PhD Projects For October 2025 Entry
Project 1
Primary Supervisor, Secondary Supervisor: Joaquina Delas, Jonathan Chubb
Project Title: Transcription factor binding integration and dynamics in developmental cell fate choice
Optical Techniques: Single molecule live cell imaging
Project Description: During development, cells specialise from multipotent to differentiated fates, building our organs. This specialisation requires the control of gene expression programs. Cis-regulatory elements drive gene expression with exquisite spatial and temporal resolution. But beyond serving as transcription factor binding platforms, how these elements act is unclear. A key feature of cis-regulatory elements is that they must integrate binding of both activating and repressive transcription factors. The tools available to date, such as ChIP-seq, have only allowed us to get snapshots of transcription factors binding to the genome. Recent developments in single molecule live cell imaging now allow us to follow individual TF molecules within the nucleus and infer their binding to chromatin. This has revealed that TFs bind chromatin only transiently. Work from our collaborator and pioneer in single molecule live cell imaging, James Liu (Janelia) has also shown that SOX2 forms clusters of high density.
The ventral spinal cord is the perfect model to address these questions. We have extensive knowledge on the regulators and cell fates, including pan-activators, cell type specific repressors and bi-functional signaling effectors. But how do these inputs integrate at cis-regulatory elements to drive gene expression or silencing? We will address this by combining cutting-edge genomics methods single molecule live cell imaging during neural differentiation. Delás, M. J. et al. (2023) Dev Cell 58, 3-17.e8. Zhang, I. et al. (2024). bioRxiv 2024.04.17.589864. Chen, J. et al. (2014) Cell 156, 1274–1285.
Project 2
Primary Supervisor, Secondary Supervisor: Christopher Stefan, Stefan Howorka
Project Title: Harnessing Optogenetics and Nanoprobes to Monitor and Modulate Membrane Tension
Optical Techniques: A key objective of this PhD project will be to develop next generation nanoprobes to monitor membrane lipid composition and organisation, as well as their effects on the biophysical properties of membrane bilayers. Most of the currently existing biosoensors simply detect a single lipid class in membranes. There is tremendous need for new probes that can simultaneously report on various changes in membrane mechano-chemical properties such as lipid composition, packing order, as well as bilayer thickness and asymmetry. In collaboration with the Howorka lab, the strategy includes the design and characterisation of DNA-based nanoprobes that can serve as co-incidence detectors of membrane composition, organisation, and biophysical properties. The novel nanoprobes are first characterised in vitro using liposomes of defined composition employing biophysical and fluorescence spectroscopy approaches including Förster resonance energy transfer (FRET) assays established in the Stefan and Howorka labs. The next generation nanoprobes will be examined in vivo using advanced quantitative imaging methods available in the LMCB (confocal and super-resolution microscopy) and model cell systems to modulate master regulators of plasma membrane tension and integrity (PI kinase and TORC2 signaling, lipid transfer proteins that function at membrane contact sites, and membrane repair pathways) currently available in the Stefan lab. The project will also develop strategies to use DNA nanoprobes as membrane tethers and force generators to monitor and manipulate membrane tension and mechanics, in collaboration with Dr. Nicholas Bell (UCL LMCB and Biophysics) using magnetic tweezers to induce and measure membrane tension and forces. Other optical technologies employed: (1) Optogenetic tools to modulate membrane tension, (2) Correlative Light and Electron Microscopy (CLEM); (3) Fluorescence-Lifetime Imaging Microscopy (FLIM), and (4) Bimolecular Fluorescence Complementation (BiFC) assays, and (5) Atomic Force Microscopy (AFM).
Project Description:
Migrating cells, including metastatic cancer cells, are constantly exposed to mechanical forces resulting in rapid alterations in plasma membrane (PM) tension. Consequently, cells have evolved robust systems to sense and respond to PM stress by adjusting the composition and biophysical properties of the PM bilayer.
Yet when considering cell mechanics, tension within the membrane bilayer is often overlooked. This is because methods to monitor membrane stress (stretch) are limiting. This project will address how cells sense and respond to membrane stress using optogenetics and novel membrane nanoprobes to modulate and monitor membrane tension.
The Stefan lab is uncovering roles of contacts between the endoplasmic reticulum and plasma membrane, ER-PM contacts, in the control of phosphoinositide (PI) kinase and target of rapamycin complex 2 (TORC2) signalling cascades necessary for PM homeostasis and repair. We are also developing new optogenetic and nanoprobe tools to modulate and measure the biophysical properties (including tension and mechanical stress) of membrane bilayers.
