Research Opportunities

Forces are important in the cardiovascular system, acting as regulators of vascular physiology and pathology. Vascular endothelial cells are constantly exposed to mechanical forces, such as shear stress, due to the flowing blood. Patterns of blood flow depend on blood vessel geometry and type and can range from uniform blood flow (which is protective) to disturbed blood flow (which is pathologic). Although we know that endothelial cells can sense and respond differently to different types of flow, the mechanisms by which they sense and respond to blood flow remain a mystery. Our laboratory has pioneered the studies of endothelial mechanosensing and has championed the use of a multi-disciplinary approach to this scientific problem. 

The focus of the proposed studentship is to identify mechanisms by which endothelial cells sense and respond to blood flow.  The student will have the opportunity to be exposed to a wide range of techniques based on the student’s individual interests that include: 
1) use of imaging and genetic approaches to characterize how mechanosensing affects disease intitiation and progression ; 
2) applying high throughput RNA sequencing and proteomics approaches to globally dissect steps involved in disease aetiology; and 
3) use of bioinformatics and biochemical experimental approaches to understand the role of blood flow forces in cardiovascular disease.

Reference:
Mehta V, Pang K, Rozbesky D, Nather K, Keen A, Lachowski D, Kong Y, Karia D, Ameismeier M, Huang J, Fang Y, Hernandez A, Reader JS, Jones EY, Tzima E. The Guidance Receptor Plexin D1 moonlights as an endothelial mechanosensor.2020 Nature Feb 5. https://pubmed.ncbi.nlm.nih.gov/32025034/

Ribosomes are fascinating protein-making factories. Ribosomes and the entourage of associated factors are responsible for translating the genetic information encoded within mRNA into amino acid sequences joined together to make proteins. Surprisingly, components of the ribosomal machinery have been recently discovered to have additional roles in eukaryotic cells, including regulation of endothelial cell health. Work from our group has identified a strong genetic link between a ribosomal factor and cardiovascular disease but we do not understand how this works. How do ribosomes regulate endothelial cell health and protection from disease? What is the connection between faulty ribosomes and cardiovascular disease? These are some of the fundamental questions that this project will tackle. Our ultimate goal is to identify novel players in cardiovascular disease and design new therapies that target these pathways.

The student will have the opportunity to be exposed to a wide range of techniques based on the student’s individual interests. The student will be supervised on a daily basis by a postdoctoral fellow and will be trained in cell culture, siRNA/CRISPR techniques, protein gels and western blotting, ribosomal and polysome profiling, subcellular fractionation, molecular cloning, confocal microscopy, protein structure analysis, and bioinformatics. 

Cell-to-cell communication by extracellular vesicles (EVs) is a growing field of investigation in basic cell biology research, biomarker discovery and therapeutic drug delivery. Our lab is investigating how different cargoes are loaded into EVs and the pathways that regulate EV biogenesis, release and uptake.  There are various chaperone proteins within the cell that aid the sorting of cargoes into EVs.  We are particularly interested in the aggregate-prone proteins that are associated with different neurodegenerative diseases (e.g. alpha-synuclein, SOD1, TDP-43, tau and huntingtin) and have shown that these proteins can be loaded into EVs and secreted from cells. We have recently identified that members of the small heat shock protein (sHSP) family can interact with various aggregate-prone proteins to facilitate their loading into EVs and their intercellular spreading.  In particular, we have demonstrated that one of the sHSP family, HSPB1, can interact with the autophagy cargo receptor p62/SQSTM1 to modulate its unconventional secretion by EVs. In cells expressing mutant huntingtin (the aggregate-prone protein associated with Huntington’s disease), these HSPB1-loaded EVs are capable of inducing the spreading of mutant huntingtin to non-expressing cells. Importantly, these findings reveal a novel mechanism for the spreading and seeding of protein aggregates, which may have wider implications for and impact the pathobiological mechanisms underlying other neurodegenerative disorders. In addition, we have identified several signalling pathways and regulatory proteins that are essential for the formation of mutant huntingtin-carrying EVs. 

This project will use a range of cell-based and in vivo assays to investigate how such signalling proteins regulate the interplay between autophagy and unconventional secretion and determine how this affects the accumulation and spreading of neurodegenerative disease-causing proteins. The first part of the project will involve over-expression and knockdown of these signalling proteins in vitro (in cell-based assays), where a range of biochemical and microscopy techniques will be deployed to look at protein interactions, localisation and spreading of these proteins. These findings will be then validated in vivo using a combination of zebrafish fluorescent reporter lines and neurodegenerative disease models. Finally, by using genetic and pharmacological activation and inhibition of signalling pathways, we will monitor EVs in vivo and characterise how perturbation of unconventional secretion can impact the disease progression. 

