NIHRDataInsights/HRCSData huggingface dataset:
Title: Single Molecule Imaging
Funder: Medical Research Council

Abstract: The overall aim of the program is to develop single-molecule and super-resolution imaging methodologies to monitor key cellular processes in real time. In recent years, cellular imaging has witnessed a remarkable revolution with the advent of single molecule spectroscopy and super-resolution microscopy. These technologies enable the direct observation of biochemical pathways in real time, and to characterize them in unprecedented detail. Specifically, our group focuses on three main topics: RNA Splicing is an essential step in the maturation of eukaryotic pre-mRNAs. Anomalous pre-mRNA splicing can have lethal effects for the cell and has been linked to numerous diseases such as breast, colorectal, epithelial and ovarian cancers, as well as neurodegenerative disorders such as Parkinsons and Alzheimers. The spliceosome is a large dynamic assembly of 5 small nuclear RNAs (snRNA) and over 100 proteins that catalyzes splicing. It undergoes several, highly conserved, conformational rearrangements. The active site of the spliceosome comprises two key snRNAs (U2 and U6) that have been shown to undergo splicing-related catalysis in absence of proteins. The structure and dynamics of the U2-U6 complex are thought to play critical roles in the mechanism of splicing in vivo. Using active spliceosomes in yeast cell extracts reconstituted with flurophore-labeled U6 snRNA, we explore the role of these dynamics in splicing assembly and catalysis. The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3-hydroxyl of the growing DNA chain. However, the mechanism by which they achieve their extraordinary accuracy remains unclear. Specifically, kinetic intermediates involved in proofreading have never before been characterized. We use single-molecule approaches to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. The APOBEC family of enzymes comprises single-stranded ssDNA cytosine deaminases that play important roles in eliminating retroviral infectivity and somatic hypermutation (SHM). For example, APOBEC3G eliminates HIV1 infectivity by converting C→U in numerous small target motifs on the minus viral cDNA, wheras AID generates advantageous mutations in the variable region of immunoglobulin genes in B-cells that increase the affinity of antibodies for antigenes. Compared with dsDNA scanning enzymes (e.g., DNA glycosylases) that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. We investigate ssDNA scanning and motif-targeting mechanisms for Apo3G and AID using single molecule fluorescence. We address specific issues of deamination activity within the general context of ssDNA scanning mechanisms.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: GENERIC HEALTH RELEVANCE
Title: High-throughput imaging and quantitative analysis for behavioural genomics in C. elegans
Funder: Medical Research Council

Abstract: The goal of behavioural genomics is to determine how genome variation in natural populations and genetic perturbation in laboratory populations affect how organisms behave. Most research has been limited to mapping genetic perturbations to particular human-defined behaviours. However, recent advances in genome sequencing and editing are not having the impact on the study of behaviour in model organisms that they should because manual observation is insufficient to properly assess behavioural phenotypes. To continue making progress we require organism-scale phenotyping methods that are commensurate with modern genomics technology in terms of their speed and applicability across organisms. We have used tracking microscopes to collect comprehensive datasets of freely behaving worms covering more than 10,000 individuals from more than 300 mutant strains. By developing new quantitative analysis algorithms we have described precise behavioural phenotypes for all of these strains, including 76 with no previously reported phenotype. We have also shown that worms with related phenotypes can be meaningfully clustered into classes to generate hypotheses for gene function. To achieve truly genome-scale behavioural experiments, we will build multi-worm trackers with megapixel cameras to increase throughput without sacrificing resolution and analysis algorithms to understand the nature of the many-to-many mapping between gene networks and complex behaviours. We will initially focus on the nematode worm C. elegans because of its tractable behavioural repertoire and well-characterised nervous system. As we develop better representations of behaviour that are unbiased, quantitative, and exhaustive, we will extend our analysis to other model organisms. In addition to advancing our understanding of locomotion and nervous system function, a systems biology of behaviour will advance the study of emerging worm models of human diseases and allow more sophisticated phenotype-based drug screens for compounds that modulate nervous system function—currently a particularly challenging area of drug discovery. The tools we will develop will eventually be applied to other model organisms and even humans. For example, it has recently been shown that gait analysis can be used for early diagnosis of disorders affecting human locomotion such as Parkinson's disease. More sensitive and general methods will push detection earlier and find application in a wider variety of diseases.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Quantitative Cell Biology
Funder: Medical Research Council

