Coupling optical tweezers with light microscopy to unravel the mechanical forces acting on cellular organelles

机械力影响细胞行为的许多方面。虽然已经有很多关于形成焦点粘连的蛋白质控制细胞力学的作用的研究，但机械力如何穿过细胞以及如何影响几个细胞器的结构和动力学最终到达核膜(NE)，在很大程度上仍然难以捉摸。主要的限制是当我们在受控的机械载荷下工作时，我们无法直接‘看到’不同的机械传感器的作用。在这里，我们将使用原子力显微镜(AFM)、磁钳和光钳(首次与荧光显微镜相结合)和质谱学(MS)相结合的方法来剖析控制单个细胞和分离的细胞核内ER和NE的机械稳定性的分子决定因素(蛋白质和脂)。我们最近发现，当细胞被电镀在坚硬的底物上时，NE可以显著地变形，导致核孔复合体的物理开放，从而显著增加机械感受转录因子的核输入速率。哪些蛋白质起到了拉力？使原子核变形需要多大的力？个别脂类在决定膜变形中的作用是什么？所有这些基本问题在很大程度上仍是未知的。同样，机械力对其他膜结合细胞器的影响也是完全未知的，主要是由于缺乏实验技术。我们提出了一个多学科项目，在这个项目中，学生将获得纳米机械技术的专业知识，与MS互补，并与细胞和分子生物学技术相结合。

Mechanical forces influence many aspects of cell behavior. While much has been studied about the roles of the proteins forming focal adhesions to control cell mechanics, how mechanical forces travel across the cell and impact on the structure and dynamics of several organelles to eventually reach the nuclear envelope (NE), has remained largely elusive. The main limitation is our technical inability to directly'see' the different mechanosensors in action, when working under controlled mechanical load.Here we will employ a combination of Atomic Force microscopy (AFM), Magnetic and Optical tweezers (for the first time coupled with fluorescence microscopy), combined with Mass Spectrometry (MS), to dissect the molecular determinants (proteins and lipids) that govern the mechanical stability of the ER and the NE within individual cells and isolated nuclei.We recently discovered that the NE can be dramatically deformed when cells are plated on stiff substrates, resulting in the physical opening of the nuclear pore complex, thus drastically increasing the rate of nuclear import of mechanosenstive transcription factors. Which proteins exert the pulling force? How much force is required to deform the nucleus? What is the role of the individual lipids in determining membrane deformation? All these fundamental questions remain largely unknown. Similarly, the effect of mechanical forces on other membrane-bound organelles are completely unexplored, mostly due to the lack of experimental techniques.We propose a multidisciplinary project where the student will gain expertise in nanomechanical technologies, complemented with MS, and combined with cell and molecular biology techniques.