Aggregations of the cytoskeletal protein Tau into neurofibrillary tangles and cleavage products of the Amyloid Precursor Protein (APP) into amyloid plaques are strongly associated with the progression of Alzheimer’s Disease (AD). However, it remains unclear whether these aggregates initiate and cause disease or are primarily by-products of more fundamental defects. Furthermore, the physiological and cellular roles of Tau and APP, which might be disrupted in AD, are not well understood.

We have developed a new cell model in the fruit fly, Drosophila melanogaster, to study the normal functions of fly APP and Tau. Remarkably, we find that they are both involved in the biogenesis and secretion of two multimolecular signalling complexes, which are formed in endosomes: exosomes, which are small secreted vesicles, and supermeres, newly identified aggregates of protein and RNA. Their secretion is disrupted when pathological versions of Tau or cleaved APP are expressed in these cells. Using the fly model, we have already identified multiple additional regulators of these APP- and Tau-dependent processes, some of which are implicated in other neurodegenerative diseases. This project will employ neuronal and cancer cell lines to investigate which of these mechanisms are conserved in human cells and how they affect both exosome and supermere signalling. Again, informed by genetic screens in flies, we will then test whether Tau- and APP-induced defects in secretion are suppressed by genetic manipulations of other genes, which might provide new therapeutic targets going forward.

Progression of arterial disease is critically determined by the response of smooth muscle cells (SMCs) within the medial layer. In their fully differentiated, contractile state, SMCs confer stability and regulate vascular tone. However, disease induces a ‘contractile-synthetic’ phenotypic switch which impairs function, leads to vascular stiffness and exacerbates inflammation, to promote atherosclerosis and susceptibility to abdominal aortic aneurysm (AAA) (1). In animal studies, we have identified candidate pathways, based on knowledge of embryonic SMC differentiation, with the potential to protect against AAA1 and atherosclerosis (2) by preserving contractile SMC phenotype. However, the low success rate in translation from animal studies to the clinic highlights the need to determine whether similar mechanisms serve to protect the human vasculature, and how they may be targeted to alleviate disease.

The aim of the project is to establish human-relevant SMC models in which to study phenotypic switching and disease: i) a monoculture of human coronary artery SMCs; the simple monoculture model will permit evaluation of factors that directly impact SMC modulation, without confounding influences of other cell types.  ii) a more physiologically relevant model of hcSMCs co-cultured with coronary arterial endothelial cells. EC-SMC interaction crucially maintains vascular tone and functionality and dysregulation of this crosstalk underpins pathological remodelling e.g. in intimal hyperplasia. These models will then be used for high genetic and pharmacological screening to reveal disease-modifying targets. The most promising will be explored in murine disease models.
 

1). Munshaw et al. (2021) J Clin Invest 131.(10):e127884.

2). Munshaw, Redpath, Pike & Smart. bioRxiv, 2021.2011.2030.470548 (2021).

In contrast to patients after myocardial infarction, fish can fully regenerate their hearts. However, not all fish are able to regenerate to the same extent, allowing comparative inter- and intra-species analysis to identify novel mechanisms controlling successful heart regeneration. We have compared the response of seven different wild type zebrafish strains as well as Astyanax mexicanus surface and cavefish to cryo-injury. Preliminary data shows that there are large differences in regeneration within each species. Using RNAseq, we have identifed OXPHOS as a crucial regulator of this difference with increased OXPHOS being beneficial to long term regeneration. This finding indicates that the current stance in the field, that OXPHOS is damaging the ability for heart regeneration, needs to be re-evaluated.

In this project, we will investigate the mechanisms underlying the beneficial role of OXPHOS during regeneration and the techniques you will perform are targeted metabolomics, QTL analysis, RNAseq and Electron Microscopy. As the human heart relies on OXPHOS for energy, the findings from this project could help identify therapies that can direct OXPHOS to enhance cardiomyocyte proliferation and harness the potential of the human heart to regenerate.

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of cardiovascular diseases such as atherosclerosis, which cause heart attack and stroke. In healthy vessels, VSMC contraction regulate blood flow and blood pressure but loose their contractile function and undergo extensive transformation upon vasuclar insult. This process results in the generation of a wide spectrum of phenotypically changed cells within atherosclerotic lesions, which are predicted to impact differently on disease progression. Using clonal lineage tracing in mouse models of atherosclerosis, we demonstrated that disease-associated cell accumulation result from extensive proliferation of a small subset of VSMC that can generate the full range of distinct cells. By combining lineage tracing with single cell RNA sequencing (sc-RNAseq) in mouse models, we have identified signatures of VSMC-derived cells subpopulations. Interestingly, cells displaying mesenchymal stem cell character are rare in healthy vessels and their numbers increase in disease models. The aim of this project is to understand how specific VSMC-derived cell populations in human disease arise, using a combination of genomics and functional assays, in order to allow efficient cell targeting in atherosclerotic lesions.