Abstract: During cell decision-making signal transduction networks dynamically change in time and space in response to cues, and thereby trigger different cellular states. The decision to divide is one of the most fundamental cellular responses and the evolutionarily conserved networks that control cell division adapt and remodel in a variety of biological contexts - during development and homeostasis, infections and malignancy, in response to drugs and stresses. A striking example of this versatility occurs during development where the same core regulators drive structurally different divisions. Divisions in the embryo are clock-like, fast, short and synchronous with no checkpoints or gap phases. With time, these divisions become longer and asynchronous. The resulting somatic like cycles have checkpoint control and gap phases, and the initiation of events is dependent on completion of early events, just like a falling domino. The question, thus, arises on how do the same cell cycle regulators self-organize and remodel in time and space to generate structurally different cell division cycles? The quantitative cell biology group addresses this question by using embryonic stem cells as a model for the embryonic cell cycle and monitors the activities, concentrations and spatial distribution of key cell cycle regulators in single cells, during differentiation into somatic cardiomyocytes. Our research program combines experimental and theoretical approaches, based on single cell advanced live cell imaging and mass spectrometry, reverse engineering, modelling and computations.. These approaches will allow us to quantitatively derive topologies of cell cycle networks under embryonic and somatic cellular contexts and uncover how remodelling happens during embryonic to somatic divisions. Multidisciplinary approaches such as those used in our group have revolutionized the way we ask biological questions and perform experiments and such a combination of approaches to understand transition states in signalling networks is likely to have profound implications for understanding normal development and disease states. More globally our research program will shed light into the organizing principles within signalling networks that emerge during cell decision-making.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: The application of bioinformatics to big data in molecular biology
Funder: Medical Research Council

Abstract: The work of this programme of research is to apply computational and mathematical technology for both the development and application of algorithms and to analyse molecular biology data. The aim in doing this, via the scientific principle of using experimental data predictively, is to either answer fundamental questions in molecular biology or to produce something of practical utility to medical science. Two specific threads of this research include the prediction of phenotype from genotype based on causal molecular biology and the prediction of factors for reprogramming one fully differentiated mature cell type into another. The prediction of phenotype from genotype combines the assessment of genomic variant consequence (e.g. our published FATHMM approach) with an association of proteins and domains with biomedical ontologies including phenotype and disease (e.g. our published dcGO resource). By bringing these together with human genome sequence data we aim to make links between genetic variants and the phenotypes that they affect through understanding of the molecular function and pathways of proteins and non-coding elements. There is a growing abundance of human genetic data, that will eventually exist for most people in our society, and a great need for increasing and improving the ways in which we can take advantage of this important step the world is taking into our future. The implications of being able to understand in detail the differences between people in their biological makeup range from medicine to sports, lifestyle and society. By knowing of characteristic differences in our individual biology, we will be able to increasingly personalise all these things. The research on the reprogramming of human cell types involves analysing the network of protein interactions alongside cell-type specific transcriptome data to predict factors that, in combination, may induce trans-differentiation of cells. Our published method called Mogrify has led to some initial experimental successes but there are numerous further developments required to make this more general and to push the boundaries of what is possible in cell fate control, and to better understand the processes which underly cell differentiation. In the long term, the ability to reprogram human cells in vivo would open up an entire field of medicine for manipulating diseased tissues within the body, and for regenerating depleted or injured materials that have been damaged. More immediately, there are already some stem-cell based treatments which may be replaced or improved on by trans-differentiating adult cells without the need for stem cells.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Developing instruments and methods for sub-nanometer resolution imaging of biological molecules by electron and photon cryomicroscopy
Funder: Medical Research Council

