Applying single-molecule localisation microscopy to achieve virtual optical sectioning and study T-cell activation

Single-molecule localisation microscopy (SMLM) allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. As a single-molecule technique, it has also introduced a new quantitative approach to fluorescence microscopy. In the Part A of this thesis, the design and building of three SMLM instruments, the implementation of a custom-developed image analysis package and the characterisation of the photo-physical properties of the photo-activable fluorescent protein used in this thesis (mEos), are discussed. Then, a new post-processing method for SMLM analysis is characterised: axial optical sectioning of SMLM images is demonstrated by thresholding fitted localisations using their fitted width and amplitude to reject fluorophores that emit from above or below a virtual ‘light-sheet’, a thin volume centred on the focal plane of the microscope. This method provides qualitative and quantitative improvements to SMLM. In the Part B of this thesis, SMLM is applied to study T cell activation. Although the T cell receptor plays a key role in immunity, its stoichiometry in the membrane of resting T cells is still a matter of debate. Here, single-molecule counting methods are implemented to compare the stoichiometry of TCRs fused with mEos2 in resting T cells to monomeric and dimeric controls. However, because of the stochasticity of mEos2 photo-physics, results are inconclusive and new counting techniques based on structural imaging are discussed. In addition to TCR triggering, T cells require the co-stimulatory triggering of the CD28 transmembrane receptor to become fully activated. However, some immobilised anti-CD28 antibodies, referred to as super-agonists (SA), can directly activate T cells without triggering the TCR. In this thesis, single-molecule tracking techniques are used to investigate the molecular mechanism of CD28 super-agonism in live T cells. The results indicate that the diffusion of CD28 is slowed by SA binding. This effect is further discussed in light of the kinetic-segregation model proposed for TCR triggering. Quantitative SMLM as implemented and further developed in this work offers new tools to investigate the molecular mechanisms initiating T cell activation, ultimately facilitating the discovery of novel approaches to target these pathways for therapeutic purposes.This work was supported by the Wellcome Trust [studentship number 093756/B/10/Z]

Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.We thank the Wellcome Trust for the PhD studentship of MP (093756/B/10/Z), and the Royal Society for the University Research Fellowship of SFL (UF120277). The work by SB and DL was also funded by the Wellcome Trust (082010/Z/07/Z). UE and MH acknowledge funding by the German Science Foundation (grants EXC 115 and SFB 902). SB is funded by a BBSRC grant (BB/K013726/1). AMC acknowledges ERC Award 268788-SMI-DDR. We also thank the European Commision for support through the 4DCellFate project (EC FP7 CP 277899).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.012543

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.This work was funded by The Wellcome Trust, the UK Medical Research Council, the UK Biotechnology and Biological Sciences Research Council and Cancer Research UK. We thank the Wolfson Imaging Centre, University of Oxford, for access to their microscope facility. We would like to thank the Wellcome Trust for the Sir Henry Dale Fellowship of R.A.F. (WT101609MA), the Royal Society for the University Research Fellowship of S.F.L. (UF120277) and acknowledge a GSK Professorship (D.K.). We are also grateful to Doug Tischer (UCSF, US) and Muaz Rushdi (Georgia Tech, US) for their critical comments on the manuscript

The remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

The αβ T-cell co-receptor CD4 enhances immune responses more than one million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native co-receptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in two dimensions (2D) using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D dissociation constant, Kd, of ~5000 molecules/μm2. This value is 2-3 orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by three-fold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore appears to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.This work was supported by the Wellcome Trust and the UK Medical Research Council. PJ was supported by grants from the Swedish Research Council (number: 623-2014- 6387 and 621-2014-3907). OD is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number: 098363)

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.The authors thank R.A. Cornall, M.L. Dustin and P.A. van der Merwe for comments on the manuscript and S. Ikemizu for useful discussions about the structure. We also thank W. Lu and T. Walter for technical support with protein expression and crystallization, the staff at Diamond Light Source beamlines I02, I03 and I04-1 (proposal mx10627) and European Synchrotron Radiation Facility beamlines ID23EH1 and ID23EH2 for assistance at the synchrotrons, G. Sutton for assistance with MALS experiments, and M. Fritzsche for advice on the calcium analysis. This work was funded by the Wellcome Trust (098274/Z/12/Z to S.J.D.; 090532/Z/09/Z to R.J.C.G.; 090708/Z/09/Z to D.K.), the UK Medical Research Council (G0700232 to A.R.A.), the Royal Society (UF120277 to S.F.L.) and Cancer Research UK (C20724/A14414 to C.S.; C375/A10976 to E.Y.J.). The Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust Core Award Grant Number 090532/Z/09/Z. We acknowledge financial support from Instruct, an ESFRI Landmark Project. The OPIC electron microscopy facility was funded by a Wellcome Trust JIF award (060208/Z/00/Z).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ni.339

The changing point-spread function: single-molecule-based super-resolution imaging

Over the past decade, many techniques for imaging systems at a resolution greater than the diffraction limit have been developed. These methods have allowed systems previously inaccessible to fluorescence microscopy to be studied and biological problems to be solved in the condensed phase. This brief review explains the basic principles of super-resolution imaging in both two and three dimensions, summarizes recent developments, and gives examples of how these techniques have been used to study complex biological systems

Innovative molecular-based fluorescent nanoparticles for multicolor single particle tracking in cells

Based on an original molecular-based design we present bright and photostable fluorescent organic nanoparticles (FONs) with an excellent colloidal stability in various aqueous environments. Near-infrared emitting and green emitting FONs were prepared using a simple and robust protocol. Both types of FONs could be simultaneously imaged at the single-particle level in solution as well as in biological environments using a monochromatic excitation and a dual-color fluorescence microscope. No evidence of acute cytotoxicity was found upon incubation of live cells with mixed solutions of FONs, and both types of nanoparticles were found internalized in the cells where their movements could be simultaneously tracked at video-rate up to minutes. These fluorescent FONs open a novel non-toxic alternative to existing nanoparticles for imaging biological structures, compatible with live-cell experiments and specially fitted for multicolor single particle tracking

Innovative molecular-based fluorescent nanoparticles for multicolor single particle tracking in cells

International audienceBased on an original molecular-based design we present bright and photostable fluorescent organic nanoparticles (FONs) with an excellent colloidal stability in various aqueous environments. Near-infrared emitting and green emitting FONs were prepared using a simple and robust protocol. Both types of FONs could be simultaneously imaged at the single-particle level in solution as well as in biological environments using a monochromatic excitation and a dual-color fluorescence microscope. No evidence of acute cytotoxicity was found upon incubation of live cells with mixed solutions of FONs, and both types of nanoparticles were found internalized in the cells where their movements could be simultaneously tracked at video-rate up to minutes. These fluorescent FONs open a novel non-toxic alternative to existing nanoparticles for imaging biological structures, compatible with live-cell experiments and specially fitted for multicolor single particle tracking