126 research outputs found
Fluorescence lifetime spectroscopy and imaging of FRET probes for the study of cell signalling
This thesis presents the development and application of fluorescence lifetime spectroscopy and imaging to the readout of cellular processes using FĂśrster resonant energy transfer (FRET). For quantitative solution-based studies, a multidimensional fluorometer was refined and applied to characterise two genetically encoded calcium FRET biosensors based on Troponin-C, including a fluorescence lifetime-resolved titration study leading to a quantitative calibration of their calcium response. A study of their time-resolved fluorescence anisotropy was also undertaken to explore the potential to probe molecular conformational changes.
For the study of signalling processes in live cells, a novel optically sectioning FLIM microscope was developed to provide multiplexed fluorescence lifetime readouts of different FRET probes in order to facilitate the observation of different components of biological signalling networks by realising FLIM interleaved in different spectral channels.
This PhD project was motivated to study the AMP-activated protein kinase (AMPK) cascade, which has a central role in the regulation of the energy level in mammalian cells and is now being studied as a potential drug target for type II diabetes. One pathway leading to activation of AMPK is triggered by an increase in intracellular calcium level leading to activation of calcium-calmodulin dependent protein kinase kinase β (CaMKKβ), an upstream activator of AMPK. This was investigated through a novel inter-molecular FRET system looking at the direct interaction of AMPK and CaMKKβ. The ultimate goal was to multiplex readouts of AMPK activation and intracellular calcium levels, for which new FRET biosensors are required. To this end, work was also undertaken for the design and production of novel FRET sensors in the red part of the spectrum, which are desirable for multiplexing with the common CFP/YFPâbased FRET probes and also for use in in vivo imaging applications.Open Acces
From single-molecule spectroscopy to super-resolution imaging of the neuron: a review.
For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research.This work was supported by grants from the UK Engineering and Physical Sciences Research Council (EPSRC), The Wellcome Trust, Alzheimerâs Research UK, the Medical Research Council (MRC), and the Biotechnology and Biological Sciences Resesarch Council (BBSRC)
Recommended from our members
HSV-1 glycoprotein endocytosis
Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.This work was supported by grants from the Leverhulme Trust (grant RPGâ2012â793), the Royal Society (University Research Fellowship UF090010), the Engineering and Physical Sciences Research Council, UK (grant EP/H018301/1, EP/L015889/1) and by the Medical Research Council (grant MR/K015850/1)
Imaging in focus: An introduction to denoising bioimages in the era of deep learning
Fluorescence microscopy enables the direct observation of previously hidden dynamic processes of life, allowing profound insights into mechanisms of health and disease. However, imaging of live samples is fundamentally limited by the toxicity of the illuminating light and images are often acquired using low light conditions. As a consequence, images can become very noisy which severely complicates their interpretation. In recent years, deep learning (DL) has emerged as a very successful approach to remove this noise while retaining the useful signal. Unlike classical algorithms which use well-defined mathematical functions to remove noise, DL methods learn to denoise from example data, providing a powerful content-aware approach. In this review, we first describe the different types of noise that typically corrupt fluorescence microscopy images and introduce the denoising task. We then present the main DL-based denoising methods and their relative advantages and disadvantages. We aim to provide insights into how DL-based denoising methods operate and help users choose the most appropriate tools for their applications
In Situ Visualization of Block Copolymer Self-Assembly in Organic Media by Super-Resolution Fluorescence Microscopy
Analytical methods that enable visualization of nanomaterials derived from solution selfâassembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallizationâdriven block copolymer (BCP) selfâassembly in organic media at the subâdiffraction scale. Four different dyes were successfully used for singleâcolour superâresolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dualâcolour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although wellâestablished for aqueous systems, the results highlight the potential of superâresolution microscopy techniques for the interrogation of selfâassembly processes in organic media
Structural analysis of herpes simplex virus by optical super-resolution imaging.
Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.This work was supported by grants from the Leverhulme Trust (grant RPG-2012-793),
the Royal Society (University Research Fellowship to C.M.C.), the Engineering and
Physical Sciences Research Council, UK (grant EP/H018301/1) and by the Medical
Research Council (grant MR/K015850/1).This is the final published version. It first appeared at http://www.nature.com/ncomms/2015/150122/ncomms6980/full/ncomms6980.html
A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.
FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.This work was funded by grants from the Medical Research Council, the Wellcome Trust, the Alzheimer Research UK Trust, and the Engineering and Physical Sciences Research Council. W.Y.C. is funded by a China Scholarship Council-Cambridge Scholarship. D.R. is a Principal Research Fellow of the Wellcome Trust.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0006349515000752#
Nanoscopic insights into seeding mechanisms and toxicity of Îą-synuclein species in neurons.
New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of Îą-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.We thank Dr Q. Jeng and Dr A. Stephens for technical assistance and Dr J. Skepper for TEM imaging. This work was funded by grants from the U.K. Medical Research Council (MR/K015850/1 and MR/K02292X/1), Alzheimerâs Research UK (ARUK-EG2012A-1), U.K. Engineering and Physical Sciences Research Council (EPSRC) (EP/H018301/1) and the Wellcome Trust (089703/Z/09/Z). D.P. wishes to acknowledge support from the Swiss National Science Foundation and the Wellcome Trust through personal fellowships. A.K.B thanks Magdalene College, Cambridge and the Leverhulme Trust for support.This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.1516546113
The effects of baryon physics, black holes and AGN feedback on the mass distribution in clusters of galaxies
The spatial distribution of matter in clusters of galaxies is mainly
determined by the dominant dark matter component, however, physical processes
involving baryonic matter are able to modify it significantly. We analyse a set
of 500 pc resolution cosmological simulations of a cluster of galaxies with
mass comparable to Virgo, performed with the AMR code RAMSES. We compare the
mass density profiles of the dark, stellar and gaseous matter components of the
cluster that result from different assumptions for the subgrid baryonic physics
and galaxy formation processes. First, the prediction of a gravity only N-body
simulation is compared to that of a hydrodynamical simulation with standard
galaxy formation recipes, then all results are compared to a hydrodynamical
simulation which includes thermal AGN feedback from Super Massive Black Holes
(SMBH). We find the usual effects of overcooling and adiabatic contraction in
the run with standard galaxy formation physics, but very different results are
found when implementing SMBHs and AGN feedback. Star formation is strongly
quenched, producing lower stellar densities throughout the cluster, and much
less cold gas is available for star formation at low redshifts. At redshift z =
0 we find a flat density core of radius 10 kpc in both of the dark and stellar
matter density profiles. We specu- late on the possible formation mechanisms
able to produce such cores and we conclude that they can be produced through
the coupling of different processes: (I) dynamical friction from the decay of
black hole orbits during galaxy mergers; (II) AGN driven gas outflows producing
fluctuations of the gravitational potential causing the removal of
collisionless matter from the central region of the cluster; (III) adiabatic
expansion in response to the slow expulsion of gas from the central region of
the cluster during the quiescent mode of AGN activity.Comment: Published on MNRAS - 13 pages, 4 tables, 9 figure
Fluctuation-Based Super-Resolution Traction Force Microscopy
Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions
- âŚ