| 研究実績の概要 |
In the pass year, I investigated whether Rab32/Rab38 is involved in macroautophagy against GAS.
Autophagic core proteins, ATGs, were important for macroautophagy induction even under GAS infection. It has been reported that LC3, an autophagosome marker, recruited to GAS in macrophage. I generated Atg7KO or FIP200KO macrophages generated by CRISPR Cas9 system, in which GFP-Rab32/Rab38 consist expressing. I found even without Atg7 or FIP200, Rab32 and Rab38 still strongly surrounding GAS under confocal microscope observation. LC3, an autophagy marker still recruit to GAS in macrophage, which indicated as an LC3 associated phagosome, instead of macroautophagy. I assume Rab32/38-mediated LAP, which containing lysosome fusion, is much important than macroautophagy in macrophage.
Furthermore, Rab32/38 double KO cells showed that GAS-phagosome are required with lysosome (LAMP1), however, acidification seems defective inside this phagosome.
Finally, I also performed EM images, as results showed that there are condensed intensity of lysosome-fused GAS-containing phagosome and a classical double membrane surrounding GAS in Wild-type macrophage. In contrast, a low intensity of GAS-containing phagosome, many free or empty space, was majorly observed in Rab32/Rab38 double KO macrophage. We conclude that Rab32/Rab38 is very important for LAP pathway, especially in acidification.
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| 今後の研究の推進方策 |
In the new year, I aim to investigate whether microautophagy target GAS and whether Rab32/Rab38 is involved in this lysosome wrapping mechanism (LWM). This phenotype will required more clear EM images analysis. Lysosome has potential to change into a long-shapes in macrophage. This active movement, named lysosome protrusion, is able to surround an organelle bigger than lysosome itself. I plan to find out whether cytosol GAS can be surrounded by lysosome directly. The structure of lysosome protrusion along GAS will also be confirmed by correlative light and electron microscope (CLEM). I hope to show a direct evidence of microautophagy against bacteria.
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