was stained by mitotracker. The columns showed the mitotracker, GFP, and merged images from left, respectively.