Abstract: The research programme is aimed at understating the fundamental limits of imaging in biology and then designing instruments, methods and techniques to improve the power of microscopy in biology. The goal is to be able to rapidly determine the structure of any biological molecule or complex, and to be able to identify and image any structure within a cell. To reach this goal, we work on studying the fundamental physical phenomenon that limit what it is possible to image in a microscope. In particular, we study radiation damage to biological specimen by electrons, photons and ions to determine an accurate theory of information per unit damage vs. specimen temperature and particle energy. This will then guide electron, ion and photon fluorescence biological microscopy by allowing us to construct cryomicroscopes capable of imaging and identifying any biological complex within a cell. We are also interested in the interaction of biological molecules with surfaces, as this often limits our ability to prepare purified specimen for high resolution imaging and structure determination. In particular, we work on preparing new specimen supports that use graphene as the starting material for controlling the absorption of biological complexes. This involves the engineering of new methods of micro and nano scale lithography and the study of protein surface interactions by various techniques. This work is intended to allow one to rapidly determine structures without the need for extensive screening and minimal data processing and storage. Ultimately, this research will improve the power of microscopy in biology aid in our understanding of human disease.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Core - Structure and Assembly Mechanisms of Centrioles
Funder: Medical Research Council

Abstract: Centrioles are barrel shaped, 9-fold symmetric cell organelles that constitute the base of cilia and flagella and are essential for their formation. Centrioles also form the core of centrosomes that they help to structure. Due to their important cellular roles, centrioles and their dysfunctions are associated with numerous human diseases ranging from microcephaly and ciliopathies to cancer and infertility. In the past, successful efforts have been made to identify the key proteins involved in the assembly and function of centrioles. However, what is so far largely lacking is a detailed understanding of how these components are structurally organized within the centriole, what their exact assembly mechanisms are and how their assembly leads to a faithful formation of functional centrioles. My lab aims to shed light on these questions by combining biochemical and biophysical approaches with the structural characterisation of centriolar components using X-ray crystallography and electron microscopy. Specifically, we try to make key centriolar components or fragments of these recombinantly using different expression systems. The purified components are characterised biochemically and biophysically to obtain information on their ability to self-associate and associate with each other. The components or their complexes are also subjected to protein crystallographic methods to derive high-resolution structures or, if diffraction grade crystals cannot be obtained, are studied by electron microscopy. The combined information from these approaches is then used to derive detailed models of the self-association and mutual interactions of the centriolar components. Subsequently, the significance of these interactions for the formation of centrioles is tested using targeted mutations of these components in vivo and the effect of these mutations on the architecture and function of centrioles is assessed using light microscopy and electron microscopy. The nature of the resulting assembly defects and specifically the resulting presence or absence of other centriolar components will then reveal the hierarchy of interactions that make up centrioles. The power of this approach is highlighted by our previous characterization of the conserved centriolar protein SAS-6. We could show that SAS-6 self-associates to organise a key assembly intermediate in centriole formation, elucidate its assembly mechanism and show how it contributes to the establishment of centrioles of the right symmetry in vivo. A detailed study of other key centriolar components can therefore be expected to yield information about how this assembly intermediate is extended and how the downstream assembly steps of centrioles are achieved on a molecular level.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Cellular rhythms, signalling and metabolic regulation
Funder: Medical Research Council

Abstract: Circadian (about daily) rhythms are cell-intrinsic phenomena that permeate every level of biology, affecting many aspects of health and disease. In humans the sleep/wake cycle is overtly circadian-regulated, as are numerous aspects of physiology and metabolism. Circadian disruption, as occurs in shift work, increases the risk of chronic illnesses such as type II diabetes, cardiovascular disease and cancer. The mechanism and significance of cellular clocks is therefore an important biological question. In previous work we have shown that, whilst cycles of nascent ‘clock gene’ expression are required for rhythmic behaviour and physiology, they are insufficient to account for circadian rhythms at the cellular level. To better understand the underlying clock machinery therefore, we break the problem down into three complementary questions: Cause – What is the cellular clock mechanism? Using pharmacological, metabolic and genetic manipulations, with real-time reporters and mass spectrometry, we perturb the cellular clock in order to identify the minimal set of components they require. Consequence – How do cellular clocks interact with other biological systems? Many biological processes are circadian-regulated but in few cases have the causal relationships, from cellular rhythm to phenotype, been clearly delineated. We investigate how internal timekeeping influences processes such as wound healing and neuronal function from a bottom-up perspective. Comparative Chronobiology – How did biological clocks evolve? By comparing the daily rhythms in mammalian cells with circadian or ultradian rhythms in evolutionary distant eukaryotes, we can distinguish fundamental timekeeping components from species-specific adaptations and so gain insight from highly conserved molecular mechanisms.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Structure and mechanism of the anaphase promoting complex and its regulation by the spindle assembly checkpoint
Funder: Medical Research Council

