Perineuronal nets (PNNs) are specialised extracellular matrix structures on the neuronal surface that control neuronal plasticity. The key components of PNNs are flexible carbohydrate polymers including the glycosaminoglycans chondroitin sulphate (CS) and hyaluronan. In the adult brain, PNNs have a reticular structure, effectively defining holes through which neighbouring neurons can establish synaptic connections (somewhat akin to a microscale ‘print board’). Current imaging technologies provide a rather crude visualisation of the PNN morphology. In order to understand how PNNs regulate neuronal plasticity, a better knowledge of the topography, permeability and mechanical properties of PNNs is required.
We will employ a scanning probe technique based on nanopipettes, called Scanning ion conductance microscopy (SICM) to investigate, for the first time, the dynamics of PNNs. We will simultaneously map the topography and biophysical properties (e.g. permeability and elasticity) of PNNs with a spatial resolution down to 100 nm and less, and explore how external stimuli such as additional PNN crosslinking proteins affect those properties.
Nanopipettes are also attractive tools for the analysis of macromolecules, as they can detect single molecules travelling through the nanopore tip. The SICM analysis of PNNs on neurons will be complemented with the nanopipette analysis of the key molecular components of PNNs: glycosaminoglycans (GAGs) and their covalent complexes with proteins.
This project will explore a new application area for nanopipettes and SICM for the analysis of glycan-rich cell coats and their molecular components. In the long term, the results of this multidisciplinary project may also provide invaluable insights to facilitate the design of PNN modulators to improve the retention of long-term memory, and to repair neuronal connections after injury. Full description Perineuronal nets (PNNs) are specialised extracellular matrix structures on the neuronal surface that control neuronal plasticity. The key components of PNNs are flexible carbohydrate polymers including the glycosaminoglycans chondroitin sulphate (CS) and hyaluronan. Protein molecules in the PNN cross-link these glycosaminoglycans and promote supramolecular self-organisation into soft and hydrated coats with distinctive morphologies on the neuronal surface. In the adult brain, PNNs have a reticular structure, effectively defining holes through which neighbouring neurons can establish synaptic connections (somewhat akin to a microscale ‘print board’). Current imaging technologies provide a rather crude visualisation of the PNN morphology. In order to understand how PNNs regulate neuronal plasticity, a better knowledge of the topography, permeability and mechanical properties of PNNs is required. To this end, new methods are needed to analyse PNNs and their molecular components.
Nanopipettes with nanopores at their tips as small as 10 nm in diameter can be easily fabricated at the bench starting from inexpensive quartz capillaries. Nanopipettes can be integrated with nanomanipulators to comprise a scanning ion conductance microscope (SICM), a scanning probe enabling the non-contact analysis of the surface of living cells with nanoscale resolution. We will employ SICM imaging to investigate, for the first time, the dynamics of PNNs. We will simultaneously map the topography and biophysical properties (e.g. permeability and elasticity) of PNNs with a spatial resolution down to 100 nm and less, and explore how external stimuli such as additional PNN crosslinking proteins affect those properties. We will complement the experimental data acquired via SICM on neurons with fluorescence micrographs of PNN markers such as the lectin Wisteria floribunda agglutinin (WFA), so that we can correlate biophysical properties with biochemical composition. We will also create molecularly defined model PNNs by directed assembly of PNN components on solid surfaces, and use these as well-defined test specimen to validate that sensitivity and resolution of SICM with PNN-like materials. Nanopipettes are also attractive tools for the analysis of macromolecules, as they can detect single molecules travelling through the nanopore tip. The SICM analysis of PNNs on neurons will be complemented with the nanopipette analysis of the key molecular components of PNNs: glycosaminoglycans (GAGs) and their covalent complexes with proteins (proteoglycans, PGs). GAGs are highly anionic linear polysaccharides. GAGs and PGs are expressed on the cell surface and in the extracellular matrix; they have prominent roles in a variety of physiological and pathological processes and are considered promising pharmacological targets. We have recently discovered a pronounced enhancement for the single molecule detection of DNA, globular proteins and protein aggregates with nanopipette induced by the commonly used macromolecular crowding agent PEG (Chau et al, Nano Letters, 2020). Here, we will investigate the application of nanopipettes as single molecule sensors of GAGs and PGs. We aim to fingerprint the molecular mass and morphology of these molecules, as well as GAG sulphation patterns.
This project will explore a new application area for nanopipettes and SICM for the analysis of glycan-rich cell coats and their molecular components. In the long term, the results of this multidisciplinary project may also provide invaluable insights to facilitate the design of PNN modulators to improve the retention of long-term memory, and to repair neuronal connections after injury.
Formal applications for research degree study should be made online through the University's website. Please state clearly in the Planned Course of Study section that you are applying for EPSRC Bragg Centre DTP and in the research information section that the research degree you wish to be considered for is Analysing the glycan coat of neurons with nanopipettes as well as Dr Paolo Actis as your proposed supervisor.
If English is not your first language, you must provide evidence that you meet the University's minimum English language requirements (below).
As an international research-intensive university, we welcome students from all walks of life and from across the world. We foster an inclusive environment where all can flourish and prosper, and we are proud of our strong commitment to student education. Across all Faculties we are dedicated to diversifying our community and we welcome the unique contributions that individuals can bring, and particularly encourage applications from, but not limited to Black, Asian, people who belong to a minority ethnic community, people who identify as LGBT+ and people with disabilities. Applicants will always be selected based on merit and ability.
Applications will be considered on an ongoing basis. Potential applicants are strongly encouraged to contact the supervisors for an informal discussion before making a formal application. We also advise that you apply at the earliest opportunity as the application and selection process may close early, should we receive a sufficient number of applications or that a suitable candidate is appointed.
Please note that you must provide the following documents in support of your application by the closing date of 21 April 2023:
Entry requirements Applicants to research degree programmes should normally have at least a first class or an upper second class British Bachelors Honours degree (or equivalent) in an appropriate discipline. The criteria for entry for some research degrees may be higher, for example, several faculties, also require a Masters degree. Applicants are advised to check with the relevant School prior to making an application. Applicants who are uncertain about the requirements for a particular research degree are advised to contact the School or Graduate School prior to making an application.
English language requirements The minimum English language entry requirement for research postgraduate research study is an IELTS of 6.0 overall with at least 5.5 in each component (reading, writing, listening and speaking) or equivalent. The test must be dated within two years of the start date of the course in order to be valid. Some schools and faculties have a higher requirement.
Funding on offer A highly competitive EPSRC Bragg Centre Doctoral Training Partnership Studentship consisting of the award of fees with a maintenance grant (currently £17,668 in academic session 2022/23) for 3.5 years.
This opportunity is open to UK applicants only. All candidates will be placed into the EPSRC Bragg Centre Doctoral Training Partnership Studentship Competition and selection is based on academic merit.
Please refer to the UKCISA website for information regarding Fee Status for Non-UK Nationals.
Contact details For further information about this project, please contact Dr Paulo Actis: e: P.Actis@leeds.ac.uk or the Bragg Centre: e: braggcentre@leeds.ac.uk
For further information bout your application, please contact Doctoral College Admissions by email: phd@engineering.leeds.ac.uk.