Cell & Developmental Biology projects

This project will evaluate the implication of IGFN1 in skeletal muscle mechanotransduction and selective autophagy by: 1) Generating an Igfn1 loss of function allele in single adult muscles by CRISPR/CAS mediated genome editing; 2) Measuring the elastic properties of purified IGFN1 by atomic force microscopy. Cytoskeletal protein crosslinkers have been recently placed at the forefront of cellular mechanotransduction. When mechanical strain causes these linkers to unfold, it is proposed that an inducible selective autophagy mechanism is triggered to dispose of damaged proteins. This pathway contributes to protein quality control and is essential for maintaining muscle integrity. However, the role of this pathway in processing the variety of cytoskeletal protein crosslinkers that are required to maintain the sarcomere is not clear. IGFN1 is a large muscle-specific protein conserved in all sequenced mammals with a typical domain composition similar to other proteins that organize and support the sarcomere structure by crosslinking the cytoskeleton. IGFN1 localizes to the Z-disc and is therefore a strong candidate for a mechanotransduction sensor in skeletal muscle. The Igfn1 gene will be targeted using CRISPR/CAS customized vectors previously validated in vitro to generate loss of function mutations in hindlimb muscles of adult mice in vivo. The loss of function effects on muscle structure and function will be characterized in detail by histology, immunofluorescence with autophagy markers and electron microscopy. The lengths obtained by AFM will be related to the physical dimensions of the sarcomeric structures during the cycles of contraction and relaxation. These experiments will provide mechanistic insights into the role of IGFN1 as a structural stabilizer and client of selective autophagy.

Supervisors: Dr Gonzalo Blanco & Dr Christoph Baumann

To discuss your suitability for this project please email: gonzalo.blanco@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

Autophagy (literally ‘self- eating) is the process by which cells clear unwanted material. The autophagic pathway is upregulated at times of stress (e.g. starvation), but is also essential under normal physiological conditions, for example to regulate levels of certain macromolecules. Autophagy involves formation of a phagophore, a membranous structure that engulfs cellular components and delivers them to the lysosome for degradation. The project will investigate a novel mechanism that regulates autophagy and the crosstalk between autophagy and apoptosis. A better understanding of the molecular mechanisms underlying the normal role of these processes will aid in our understanding of processes such as ageing. Training will be provided in cell biology techniques including nucleic acid manipulations, yeast genetics, mammalian cell culture, in silico analyses of species-specific orthologues and fluorescence microscopy.

Supervisors: Prof. Nia Bryant & Dr Paul Pryor

To discuss your suitability for this project please email: nia.bryant@york.ac.uk

Please read the 'How to apply' tab before submitting your application. 

The circadian clock is required to set metabolism and development to the appropriate time of day. As the rising of the morning sun changes by up to 7 minutes a day, mechanisms exist to reset this clock so that it is in harmony with prevailing conditions, and this process is called entrainment. The mechanism of plant photoreceptor signalling to the circadian oscillator remains unknown. Our previous identification of ELF4 and ELF3 as components of the key complex required for the oscillator to cycle and our genetic finding that this complex is the target of phytochrome photoreceptor entry to this clock sets up a series of programmes to mechanistically resolve an entrainment mechanism and this answers a long-standing question in circadian chronobiology, termed Aschoff's Rule, whereby increases in light intensity lead to periodicity acceleration.

The proposed project starts from a systems-genomic approach i) to define when and where the ELF4 / ELF3 complex target transcriptional repression, ii) moves to examine the roles of timing in said repression and then iii) to describe the role of light perception in modulating their target association. New technical approaches to find chromatin targets facilitate these experiments. From there, molecular-biological approaches would be used to improve the understanding of the structure-function relationship of the ELF4 and ELF3 repression cycle through characterisation of their timed complex assembly n the living plant cell. Novel cellular-imaging and luciferase-imaging approaches will be employed to study the kinetics of these events, and determine photoreceptor occupancy in their complex. Efforts to solve by crystallography the structure of the ELF4 / ELF3 repression complex will be undertaken and, in parallel, associated in vitro biophysics of complex assembly will be elucidated. All of these will be connected back to the molecular-physiological action of oscillator behaviour and the cellular-movement events that coincide with regulated complex formation. Taken together this project is envisaged to provide the mechanistic basis of a long-standing problem and be the first description of a clock-resetting mechanism in plants.