Abstract: The anaphase promoting complex (APC/C) is a large multi-subunit complex that functions as an E3 ubiquitin ligase to regulate progression through the cell cycle via controlled degradation of specific cell cycle regulatory proteins by the ubiquitin proteasome system. The APC/C becomes activated in mitosis through phosphorylation of the core complex and the association of co-activator subunits (either Cdc20 or Cdh1). Co-activators promote substrate recognition by interacting with degrons present in APC/C substrates. These degrons are mainly the D-box and KEN box motifs. The key role of the APC/C at metaphase is to promote sister chromatid segregation, achieved through the ubiquitin-dependent degradation of securin and cyclin B. To avoid premature chromatid segregation, i.e. before chromatids are correctly attached to the mitotic spindle, the APC/C is controlled by the spindle assembly checkpoint that is activated by unattached kinetochores. Our aim is to determine a high-resolution structure of the APC/C to address questions of its assembly and molecular basis for substrate specificity, regulation and the process of processive ubiquitination. To address these questions we are determining atomic resolution structures of various functional states of the APC/C using single particle cryo-electron microscopy. Progress towards high-resolution analysis of the APC/C has been facilitiated by our abilities to reconstitute the entire complex (including coactivator substrate complexes) using the baculovirus/insect cell expression system. To provide a full mechanistic understanding of the APC/C, we will investigate the structures of the APC/C in complex with E2~ubiquitin, the mitotic checkpoint complex, and intact protein substrates and ubiquitinated substrates, and different phosphorylation states. In the longer term we plan to reconstitute the spindle assembly checkpoint (SAC) signalling system in vitro to understand how unattached kinetochores detect the lack of tension and then generate the mitotic checkpoint complex – the effector complex of the SAC. This will include generating functional kinetochores that simulate the mitotic checkpoint signal.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.8 COMPLEMENTARY
Health categories: GENERIC HEALTH RELEVANCE
Title: Mechanisms of mitochondrial dysfunction during stress, toxicity and disease
Funder: Medical Research Council

Abstract: The MRC toxicology unit aims to understand molecular mechanisms of toxicity as to guide the development of safer new medicines and devise strategies to diminish/alleviate off-target toxicity of existing treatments. Mitochondria maintain cellular energy homeostasis and also govern apoptotic susceptibility; unsurprisingly mechanisms of toxicity frequently disrupt these vital functions. Mitochondria are also morphologically dynamic undergoing fission and fusion reactions which maintain organelle function. Accordingly, fission-fusion dysregulation causes and/or contributes to perturbed cellular homeostasis and toxicity. Morphological remodeling is typically driven by a plethora of stress-induced post-translation modifications (PTMs) and occurs during cardiotoxicity, hepatotoxicity and neurotoxicity and contributes to atrophy, degeneration, organ failure and ultimately death. Using biochemical, cell biological and in vivo approaches we aim to define and delineate how toxicity affects mitochondrial dynamics and function and establish mechanistic frameworks of conserved toxicity to facilitate future drug development and aid strategies to mitigate existing toxicology.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE
Title: Frozen Embryo and Sperm Archive
Funder: Medical Research Council

Abstract: The MRC’s Frozen Embryo and Sperm Archive (FESA) offers a free mouse archiving service to the scientific community. This service includes long-term storage and the presentation of stock details on MouseBook, IMSR and INFRAFRONTIER/EMMA websites. We provide the following services: • Cryopreservation and long term storage free for academic users • Distribution of frozen embryos/sperm or SPF mice • Cryo-recovery from frozen material • Importation and rederivation of mouse strains from around the world • Training in cryopreservation and relevant assisted reproductive technologies Stocks held within the archive are only made available to the community once a Material Transfer Agreement has been executed, designed to protect the beneficial rights of the original owner/depositor. Our archive aims to collate a wealth of useful mouse mutants that will be publicly available and accessible to the whole scientific community. Together with the scientists who deposit at our archive, we are looking to establish a cooperative future in mouse research that will benefit the scientists, funders and ultimately society as a whole through a better understanding of human disease.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE