Artificial induction of autophagy around polystyrene beads in nonphagocytic cells
Artificial induction of autophagy around polystyrene beads in nonphagocytic cells
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DOI：
10.4161/auto.6.1.10324
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发表时间：
2010-01-01
期刊：
影响因子：
13.3
通讯作者：
Haraguchi, Tokuko
中科院分区：
文献类型：
--
作者：
Kobayashi, Shouhei;Kojidani, Tomoko;Haraguchi, Tokuko
自噬是一种细胞内事件，作为一种固有的细胞防御机制，在非吞噬上皮样细胞中杀死入侵的细菌，如A组链球菌。细菌入侵时自噬小体形成的潜在细胞事件尚不清楚，这是因为与未知细菌成分相关的生化复杂性，以及预测自噬小体形成的地点和时间的困难。为了克服这些问题，我们通过诱导人造微米大小的珠子而不是细菌周围的自噬来监测活的非吞噬细胞中自噬小体的形成。与生物活性分子结合的微球为体外检测生化特性提供了强有力的工具。然而，这项技术还没有应用于活细胞，除了吞噬细胞，因为珠子不容易被结合到非吞噬细胞中。在这里，我们报道微米大小的聚苯乙烯微珠包裹着含有阳离子脂类的转染剂，可以被整合到非吞噬细胞中，并且在这些细胞中的微球周围可以有效地诱导自噬。通过荧光活细胞成像结合相关光镜和电子显微镜监测pH敏感荧光染料(PHrodo)偶联小球的自噬小体形成过程，我们发现在小球内膜部分破裂后，小球周围形成了自噬小体。此外，这些珠子随后在几小时内被转移到溶酶体中。我们的发现证明了在病原体入侵时导致自噬的细胞反应。
Autophagy is an intracellular event that acts as an innate cellular defense mechanism to kill invading bacteria such as group A Streptococcus in nonphagocytic epithelial-like cells. The cellular events underlying autophagosome formation upon bacterial invasion remain unclear due to the biochemical complexity associated with uncharacterized bacterial components, and the difficulty of predicting the location as well as the timing of where/when autophagosome formation will take place. To overcome these problems, we monitored autophagosome formation in living nonphagocytic cells by inducing autophagy around artificial micrometer-sized beads instead of bacteria. Beads conjugated with bio-reactive molecules provide a powerful tool for examining biochemical properties in vitro. However, this technique has not been applied to living cells, except for phagocytes, because the beads cannot be easily incorporated into nonphagocytic cells. Here we report that micrometer-sized polystyrene beads coated with transfection reagents containing cationic lipids can be incorporated into nonphagocytic cells, and that autophagy can be efficiently induced around the beads in these cells. Monitoring the process of autophagosome formation for pH-sensitive fluorescent dye (pHrodo)-conjugated beads by fluorescence live cell imaging combined with correlative light and electron microscopy, we found that autophagosomes are formed around the beads after partial breakdown of the endosomal membrane. In addition, the beads were subsequently transferred to lysosomes within a couple of hours. Our findings demonstrate the cellular responses that lead to autophagy in response to pathogen invasion.