Cell Biophysics draws upon the physical sciences to develop novel avenues for investigation of biological processes such as the mechanisms involved in the regulation of nuclear envelope assembly and how selective variations in lipid species composition will be related to the physical properties that characterise membrane structure and dynamics. Modulation of lipid species composition defines both the conformational changes of the membrane and the 'local signal'. Perturbations caused by changes in membrane curvature and phospholipid composition affect the affinity of proteins to be targeted to appropriate membrane compartments.
The regulation of membrane protein-lipid and protein-protein interactions, both inter- and intramolecular, will be affected by the physical and compositional properties of the membrane bilayer. Therefore we investigate how lipid activated AGC kinases and lipases are affected by membrane dynamics, upon local modification of phospholipids.
Our main focus is to study and link, structural regulation and signalling during membrane fusion. Nano-analytical tools such as fluorescence lifetime imaging microscopy (FLIM) and other precise tools such as NMR spectroscopy and liquid chromatography tandem mass spectrometry (LC-MS/MS) are used to provide increased insight into molecular composition and associations in the cell and various sub-cellular compartments.
Proteo-lipid regulation in nuclear envelope assembly
Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion and fission. The nuclear envelope is disassembled and reassembled at each mitosis in typical animal cells. It is not a passive membrane in that its reformation is central to proper cell functioning. To understand the complex architecture of the nuclear envelope is important, as its correct formation is a requirement for all cells that breakdown their nuclear envelope at mitosis. The dysfunction of nuclear envelope assembly leads to different forms of human disease from various types of cancer to Emery-Dreifus muscular dystrophy. In the past years there has been much debate on the mechanism of nuclear envelope assembly, and to date this complex issue has not been fully addressed. In contrast to the typical focus on thestructural proteins we have focused our investigations on the role of lipids and their modifying enzymes in nuclear envelope assembly.
In conjunction, we have used nuclear envelope formation as a model for characterising the molecular dynamics of membrane fusion. In collaboration with Dominic Poccia (Biology Dept., Amherst College, USA) we have shown the involvement of lipids in the regulation of nuclear envelope assembly. A non-endoplasmic reticulum membrane fraction enriched in PLCγ both in vivo and in vitro has elevated amounts of phosphoinositides. Its involvement is critical for nuclear envelope formation and perhaps for other localised membrane fusion events. The discovery of this non-endoplasmic reticulum membrane fraction with atypical levels of phosphoinositides (60mol%) has led to the hypothesis that this family of lipids are not only transient signals but they affect membrane dynamics rendering them both the property of signalling and membrane modifying molecules. To provide evidence for this hypothesis we have developed solid-state nuclear magnetic resonance spectroscopy methods in collaboration with EJ Dufourc (CNRS- Institut Européen de Chimie et Biologie, (IECB) Bordeaux, France), to define the structure and dynamics of the natural membrane domains in our model. Our future work in the regulation of nuclear envelope assembly encompasses the role of lipids and their modifying enzymes in both somatic and non-somatic cells.
3-phosphoinositide dependent protein kinase 1 (PDK1) and Protein kinase B (PKB/Akt) activation mechanism: from structure to function
The serine/threonine kinases PDK1 and PKB/Akt have received considerable attention over recent years and have become the focus of drug targeting for cancer therapy since a major role for these proteins in tumour progression has emerged. Our recent results provide novel insights into the mechanism involved in the in vivo conformational change of theses protein kinases and their association with one another. The full-length structures of PKB/Akt and PDK1 have not been resolved and the key aim of our most recent investigations was to elucidate their three dimensional structure in relation to their function. By using a multi-disciplinary approach including molecular dynamic simulations, classical biochemical assays, FRET/two-photon FLIM and in vitro anisotropy, we illustrate that the homo-dimerisation of PDK1 is a dynamic mechanism spatially and temporally regulated in vivo. FRET/two-photon FLIM was employed to observe this signalling event in live cells. A PH-domain dependent basal dimeric interaction of PDK1 was increased upon stimulation of the cells. The increased dimerisation was prevented by pre-treatment with a PI 3-kinase inhibitor as well as by a mutation or a complete deletion of the PDK1 pleckstrin homology domain, demonstrating the key role of 3-phosphorylated lipids in promoting this interaction. The distinct spatial distribution of PDK1 homodimers compared to PDK1-PKB heterodimers and the fact that monomeric mutants of PDK1 retained full kinase activity toward PKB suggested that the monomer would be the active conformation of the kinase.