The student will determine i) how ER-PM crosstalk and the PI kinase/TORC2 signalling nexus modulate PM tension, ii) how alterations in these pathways impact membrane integrity, and iii) develop next-generation fluorescent nanoprobes to monitor membrane tension. The project will employ state-of-the-art imaging approaches (super resolution microscopy, FRET), biophysical approaches (optical and magnetic tweezers, AFM), and computational approaches to model membrane tension.
We expect to uncover fundamentally important regulatory mechanisms for membrane homeostasis and to develop new technologies and applications relevant to health and disease including innovative strategies to target membrane stress in cancer cells.
Project 3
Primary Supervisor, Secondary Supervisor: Antonella Riccio, Marousa Darsinou
Project Title: The role of RNAs in axons
Optical Techniques: Confocal microscopy, live imaging, superresolution, EM
Project Description: The goal of the project is to discover how localised RNA transcripts regulate neuronal development and survival. We found that in axons of sympathetic neurons, RNA are rapidly transported and m6A methylated in response to growth factors. We now want to explore the RNA binding proteins (RBPs) that are recruited to the 3'UTRs of axonal transcripts and the role of RNA epigenetic modifications in mediating the recruitment of specific RBP complexes. The project will employ cutting-edge molecular, cellular and optical techniques to study the fate of RNAs in developing neurons, and their role in axon growth and cell survival.
Project 4
Primary Supervisor, Secondary Supervisor: Yanlan Mao, Simon Walker-Samuel
Project Title: Developing machine learning methods for high throughput acquisition and analysis of 4D imaging data to understand organ development in vitro.
Optical Techniques: Deep tissue confocal imaging, high content imaging, machine learning image analysis
Project Description: To fully understand how organs grow, develop, and change shape requires detailed understanding of how individual cells in that organ coordinate their cell shape changes dynamically. This requires high resolution live imaging of organ development, capturing at the single cell level, cell shape changes, but in the context of a whole organ where thousands of cells are tightly packed together.
Organoids are 3D multicellular model systems derived from stem cells and can provide insights into tissue development during health and disease, bridging the gap between basic and clinical research.
Despite advances in culturing organoids, their growth still heavily relies on the use of animal-derived matrices. These matrices are highly heterogenous in composition, affecting reproducibility of organoid experiments and limiting their translational potential to clinics. Synthetic matrices are alternative platforms that allow for control of matrix composition and mechanics, but their use is not widely adopted yet due to the complexity to optimise their properties for different organoid systems.
In this project, you will:
1. Develop a high throughput confocal imaging pipeline using Opera Phenix platform, to live image 3D organoid develop in different synthetic matrices, to define robust synthetic-gel-based growth conditions. (Mao Lab and High content screening lab).
2. Develop and use machine learning methods for image analysis of the imaging data from (1). This quantitative data will feed into part 1 to iteratively assess what the optimal growth conditions are. Together, part 1 and 2 will develop a library of 4D organ develop images at high cellular resolution. You will initially use liver and intestinal organoids. (Mao and Walker-Samuel labs).
3. The image library obtained above will be used to train an AI-assisted computational simulation model to generate interpretable models or ‘digital twins’ of organ growth, useful for predictive therapies in the future. (Mao and Walker-Samuel labs).
Project 5
Primary Supervisor, Secondary Supervisor: Rob de Bruin, David Selwood
Project Title: Proximity-induced autophagy: expanding the universe of clinical targets.
Optical Techniques: High content image based analysis and live cell imaging
Project Description: Targeted protein degradation (TPD) has opened new opportunities to target any single protein within the cell to investigate signalling pathways as a research tool, and as a unique therapeutic strategy. We developed a novel TDP technology, AUTOCURE, which is based on proximity-induced autolysosomal degradation. Our technology allows not just the targeting of any single proteins, but protein complexes all the way to entire cellular organelles. Our technology is based on a novel mechanism that creates autophagosomes de novo at the site of the target by using an activating ligand for the autophagy initiator kinase ULK1 (ULK1-Targeting chimera, ULKTAC). Our novel AUTOCURE degradation technology platform has been designed, using click chemistry, in a modular way, allowing the binding ligands to be easily swapped in and out. Establishing our platform will provide a versatile drug development system that is robust and easy to use.
To advance the technology further and implementing it as a research tool and precursor for drug development, the following key question need to be addressed: What are the limits and scope of this technology and how can we optimise the chemical structure of ULK1-Targeting chimera to optimise its efficacy?
Completion of the project will establish AUTOCURE as a drug development platform technology to maximise its clinical and commercial impact.
