Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes.

We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail, while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion-beam scanning electron microscopy, and machine learning image segmentation.

Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures, located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms.

Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.

肌动蛋白应激纤维在培养细胞中含量丰富，但在体内对它们知之甚少。在足细胞中，许多证据表明，机械生物学机制是健康和损伤足细胞形状和粘附的基础，肌动蛋白应力纤维的结构变化可能导致细胞形态的病理变化。然而，由于可视化的挑战，这一假设很难在体内进行严格的测试。需要一种高分辨率成像肌动蛋白细胞骨架的技术，以更好地了解足细胞中肌动蛋白应力纤维等结构的作用。

我们开发了第一种可视化技术，能够详细解析小鼠足细胞中的三维细胞骨架网络，同时明确识别构成该网络的蛋白质。该技术集成了膜提取、聚焦离子束扫描电子显微镜和机器学习图像分割。

使用来自健康动物的分离小鼠肾小球，我们观察到肌动蛋白电缆和中间丝将指状足细胞足突连接到新描述的位于足细胞细胞体外围的收缩肌动蛋白结构。足突内的肌动蛋白电缆形成了一个连续的、网状的、电子致密的薄片，其中包含狭缝隔膜。

我们的新技术首次揭示了健康足细胞中肌动蛋白网络的详细三维组织。除了与凝胶压缩假说一致外，该假说假设由狭缝隔膜连接的足突共同作用以抵消穿过肾小球滤过屏障的水动力，我们的数据还提供了足细胞如何对其周围环境的机械信号作出反应的见解。