In vitro fluorescence intensity and anisotropy measurements combined with existing crystal structures and computational molecular modelling enabled the prediction of the geometrical arrangement of the homodimer. This multidisciplinary approach allowed the calculation of the in vitro and in vivo dimerisation populations of PDK1, and discovery of a new regulatory mechanism of PDK1 activation. Based on our studies a new model depicting the conformational dynamics of PDK1 and its activation of PKB has been developed. This has important implications not only in extending our understanding of this 'master' signalling protein kinase, but also in opening up distinct opportunities for therapeutic intervention. Our recent work on PDK1 has been performed in collaboration with Angus Bain (Department of Physics and Astronomy, and CoMPLeX, UCL) and Michel Laguerre (CNRS, Institut Européen de Chimie et Biologie, University Bordeaux I- France).
Prognostic value of an activation state marker for downstream pathways of growth factor receptors in tumour microarrays
Current methods for assessing patient's entry for treatment of signal transduction inhibitors are limited. A very low percentage of breast cancer patients, with a 3+ over-expression of growth factor receptors, respond to molecular therapy. This indicates the limited capacity of the immunohistochemistry alone in predicting treatment success of these patients as the over expression of growth factor receptors may not correlate with the functional status of these receptors. A methodology to improve assessment of not just concentration of signal transduction targets but their status is required. We have developed an innovative analytical approach to measure functional status of these signal transductions using FRET efficiency by high throughput frequency domain-fluorescence lifetime imaging microscopy (fFLIM) in tumour arrays. We have validated this two-site FRET assay to assess EGFR1 functional status in cells and head and neck tumour and various breast cancer cell lines (in collaboration with Peter Parker's laboratory (Protein Phosphorylation) and Adrian Harris's laboratory (IMM-Oxford University).
The FRET efficiency by fFLIM has great potential to be a prognostic marker for molecular therapy. Current work is being done on breast and other solid tumours in collaboration with Peter Parker's laboratory. The high throughput screening system has been implemented for tumour array analysis in collaboration with Pierre Leboucher (College de France-CNRS-Paris).
Figure 1. Proposed scheme for SFK activation (src kinase) of PLCγ during nuclear envelope assembly in the Fusion model. Top left panel: MV1- (non endoplasmic reticulum derived membrane vesicles) (green hollow circles) and MV2 (endoplasmic reticulum derived membrane vesicles) (green circles) bind to the surface of the sperm nucleus in the presence of ATP. The lower left panel shows a blow up of the surface of MV1, with PLCγ and SFK co-localised on the cytoplasmic facing leaflet. PLCγ will be in the vicinity of its substrate PtdIns (4,5)P2 due to the high abundance of both in MV1 (Byrne et al., 2007). Upon addition of GTP to the cell-free system membrane vesicles fuse to form a double membrane (top right panel). This fusion process is driven at the molecular level by PLCγ (lower right panel), phosphorylated on the equivalent Tyr783 residue by SFK in response to GTP. Active PLCγ subsequently hydrolyses PtdIns(4,5)P2 to the fusogenic lipid DAG, promoting membrane vesicle fusion. NERs are nuclear envelope remnants - conserved regions of the sperm nuclear membrane essential for nuclear envelope assembly.
For a list of refereed research papers, see Publications (in navigation on left).