Project 6
Primary Supervisor, Secondary Supervisor: Franck Pichaud, Sabrina Simoncelli
Project Title: Mechanisms of Peg-and-Socket Junction Formation and Function in Paracellular Barrier Integrity
Optical Techniques: Employed: Employed: Airyscan confocal microscopy, SoRA spinning disc confocal microscopy, lattice light sheet, and DNA-PAINT (Zaza C, Joseph DJ, Dalby OPL, Walther RF, Kołątaj K, Chiarelli G, Pichaud F, Acuna GP, Simoncelli S. Super-Resolution Simplified: Sub-10nm Imaging Over Large Areas and Deep Penetration via SDC-OPR and DNA-PAINT bioRxiv https://doi.org/10.x/2024.08.26.609760). Laser ablation of subcellular structures, Optogenetic perturbation of the F-actin and microtubule cytoskeleton. Volume electron microscopy. To develop: i) 3D DNA-PAINT for thick sample imaging: extending 2D DNA-PAINT using SoRA to 3D in consultation with Nikon. q-PAINT for thick samples: single protein quantitative imaging (i.e. precise quantification of number of protein copies). ii) Develop optogenetic manipulation (blue light) combined to lattice light sheet imaging in consultation with intelligent imaging (3i). iii) Correlative DNA-PAINT – volume electron microscopy in collaboration with Prof Jemima Burden (LMCB electron microscopy platform).
Project Description: The goal of this PhD project is to characterize the peg-and-socket junction, a poorly understood intercellular junction essential for the integrity of the vascular system, including the blood-retinal/brain barriers. Peg-and-socket junctions are specialized structures where finger-like projections from one cell, called pegs, fit into complementary indentations, or sockets, in the neighboring cell. These intercellular junctions have been observed in many tissues, including in the blood-brain and blood-retinal barriers. While they are thought to be vital for barrier integrity, little is known about their establishment, maintenance, and function.
Our lab recently identified peg-and-socket junctions in the developing Drosophila retina. This animal model system allows for precise genetic manipulation of single cells and intravital imaging from molecular-to-tissue levels, to study the consequence of the manipulations. The PhD student will capitalise on this discovery to (i) characterize peg-and-socket formation, (ii) establish the molecular pathways underlying their development and maintenance, and (iii) assess their roles in barrier development, maintenance.
To this end, they will develop and use lattice light-sheet microscopy for live imaging and super-resolution microscopy (SoRA spinning disk, Airyscan) with DNA-PAINT for detailed structure analysis. She/he will investigate the molecular composition of these junctions, exploring the presence of surface receptors, organelles, and cytoskeletal structures. Genetic and optogenetic tools will allow targeted perturbations to examine junction function. We expect this work will advance our understanding of how cellular interactions maintain barrier integrity and contribute to disease susceptibility, including inflammation, infection, and neurodegenerative diseases such as Alzheimer’s disease, stroke, and multiple sclerosis.
Project 7
Primary Supervisor, Secondary Supervisor: Nicholas Bell
Project Title: Single-molecule microscopy for understanding DNA repair enzyme dynamics and activation
Optical Techniques: Single-molecule TIRF microscopy, Single-molecule FRET, Magnetic force spectroscopy
Project Description: The DNA in our cells is constantly subjected to a variety of types of damage such as strand breaks and base modifications. DNA repair enzymes scan the genome for damage and repair them through a number of different pathways. In this project we will use TIRF microscopy and single-molecule FRET to track the dynamics and activation of human DNA damage sensor proteins. Our studies will focus on two classes of strand break sensors - Poly(ADP-ribose) polymerase (PARP) proteins and Ku proteins which are highly abundant in the cell nucleus and are targets for cancer therapeutics (1). There are many open questions concerning how these proteins function including what types of DNA damage or alternative DNA structures they are activated by and how they efficiently search for damaged DNA.
To address these questions, we will develop single-molecule microscopy techniques for high throughput imaging of individual proteins on arrays of DNA molecules. Building on our recent advances in DNA synthesis (2,3) we will begin by designing and testing a DNA array platform using TIRF microscopy and single-molecule localization. We will then investigate how different DNA damage types affect assembly of strand break sensing proteins and how small molecule drugs change their dynamics. Ultimately, we will further our understanding of the molecular mechanisms of DNA repair which will impact therapeutic interventions.
