Physique et Mécanique des Milieux Hétérogènes, ESPCI Paris, PSL University, CNRS, Univ Paris, Sorbonne Université, Paris, France.
UMR 144, Institut Curie and Institut Pierre Gilles de Gennes, PSL University, CNRS, Paris, France.
Methods Mol Biol. 2024;2800:115-145. doi: 10.1007/978-1-0716-3834-7_10.
The actin cortex is an essential element of the cytoskeleton allowing cells to control and modify their shape. It is involved in cell division and migration. However, probing precisely the physical properties of the actin cortex has proved to be challenging: it is a thin and dynamic material, and its location in the cell-directly under the plasma membrane-makes it difficult to study with standard light microscopy and cell mechanics techniques. In this chapter, we present a novel protocol to probe dynamically the thickness of the cortex and its fluctuations using superparamagnetic microbeads in a uniform magnetic field. A bead ingested by the cell and another outside the cell attract each other due to dipolar forces. By tracking their position with nanometer precision, one can measure the thickness of the cortex pinched between two beads and monitor its evolution in time. We first present the set of elements necessary to realize this protocol: a magnetic field generator adapted to a specific imaging setup and the aforementioned superparamagnetic microbeads. Then we detail the different steps of a protocol that can be used on diverse cell types, adherent or not.
肌动蛋白皮质是细胞骨架的重要组成部分，使细胞能够控制和改变其形状。它参与细胞分裂和迁移。然而，精确探测肌动蛋白皮质的物理性质一直是一个挑战：它是一种薄而动态的物质，其在细胞中的位置——紧贴质膜——使得使用标准的光学显微镜和细胞力学技术对其进行研究变得困难。在本章中，我们提出了一种使用超顺磁微珠在均匀磁场中探测皮质厚度及其波动的新方案。被细胞摄取的一个微珠和另一个位于细胞外的微珠由于偶极相互作用而相互吸引。通过纳米精度跟踪它们的位置，可以测量两个微珠之间夹着的皮质的厚度，并监测其随时间的演化。我们首先介绍了实现该方案所需的一组要素：适应特定成像设置的磁场发生器和前面提到的超顺磁微珠。然后，我们详细介绍了可用于不同细胞类型（贴壁或非贴壁）的方案的不同步骤。
