The nuclear connection to 3D cell polarity and migration

A central task of any cell migrating in dense three dimensional (3D) matrices is the directional movement of the nucleus. Cell migration and invasion in 3D depends on the cells ability to deform the nucleus to actively squeeze it through tight matrix pores. We have identified an adhesion module in migrating cells that is required for effectively transmitting actomyosin contractile forces from 3D adhesion sites to the outer nuclear membrane (ONM) and concomitantly establish cell polarity. We have developed a FRET nuclear force transmission biosensor, based on mNesprin2, an ONM protein that connects the cytoskeleton to the nuclear interior, which allows us to accurately measure forces applied on the ONM. The spring constant is known and will allow extrapolation of the pN forces on the ONM. Our unpublished findings show that the nucleus is being pulled forward from the cell front in an actin and adhesion dependent manner.
In this PhD proposal we set out to answer two questions central for these cells migrating in three dimensional environments:

1. How much force is needed to move the nucleus forward and how does directional actomyosin force contribute to it?
2. Which nuclear membrane molecules are the recipients of these forces and are they still effectively propagated into the nucleus in ageing cells or cells with mutations implicated in premature ageing, like progeria?

We have recently acquired a BBSRC funded atomic force microscope (AFM) capable of simultaneous confocal microscopy enabling usage of the AFM cantilever to pull the cell using integrin ligands (cRGD) while acquiring FRET images. We will develop novel bio-imaging methodology to be able to probe cells with defined forces, record the force transmitted to the nucleus, and correlate this with the force values recorded on the nucleus of cells migrating in 3D matrixes. This will give a detailed picture of the forces needed to move the nucleus and will give us an ideal model system to interrogate the effect of nuclear force transmission on a series of established mutants, implicated in premature ageing, where the physical connection of the nuclear membrane to either the cytoskeleton and/or the nucleoskeleton has been uncoupled. In parallel the relative contribution of each class of cytoskeletal filament will be investigated.