Mechanical forces influence many aspects of cell behaviour. While the roles of the proteins forming focal adhesions to control cell mechanics have been much studied, 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. 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 (the main gateway in and out of the nucleus), thus drastically increasing the nuclear import rate of mechanosensitive transcription factors. How much force is required to deform the nucleus? What are the roles of the individual lipids in determining membrane deformation? These fundamental questions remain largely unknown.
We propose a multidisciplinary project where the student will gain expertise in nanomechanical techniques —including atomic force microscopy (AFM), magnetic tweezers (MT), and optical tweezers (OT) coupled with fluorescence microscopy— complemented with MS, and combined with cell and molecular biology to dissect the molecular determinants (proteins and lipids) that govern the mechanical properties of the NE within individual cells and isolated nuclei.
Initially (Year1), the student will be trained in molecular and cell biology techniques, they will learn to prepare substrates of different stiffness, and probe the nanomechanical properties of individual proteins and cells using AFM and MT (in the Garcia-Manyes lab) and fluorescence-coupled OT (with LUMICKS). As the project develops (Years 2 and 3), the student will work towards understanding how the NE responds to mechanical forces. They will start by tethering the nuclear membrane of isolated nuclei to beads and measure membrane tension using MT and fluorescence-coupled OT, in parallel with using fluorescent probes to visualise changes in membrane tension or fluidity. Within a cellular context, they will then dissect the role of different cytoplasmic proteins – capable of applying mechanical perturbations to the nucleus – by selectively knocking them out and studying the resulting effect on nuclear shape. Finally, to elucidate the role of the lipids in NE deformation, the student will plate cells on substrates of different stiffnesses, and subsequently extract their nuclei. Plasma membrane and nuclear lipids will be analysed by MS (in the Eggert lab), resulting in lipidomic profiles of the different substrate conditions. Year 4 will be devoted finish data analysis, and to paper and thesis writing.
This project is in collaboration with LUMICKS, a leading company in single-molecule and single-cell analysis instrumentation. Based on the correlated optical-tweezers fluorescence-microscope developed by the Wuite Lab (VU University, Amsterdam), the LUMICKS C-Trap currently has the highest resolution and stability in the field. In terms of capabilities, in certain fields, LUMICKS have surpassed the state-of-the-art in academia, combining widefield, total internal reflection fluorescence (TIRF), and interference reflection microscopy (IRM) with optical tweezers.
Supervised by Dr. Candelli (Chief Scientific Officer at LUMICKS), the student will first receive extensive training by the relevant application scientists on the C-Trap, to then be introduced to the different R&D scientists/engineers they would be working with. The student will then continue to work alongside the application scientists and Dr. Candelli in designing efficient experimental protocols. The student will be supported by software engineers in case new functionality or data analysis scripts would be necessary, and by the relevant system architect and hardware engineer(s) in case alterations need to be made to the hardware. This allows for an effective way of developing the application, characterising the instrument, and further developing the instruments. Ultimately, the student will get both a thorough understanding of the technology (and the limitations of the instrument), as well as an in-depth insight into the process of further developing the instrument for a new user segment.
Applications Applications must be complete, including both references, by 24th January 2020
Fully funded place including home (UK) tuition fees and a tax-free stipend in the region of £17,009. Students from the EU are welcome to submit an application for funding, any offers will be subject to BBSRC approval and criteria.
Infante, E.*; Stannard, A.*; Board, S.J.; Rico-Lastres, P.; Rostkova, E.; Beedle, A.E.M.; Lezamiz, A.; Wang, Y.J.; Gulaidi Breen, S.; Panagaki, F.; Sundar Rajan, V.; Shanahan, C.; Roca-Cusachs, P.; Garcia-Manyes, S. The mechanical stability of proteins regulates their translocation rate into the cell nucleus. Nature Physics 2019; 15, 973-981.
Beedle, A.E.M.*; Mora, M.*; Davis, C.; Snijders, A.P.; Stirnemann, G.; Garcia-Manyes, S. Forcing the reversibility of a mechanochemical reaction. Nature Communications 2018; 9, 3155.
Elosegui-Artola, A.; Andreu, I.; Beedle, A.E.M.; Lezamiz, A.; Uroz, M.; Kosmalska, A.; Oria, R.; Trepat, X.; Navajas, D.; Garcia-Manyes, S.; Roca-Cusachs, P. Force triggers YAP nuclear entry by mechanically regulating transport across nucleopores. Cell 2017; 171, 1397-1410.
Beedle, A.E.M.; Mora, M.; Lynham, S., Stirnemann, G.; Garcia-Manyes, S. Tailoring protein nanomechanics with chemical reactivity. Nature Communications 2017; 8, 15658.
Atilla-Gokcumen, G.E.*; Muro, E.*; Relat-Goberna, J.; Sasse, S.; Bedigian, A.; Coughlin, M. L.; Garcia-Manyes, S.; Eggert, U.S. Dividing cells regulate their lipid composition and localization. Cell 2015; 156, 428-439.