von Bilderling Catalina, Caldarola Martín, Masip Martín E, Bragas Andrea V, Pietrasanta Lía I
Centro de Microscopías Avanzadas and Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Laboratorio de Electrónica Cuántica, Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Rev Sci Instrum. 2017 Jan;88(1):013703. doi: 10.1063/1.4973664.
The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.
细胞与细胞外基质的黏附是一个分级的、力依赖的多阶段过程，在多个时间尺度上演变。要理解这个复杂过程，需要精确测量力及其与活细胞中蛋白质反应的相关性。我们提出了一种定量评估活细胞对局部和特定机械刺激反应的方法。我们的方法将原子力显微镜与荧光成像相结合。使用这种方法，我们评估了通过向活细胞施加纳牛顿量级的力触发的黏附蛋白（如纽蛋白、黏着斑激酶、桩蛋白和斑联蛋白）的募集。我们实时观察到新生黏附位点的形成，这从早期黏附蛋白在施力位置的积累中明显可见。我们表明该方法可用于量化黏着斑形成过程中黏附蛋白的募集特征时间。我们还发现成熟的黏着斑蛋白斑联蛋白会随着所施加的力发生空间重塑。我们的方法允许研究细胞机械转导中涉及的各种复杂生物学过程。
