Communication between the cytoplasm and the nucleus is crucial to maintain cellular homeostasis. The nuclear pore complex (NPC) is a large multi-protein pore, embedded within the nuclear envelope membrane, that acts as the main gateway in and out from the nucleus. We have recently discovered that the nuclear pore complex is sensitive to the mechanical stability of the proteins that cross the pore; while mechanically labile proteins translocate to the nucleus fast, mechanically resilient proteins exhibit a much delayed import into the nucleus. Yet we still do not fully understand the molecular mechanisms underpinning this newly uncovered mechanoselectivity function of the nuclear pore. In particular, we do not know if the NPC exhibits mechanical directionality, implying that the mechanical stability of only the first regions of the translocating protein determine the protein’s import rate. We also do not know which biomolecule is effectively applying mechanical force to drag the protein into the pore. We speculate that either a specific Nup – unstructured and highly dynamic proteins that line the NPC – or an ATP-dependent chaperone working in the vicinity of the NPC might provide the energy required to mechanically unfold the translocating proteins. Finally, we do not know whether the lipids and/or membrane proteins directly interacting with the NUPs forming the NPC might affect the elasticity of the pore, thereby mechanically modulating protein import.
Acquiring a full understanding of the molecular players and related mechanisms regulating the mechanical selectively of the NPC will open up the unprecedented opportunity to develop molecular strategies directed to engineer nuclear mechanostransduction, with potential modulation on a myriad of biological cellular functions.
This PhD project combines single molecule and single cell mechanobiology with membrane and membrane protein chemistry to unravel the molecular determinants underpinning the mechanoselectivity of the nuclear pore complex and use these fundamental findings to design strategies able to engineer cellular mechanotransduction and function. In a largely interdisciplinary approach across different scales we will employ single molecule force spectroscopy AFM and magnetic tweezers, dynamic stretching devices, fluorescence-based optogenetic tools, confocal microscopy combined with cellular and molecular biology techniques.
Approximate time-line
Year 1. Unraveling the mysteries underpinning the mechano-directionality of the nuclear pore complex. From the single molecule to the single cell. In the first year, the student will be trained in the use of single molecule AFM and magnetic tweezers to study the nanomechanical properties of proteins that have regions of different mechanical stabilities. They will then transfer these proteins to an optogenetic vector that enables to drive proteins in or away from the nucleus inside cells upon the application of light. Through training in cell biology and confocal microscopy, the student will test whether the nanomechanical properties of proteins observed in vitro recapitulate their behaviour at the cellular level as they cross the nuclear pore complex (Garcia-Manyes lab).
Year 2. The role of membrane proteins, membrane tension and individual nucleoporin proteins on the mechanoselectivity of the nuclear pore complex. Using the optogenetic tools with which the student will have been trained in Year 1, the student will compare the rates of nuclear entry of a particular model protein upon systematically silencing (using siRNA) independent NUPs. The choice of Nups will be guided by a screen that has been already conducted in the Garcia-Manyes lab, suggesting 8 possible targets that affect cellular mechanotransduction. Using the same experimental approach, the student will then knock down specific membrane nuclear proteins that are known to associate with the NPC to test whether their absence entails a change in the mechanical deformability of the NPC, thereby changing the rate of nuclear shuttling. For those positive results we will then test whether those specific identified membrane proteins, when purified in-vitro and embedded within a lipid bilayer, also result in a global change in the mechanical stability of the membrane (Booth lab and Garcia-Manyes lab).
Year 3. Enginneering nuclear mechanotransduction. Building from the lessons obtained in year 1 and year 2, the student will (1) modify naturally occurring mechanosensitive transcription factors (such as YAP, MRTF-A or zyxin) with particular protein sequences of tailored mechanical stabilities to change (increase or decrease at will) their endogenous rate of nuclear translocation; and (2) will test whether unmodified transcription factors change their rate of nuclear entry upon down-regulating specific Nups and membrane proteins that have shown to affect the kinetics of translocation of optogenetic tools. For those experiments exhibiting clear mechanical phenotypes, we will conduct functional assays (such as wound healing or cell motility assays) to probe whether minute changes in the structure and mechanical stability of the engineered transcription factors have knock on effects at the collective cellular level (Booth lab and Garcia-Manyes lab).
Both hosting laboratories (Garcia-Manyes and Booth) have mastered the experimental assays described in this proposal, and have experienced postdocs that will train and make sure that the student acquires the training and expertise required to perform the experiments in a meaningful and paced way. The student will learn different techniques across scales (from the molecular to the cellular level) and will be trained in a number of novel mechano-related techniques – some of them uniquely available in the supervisors’ laboratories worldwide.
The candidate
Applicants should have, or expect to have, an integrated Master’s (e.g., MSci) with first-class honours or upper division second-class honours (2:1), or a BSc plus Master’s (MSc) degree with Merit or Distinction in Physics, Biophysics, Biochemistry, Cell Biology, or related subject. The successful applicant will demonstrate a strong interest and motivation for combining experiments with computational modelling. Previous research experience in an interdisciplinary research environment is desirable.
Application Notes
- King’s Apply.
- Under Programme Name, select – Centre for the Physical Science of Life Doctoral Studentship
- Enter Project Title: Engineering nuclear mechanotransduction across the nuclear pore complex (NPC) through molecular design
- Select “Yes” for “I have identified the King’s supervisor I would like to study under” and enter Garcia-Manyes.
- In the Research Proposal box, enter ’This is not required’
- Funding - select Option 5. King's College London Funding and enter code PSoL_2023
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