Guadagno Noemi A, Progida Cinzia
Department of Biosciences, University of Oslo, Oslo, Norway.
Methods Mol Biol. 2023;2608:39-50. doi: 10.1007/978-1-0716-2887-4_3.
Focal adhesions (FAs) are contact points of the cell with the extracellular matrix (ECM) and play a major role in several cellular functions including migration, proliferation, differentiation, and growth. During cell migration, FAs are continuously assembled and disassembled. It is well established that FA dynamics are regulated by the cytoskeleton, motor proteins, small GTPases, and specific kinases and phosphatases. However, more recently, the establishment of contacts between FAs and the endoplasmic reticulum (ER) has been shown to be another factor implicated in the regulation of FA dynamics. The transport of ER tubules along microtubules to contact FAs is indeed crucial to support FA growth. Alteration of such ER-FA contacts affects FA growth, dynamics, and thus cell migration. Here, we present a protocol for live-cell imaging and analysis of ER-FA contact points during cell migration. Our analysis pipeline includes two examples showing physiological conditions and disruption of ER-FA contacts upon nocodazole treatment. The described method can be adapted to different cell lines.
粘着斑（FAs）是细胞与细胞外基质（ECM）的接触点，在包括迁移、增殖、分化和生长在内的多种细胞功能中起主要作用。在细胞迁移过程中，粘着斑不断组装和拆卸。众所周知，粘着斑动力学受细胞骨架、运动蛋白、小GTP酶以及特定的激酶和磷酸酶调节。然而，最近研究表明，粘着斑与内质网（ER）之间建立的联系是另一个参与调节粘着斑动力学的因素。内质网管沿着微管运输以接触粘着斑对于支持粘着斑生长确实至关重要。这种内质网-粘着斑接触的改变会影响粘着斑的生长、动力学，进而影响细胞迁移。在这里，我们提供了一个在细胞迁移过程中对内质网-粘着斑接触点进行活细胞成像和分析的方案。我们的分析流程包括两个例子，展示了生理条件以及诺考达唑处理后内质网-粘着斑接触的破坏情况。所描述的方法可适用于不同的细胞系。
