Molecular-scale analysis of nesprin mechanics in cells

Basic data for this project

Description

The ability of the mammalian nucleus to integrate a wide range of mechanical signals is critical for many developmental and postnatal homeostatic as well as pathological processes. Nuclear mechanosensing allows cells to efficiently sense their microenvironment and respond with changes in cell signaling, nuclear positioning, chromatin organization, and epigenetic and transcriptional regulation to adapt to a changing physical environment. It has been recognized that nuclear mechanosensing relies, at least in large part, on a multimolecular structure called the LINC complex. Located at the nuclear envelope, it is thought to transmit mechanical forces from the plasma membrane and cytoskeleton to the nucleus and to act as a scaffold for signaling molecules. Although the core components of the LINC complex have been identified, such as the nesprin giant proteins at the outer nuclear envelope and the SUN proteins at the inner nuclear envelope, its precise role and regulation during the force transduction process remains poorly understood. It is still unclear where and when endogenously expressed nesprin proteins are exposed to mechanical forces, how the very large nesprin giant proteins contribute to force transmission, and how the different cytoskeletal networks and binding partners affect their ability to transmit mechanical information. A major reason for our current inability to better understand these processes is the lack of suitable methods to quantify the molecular forces processed by the LINC complex under physiological conditions. To address this important knowledge gap, we have developed and characterized a novel strategy to genomically modify nesprin genes with picoNewton-sensitive biosensors and to quantify molecular force transmission in the resulting cell lines using live-cell FLIM-FRET. Here, we propose to apply this exciting technology to elucidate the molecular mechanics of nesprin-1 giant and nesprin-2 giant in cells. In Aim-1 we will investigate nesprin-2 force transmission in keratinocytes when cells are exposed to internally or externally generated stresses. In different physiological contexts, we will address the role of cytoskeletal networks and potential modulators on nesprin force transmission, and explore nesprin mechanics during single and collective cell migration. In Aim 2 we will focus on nesprin 1 and nesprin isoform-specific force transmission in muscle cells. We will elucidate the mechanics of nesprin-1 and nesprin-2 in undifferentiated and differentiated C2C12 cells, evaluate molecular mechanisms of regulation and explore potential synergy effects using tension sensor multiplexing. Altogether, the expected results should provide unique and much needed insights into the fundamental principles of nesprin force transmission and by extension LINC complex mechanics.