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

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.