Macropinosome formation by tent pole ruffling in macrophages.

Condon et al. use lattice light-sheet microscopy to analyze live macrophages and define a new model of macropinosome formation and closure through tent pole ruffles. The ruffles, which are enhanced by LPS and regulated by Rab13, are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.

康登等人利用晶格光片显微镜分析活的巨噬细胞，并通过帐篷杆状褶皱定义了一种巨胞饮体形成和闭合的新模型。这些褶皱由脂多糖（LPS）增强并受Rab13调节，由F - 肌动蛋白帐篷杆竖起和支撑，这些帐篷杆交叉并扭转以收缩正在形成的巨胞饮体。 病原体介导的巨噬细胞激活引发先天免疫反应，包括增强的表面褶皱和巨胞饮作用，用于环境采样以及受体内化和信号传导。用细菌脂多糖（LPS）激活巨噬细胞会产生明显的背侧褶皱，它们是巨胞饮体的前体。利用晶格光片显微镜（LLSM）对活的巨噬细胞进行非常快速的高分辨率成像，揭示了背侧褶皱的新特征和作用，重新定义了巨胞饮体形成和闭合的过程。我们提出了一种新模型，其中褶皱由F - 肌动蛋白帐篷杆竖起和支撑，这些帐篷杆交叉并扭转以收缩正在形成的巨胞饮体。这个过程使得由LPS诱导形成大的巨胞饮体成为可能。我们进一步描述了活性Rab13在帐篷杆状褶皱上的富集，并表明通过CRISPR技术删除Rab13会导致异常的帐篷杆状褶皱，并阻断由LPS诱导的大巨胞饮体的形成。基于LLSM出色的时间和空间分辨率，我们能够重新定义作为先天免疫反应基础的褶皱和巨胞饮体过程。