The human placenta is the first organ of the embryo and it is functional immediately after implantation. Before fetal organogenesis, the placenta holds a multi-functional and unique role as a physical, chemical, and cellular barrier. It alone orchestrates the chemical communication between mother and fetus. Most of this communication is mediated by the secretion of specific placental peptide hormones (hCG, hPL, etc.) into the maternal bloodstream. Despite these hormones having developmental and irreplaceable functions, little is known about their intracellular life: synthesis, intracellular traffic, secretion, and degradation. More importantly, many pregnancy disorders are associated with lower expression and levels of these hormones in circulation, such as fetal growth restriction and preterm birth. The cell biology of these diseases is not well understood.

With the recent advance in human placenta organoid development and novel culture techniques of trophoblasts derived from stem cells, we can now finally interrogate the fundamental questions about placental hormones' intra and extra-cellular fate. In combination with the diverse set of advanced imagining methods available between the co-mentors of this project (advanced fluorescence and cryo-electron microscopy), innovative multi-omics techniques, and advanced biochemistry and cell biology approaches, we propose to 1) discover the diversity of secretory granules (SGs) expressed in and secreted from the human placenta; 2) implement a novel in vitro secretomics approach to determine the molecular machinery that regulates the secretion of the SGs and their content; 3) validate the mechanisms using human placenta and isolated trophoblasts from donated tissue and new placenta organoids culture.

The successful candidate will have the opportunity to train on several modern imaging techniques and learn about the fundamentals of placenta development and physiology while using a multi-disciplinary approach in a team of expert cell biologists from both institutions and generating impactful basic and applied research.

Mitochondrial diseases are caused by defects in genes required for energy production and oxidative phosphorylation (OxPhos). We find it intriguing that some patients with mitochondrial disease present late in life, with very tissue-specific phenotypes. It seems that not all cells and tissues are equally susceptible to mitochondrial disease.

We mainly study how mitochondrial dysfunction and mutations in the mitochondrial genome affect neural stem cell behaviour in Drosophila and mouse. The questions we address are:
(1) how mitochondrial dysfunction affects normal and pathological cell fate decisions in the developing brain. We previously showed that neural stem cells in the brain rely heavily on mitochondrial energy production and now study how they interact with the glial cells that make up their stem cell niche.
(2) how transcription of the nuclear genome is regulated when a cell is confronted with mitochondrial dysfunction. We employ and develop innovative DamID-based in vivo chromatin profiling technology to study metabolism of chromatin modification.
(3) how mutations in the mitochondrial genome evolve over time, during brain development and aging. We use in situ hybridisation-based methods and single-cell CRISPR screening to identify novel regulators of mitochondrial genome maintenance.

In order to study these questions in an in vivo context, in (stem) cells surrounded by their appropriate tissue environment, our primary model system is the fruit fly, Drosophila melanogaster. In addition, we actively translate our findings and the technology we develop into mammalian model systems, in particular the mouse embryonic cortex.

Relevant references
- van den Ameele J, Krautz R, Cheetham SW, et al., Reduced chromatin accessibility correlates with resistance to Notch activation. Nat Commun. 2022;13(1):2210.
- van den Ameele J, Li AYZ, Ma H, Chinnery PF. Mitochondrial heteroplasmy beyond the oocyte bottleneck. Semin Cell Dev Biol. 2020 Jan. 97:156-66.
- van den Ameele J, Brand AH. Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation. eLife. 2019;8:e47887.
- Tiberi L*, van den Ameele J*, Dimidschstein J, Piccirilli J, Gall D, Herpoel A, Bilheu A, Bonnefont J, Iacovino M, Kyba M, Bouschet T, Vanderhaeghen P. Bcl6 induces neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets. Nat Neurosci. 2012 Dec;15(12):1627-35.
- Gaspard N, Bouschet T, Hourez R, Dimidschstein J, Naeije G, van den Ameele J, Espuny-Camacho I, Herpoel A, Passante L, Schiffmann SN, Gaillard A, Vanderhaeghen P. An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature. 2008 Sep 18;455(7211):351-7.

My lab is interested in understanding how membrane-bound organelles form and acquire their distinctive proteome essential to carry their specialized functions. In particular, we focus on how organelle function is maintained through quality control processes, such as protein degradation. We are also interested in inter-organelle communications- which and how molecules are exchanged between organelles, which signals regulate those exchanges, etc.  Although my lab does mostly basic research, we are interested how these processes are disrupted in human disease.

Our lab is interested in membrane-bound organelles- how they form and acquire their distinctive proteome essential to carry their specialized functions. In particular, we focus on how organelle function is maintained through quality control processes. Our lab has been particular interested in a quality control process termed ERAD, which targets misfolded membrane proteins in the endoplasmic reticulum (ER). While some of the components of this process have been identified, the mechanisms by which diverse range of misfolded proteins are selected, ubiquitinated, extracted from the ER membrane and targeted for degradation by the proteasome remain elusive. To gain insight on the mechanisms of protein quality control our lab is taking multidisciplinary approaches. We are using CRISPR-based genome-wide genetic screens to delineate the molecular pathways involved in the degradation of disease-relevant misfolded proteins. In parallel, we use biochemical, proteomics and structural approaches to dissect mechanistically the multiple steps of ERAD. These studies will reveal the molecular basis of quality control processes by which misfolded and aggregation-prone proteins are handled by the cell both under normal and pathological situations. We are also interested in inter-organelle communications- which and how molecules are exchanged between organelles, which signals regulate those exchanges, etc.  Although we do mostly basic research, we are interested how these processes are disrupted in human disease.

Neurons are specially shaped cells that have an extremely polarized structure. They contain protrusions called dendrites and a long axon extending from cell body that connect to neighbouring cells forming a neural network. Axons and dendrites retain a dynamic plasticity throughout the whole lifespan of a neuron to ensure the ability to adjust and adapt neural network connections.  The polarity and plasticity of neurons is maintained by a cytoskeleton of actin filaments and microtubules together with associated motors and other essential proteins.

This PhD project aims to elucidate the molecular organization of axons and dendrites using in situ cryo-electron tomography (cryo-ET) of primary neurons.  We will address how cytoskeletal filaments and motors drive the branching, elongation and neural network formation. To this end we form a strong collaboration between the lab of Andrew Carter at the Laboratory of Molecular Biology (LMB), Cambridge University and Mizuno Naoko at the National Heart, Lung and Blood Institute at NIH. Mizuno’s lab’s expertise is visualizing cell shape formations controlled by remodelling of cytoskeleton and the Carter lab has a long-standing interest in how trafficking is carried out by cytoskeletal motors.

Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) bridge the resolution gap between light-microscopy and conventional structural methods for gaining information on a molecular level (X-ray crystallography/NMR).  Current technical developments further facilitate in-depth analysis of cells on a molecular level that has not been possible before. The skillset obtained in this PhD project will be highly relevant to the field of newly emerging structural cell biology.

The formation of neural network is essential for building a nerve system and to maintain its dynamic function. Neuronal cells extend their axons to connect to dendrites of partner neurons. The process is facilitated by axonal migration and controlled by the balance of the search for partner neurons vs the locking of axon-dendrite connections. This balance is maintained by two signaling pathways relying on the integrin receptor (mode of neurite migration) and the semaphorin receptor (mode of axon-dendrite locking). The inter-regulation between both pathways is mediated by the intracellular signaling factor Rap1. However, the mechanism of this interconnection is unknown.

This PhD project aims to elucidate the nature of this crosstalk using a bottom-up reconstitution of integrin, semaphorin and Rap1 by developing a cell-surface mimicking membrane system. We aim to establish a functional membrane system that allows us to control the two modes, i.e. attachment/detachment of two neighboring membranes representing dendrite and axon connections. The uniqueness of the project lies in the exact control of communication through membranes by the bottom-up strategy, which would be otherwise extremely challenging to elucidate. Moreover, the system will allow us to probe receptor interactions using biophysical, light microscopic as well as cryo-EM methods to understand the underlying principles of neural network formation and neuronal regeneration.

Our collaborative team has expertise in a wide variety of interdisciplinary techniques to facilitate the proposed PhD research, such as X-ray crystallography, cryo-EM, biophysical analysis, membrane biology and light microscopy. Mizuno lab is leading in the use of cryo-EM in combination with cellular methods to visualize cell shape formations controlled by the integrin signaling pathway and the remodeling of cytoskeleton components. Jones lab has a long-standing interest in axon guidance and structural biology of membrane proteins. Yvonne Jones co-heads the Structural Biology Division of the Wellcome Centre for Human Genetics at the University of Oxford.

This project will give a candidate a tremendous opportunity to apply cutting-edge in vitro reconstitution methods in the field of structural- and neuro- biology.