1. Pascal, J. M. Curr. Opin. Struct. Biol. 81, 102643 (2023).
2. Bell, et al Proc. Natl. Acad. Sci. 120, e2214209120 (2023).
3. Bell, et al. Nucleic Acids Res. 50, e77 (2022).
Project 8
Primary Supervisor, Secondary Supervisor: Sophie Acton, Sabrina Simoncelli
Project Title: Cell-Cell Communication in Lymphoid tissues - Examined with 3D Super-Resolution Imaging
Optical Techniques: Currently Utilized: SoRA spinning disc confocal microscopy in conjunction with DNA-PAINT imaging: A new approach developed by the Simoncelli group that enables high-resolution imaging down to sub-10nm, in depth and large field-of-view (Zaza C, Joseph DJ, Dalby OPL, Walther RF, Kołątaj K, Chiarelli G, Pichaud F, Acuna GP, Simoncelli S. “Super-Resolution Simplified: Sub-10nm Imaging Over Large Areas and Deep Penetration via SDC-OPR and DNA-PAINT,†bioRxiv, https://doi.org/10.x/2024.08.26.609760). To Develop: We aim to develop single-molecule tracking techniques integrated with 3D imaging of signaling outputs, which will allow us to study the system in both fixed and live states. This will involve extending the current 2D DNA-PAINT on SoRA imaging pipeline (bioRxiv https://doi.org/10.x/2024.08.26.609760) to 3D imaging capabilities, in collaboration with Nikon. This extension will offer more comprehensive spatial insights into molecular dynamics.
Project Description: This project aims to investigate cell-cell communication between stromal fibroblast cells (stroma-stroma interactions) and between fibroblasts and immune cells, specifically dendritic cells (stroma-immune interactions). The primary focus is on the role of gap junctions, with particular focus connexin 43, a key structural protein of gap junctions. Using connexin 43 knockout (KO) cells, we will assess how its absence affects intercellular communication and the transfer of small molecules and ions between neighbouring cells in both stroma-stroma and stroma-immune cell contexts.
Advanced imaging techniques, including SoRA spinning disc confocal microscopy coupled with DNA-PAINT super-resolution imaging, will be employed to visualize and quantify gap junction structures at the nanoscale level. In addition, we aim to develop 3D single-molecule tracking coupled to signaling output imaging, allowing us to observe dynamic interactions between fibroblasts and dendritic cells in real time. This approach will provide unprecedented insights into cellular crosstalk and the coordination of cellular responses during immune interactions and tissue repair processes.
The findings from this project are expected to shed light on the molecular mechanisms underlying stromal and immune cell communication, with potential implications for understanding inflammation, tissue homeostasis, and fibrosis.
References:
Zaza C, Joseph DJ, Dalby OPL, et al. Super-Resolution Simplified: Sub-10nm Imaging Over Large Areas and Deep Penetration via SDC-OPR and DNA-PAINT. bioRxiv https://doi.org/10.x/2024.08.26.609760. (2024).
Horsnell, H.L., Tetley, R.J., De Belly, H. et al. Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics. Nat Immunol 23, 1169–1182 (2022). https://doi.org/10.1038/s41590-022-01272-5
Project 9
Primary Supervisor, Secondary Supervisor: Jonathan Chubb, Hugh Ford
Project Title: Understanding cell decision-making using cross-scale imaging
Optical Techniques: Fluorescence reporter development, Light sheet imaging. Optogenetics. Optimisation of cross-scale live fluorescence imaging protocols for imaging sub-micron scale events (transcription) over millimetre length scales (the entire developmental niche). Coding in Matlab, Python and ImageJ for image analysis and if necessary, modelling. Deep learning protocols.
Project Description: Cell decisions are determined by processes, such as transcription, occurring at sub-micron scales within cells. However, cells make decisions based on reacting to their environment. The relationship between subcellular and external events is poorly understood, with major obstacles being the challenge of observing biological phenomena at both cell and tissue scales simultaneously, in addition to being able to monitor the full complexity of the inputs a cell receives.
Optical microscopy suffers from trade-offs between resolution and scale. Low-power objectives can survey broad fields-of-view, but lack the resolution to image processes within cells. Conversely, high-power objectives can image small structures inside cells, but are limited in their spatial range. Image stitching is too slow for observation of many subcellular events, which often occur over timescales of seconds or less.
This project will bridge this spatial coverage-resolution gap. We will directly image transcription dynamics in living cells (using technology developed by the Chubb lab) over tissue scales, together with simultaneous imaging of the multiple inputs that cells receive. You will develop imaging protocols on our new bespoke microscope for imaging simultaneously at high resolution and at the scale of the entire tissue.
By allowing large-scale imaging of transcription and extracellular signals with unprecedented speed and accuracy, you will greatly improve the ability to determine how cells make choices during development and how these choices respond to the complexity of the niche.