References: Anwer et al. eLife e02206 (2014); Herrero et al. Plant Cell 24: 428–443 (2012); Levdikov et al. Proc. Natl. Acad. Sci. USA 109, 5441-5445 (2012); Kolmos et al. Plant Cell 23: 3230-3246 (2011)

Supervisors: Prof Seth Davies (Biology) & Prof Anthony Wilkinson (Chemistry)

To discuss your suitability for this project please email: seth.davies@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

Dynamic complexity is the emerging paradigm for all G protein coupled receptors (GPCR), including chemokine receptors, and one that explains why the membrane microenvironment influences their function. We have shown for the chemokine receptor CCR5 that ligand stimulation triggers plasma membrane re-organisation before internalisation of b-arrestin-bound CCR5, and occurs in a cell type-dependent manner.

The proposed PhD project aims to unravel how the different events affecting CCR5 are orchestrated. It will use a cutting-edge biophysical approach to assess chemokine receptor dynamics in the membrane of living cells and, for the first time, in real-time. It will investigate the early steps in ligand-mediated CCR5 activation using ensemble and single-molecule fluorescence microscopy approaches developed in our research groups to probe membrane receptor dynamics. Experiments carried out initially on well-characterised CCR5 expressing cell-lines using CCR5-specific antibodies and single-molecule tracking will assess CCR5 membrane mobility and its ability to oligomerise. Over-expression of fluorescently tagged proteins and specific inhibitors will be used to establish the link between CCR5 membrane mobility, cytoskeleton scaffolds and endocytosis. We will then use human blood-isolated primary cells, which endogenously express CCR5, but differ in their ability to support ligand-mediated CCR5 internalisation, to validate our key findings and unravel cell-type specific mechanisms. This project should rapidly generate valuable findings leading to a better mechanistic understanding of how ligand stimulation affects GPCR membrane dynamic.

Supervisors: Dr Nathalie Signoret & Dr Christoph Baumann

To discuss your suitability for this project please email: nathalie.signoret@york.ac.uk

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The 2013 Nobel prize in Medicine was awarded for the discovery of the protein factors involved in intracellular vesicle transport. Vesicle transport is critical for processes such as neurotransmitter release or insulin regulated GLUT4 secretion, and therefore impacts on areas like neuroscience and diabetes. Yet the regulation of the final step of vesicle trafficking, the fusion of the two membranes is not well understood. It is well known that a SNARE complex that bridges the two membranes has to form to fuse these membranes, but how SNARE complex formation is regulated is an important open question. We would like to understand how the process of vesicle targeting regulates SNARE complex formation and thereby membrane fusion. During vesicle targeting the first contact between the vesicle and target membrane are made, and therefore this is a key site for vesicle trafficking specificity – where does a vesicle end up in the cell. Large multisubunit tethering complexes (MTCs) coordinate vesicle targeting throughout the cell, one working at the Golgi apparatus is the COG complex. COG is important for the sorting of Golgi-resident proteins, most notably glycosylation enzymes, but also Golgi SNAREs. COG is known to interact with several Golgi SNAREs, yet if this interaction is involved in regulating SNARE complex formation is not established. We will use biochemical SNARE assembly assays with purified COG to address this question. Mutant COG complexes will also be generated, which cannot bind SNAREs, and tested both in the biochemical assay and for their ability to alter sorting within the Golgi. Golgi sorting will be assessed by analyzing the glycosylation potential for cells using a mass spectrometric glycan profiling method. Thus this project will combine biochemical, cell biological, glycobiological and mass spectrometric methods for the analysis of an important cell biological problem.

Supervisors: Dr Dani Ungar & Prof Nia Bryant

To discuss your suitability for this project please email: dani.ungar@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

Autophagy (self-eating) is the process by which cells clear unwanted material. The autophagic pathway is upregulated at times of stress (e.g. starvation) and cellular differentiation. Autophagy involves formation of a phagophore, a membranous structure that engulfs cellular components and delivers them to the lysosome for degradation. This project will investigate whether a Tlg2:Vps45 interaction that regulates autophagy in yeast is conserved in Leishmania spp. parasites and necessary for lifecycle differentiation to human-infectious forms. A better understanding of the molecular mechanisms underlying the role of these processes will pave the way for development of therapeutic strategies to combat Leishmaniasis. Advanced training will be provided in cell biology techniques including nucleic acid manipulations, Leishmania spp. genetics, Cat3 cell culture, parasite infectivity and differentiation assays, Western/Northern blots, quantitative PCR, in silico analyses and live cell fluorescence microscopy.

Supervisors: Dr Pegine Walrad & Prof Nia Bryant

To discuss your suitability for this project please email: pegine.walrad@york.ac.uk

Please read the 'How to apply' tab before submitting your application.