ORGANISATION NAMEUniversity College London
ORGANISATION COUNTRYUnited Kingdom
RESEARCH FIELDNatural sciences
CAREER STAGEFirst Stage Researcher (R1) (Up to the point of PhD)
The main objective of this research project is to uncover new molecular interactions relevant to virus entry into living cells by means of precision force-sensing and fluorescence microscopy experiments at the single molecule level.
The cell membrane is the main barrier that viruses need to overcome to penetrate cells and cause disease. As part of their entry strategy, viruses interact with specific receptor proteins at the cell surface in ways which are not well understood. These cell-surface receptors are typically embedded in the membrane of the cell, where they can move randomly (via Brownian diffusion) in the membrane plane. The physical properties of cell-surface receptors, such as their mobility and anchoring to the cellular cytoskeleton (a mesh of filaments beneath the cell membrane), are likely to importantly influence virus entry events. However, our knowledge of these receptor properties and their role in virus entry is currently very limited.
This project aims at detecting and characterising molecular attachments between virus receptors and the cellular cytoskeleton. These attachments can play a crucial role in virus entry by modifying receptor mobility, enabling the clustering of receptors on the cell surface and/or stabilising virus-receptor interactions.
We will focus on the Human Immunodeficiency Virus (HIV) as a model system. HIV particles first attach specifically to receptor molecules CD4 and CCR5/CXCR4 on the surface of cells of the immune system. These receptors then redistribute and accumulate at the sites of virus attachment on the cell surface. Eventually, the virus penetrates the cell membrane and releases its genome into the cellular cytoplasm. Several recent studies have pointed towards links between the CD4, CCR5 and CXCR4 receptors for HIV and the cellular cytoskeleton. These links, together with dynamic rearrangements of the cytoskeleton upon HIV attachment, have been suggested as responsible for the receptor redistribution and clustering required for HIV entry. However, the proposed links have not been observed directly to date and the mechanisms for clustering remain unknown. We will first measure receptor mobility and receptor-cytoskeleton attachments in lymphocytes (and model cells) in the absence of viruses to understand the baseline biophysical properties of the HIV receptors. Following this, we will investigate changes in receptor properties upon virus engagement to find out more about the mechanisms and dynamics of virus entry.
Optical tweezers will be used to pull individual receptor molecules on the surface of living cells while precisely detecting forces at the picoNewton level and displacements at the nanometer scale. You will make use of optical tweezer technology and develop novel sequential data acquisition and real-time data analysis that will allow the controlled measurement of these unknown molecular interactions at the cell surface. Comparing force measurements on cells displaying HIV receptors and different candidate linker proteins will enable us to probe for the first time and understand the role that receptor-cytoskeleton interactions play in virus entry. Force spectroscopy measurements will be complemented with receptor mobility measurements using single-molecule light-sheet fluorescence microscopy and single particle tracking. Our results will form the basis of future investigations into HIV entry and into other virus-receptor systems that exhibit similar entry mechanisms, potentially opening new avenues for anti-viral drug design, generating benefits to human health and positive societal and economic impact.
For this PhD project, you will make use of and participate in the further development of our advanced multifunctional fluorescence-force microscope that combines dual-colour light-sheet fluorescence imaging with optical tweezers for force exertion/sensing in in-vivo experiments. Throughout the course of this project, you will apply several biophysical approaches such as optical tweezers for single-molecule force spectroscopy, advanced fluorescence microscopy techniques, image processing and single-particle-tracking; various biochemical and cell and molecular biology techniques, e.g., cell culture, fluorescence labelling of cell-surface receptors, particle-functionalization techniques for specific attachment of micro-beads to the receptors of interest for force sensing experiments, etc.
You will work in a multi-disciplinary environment under the supervision of Dr. Isabel Llorente-García in the Biological Physics Group within the Dept. of Physics and Astronomy at University College London, UK. You will benefit from close collaboration with Prof. Mark Marsh, MRC LMCB, and will have access to all LMCB core facilities (light microscopy, EM, super-resolution imaging, high-throughput screening, etc). Additionally, you will have the opportunity of collaborating with AFM expert Prof. Sonia Contera (Dept. of Physics, Oxford University). As a student, you will be part of the Advanced Characterisation of Materials Centre for Doctoral Training (http://cdt-acm.org/), funded by the EPSRC for a partnership between University College London (UCL) and Imperial College London, and benefit from a variety of training in a wide range of techniques.
Candidates should have a high grade point average MSci or Master’s degree (or equivalent) in physics, engineering, quantitative life sciences or similar, with physics experience. Previous experience in experimental research, optics and programming are highly desirable, along with an interest in biological physics.
The stipend is £16,785 p.a. for 3.5-4 years, and tuition fees will be covered. Funding is available to UK/EU/EEA candidates. Suitably qualified and eligible candidates should send their application with cover letter, CV and transcript of records/grades to Dr Isabel Llorente-García (email@example.com), including two references (at least one of these academic), highlighting academic excellence and previous research experience. Deadline: 31-March-2107.
Suggested background reading:
The cell biology of receptor-mediated virus entry, J. Grove and M. Marsh, J. Cell Biol. 195, 1071 (2011).