Advanced single-molecule fluorescence micropscopy
Antibiotic persistence and resistance
Artificial Neural Networks
Goudsmits, JMH, Slotboom, DJ & Van Oijen, AM 2017, 'Single-molecule visualization of conformational changes and substrate transport in the Vitamin B12 ABC importer BtuCD-F', Nature Communications, vol. 8, no. 1.View/Download from: Publisher's site
© 2017 The Author(s). ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins. Binding and hydrolysis of ATP by their highly conserved nucleotide-binding domains drive conformational changes of the complex that mediate transport of substrate across the membrane. The vitamin B12 importer BtuCD-F in Escherichia coli is an extensively studied model system. The periplasmic soluble binding protein BtuF binds the ligand; the transmembrane and ATPase domains BtuCD mediate translocation. Here we report the direct observation at the single-molecule level of ATP, vitamin B12 and BtuF-induced events in the transporter complex embedded in liposomes. Single-molecule fluorescence imaging techniques reveal that membrane-embedded BtuCD forms a stable complex with BtuF, regardless of the presence of ATP and vitamin B12. We observe that a vitamin B12 molecule remains bound to the complex for tens of seconds, during which several ATP hydrolysis cycles can take place, before it is being transported across the membrane.
Goudsmits, JMH, van Oijen, AM & Slotboom, DJ 2017, 'Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy', vol. 594, pp. 101-121.View/Download from: Publisher's site
© 2017 Elsevier Inc. Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data.
Goudsmits, JMH, Van Oijen, AM & Robinson, A 2016, 'A tool for alignment and averaging of sparse fluorescence signals in rod-shaped bacteria', Biophysical Journal, vol. 110, no. 8, pp. 1708-1715.View/Download from: Publisher's site
© 2016 Biophysical Society. Fluorescence microscopy studies have shown that many proteins localize to highly specific subregions within bacterial cells. Analyzing the spatial distribution of low-abundance proteins within cells is highly challenging because information obtained from multiple cells needs to be combined to provide well-defined maps of protein locations. We present (to our knowledge) a novel tool for fast, automated, and user-impartial analysis of fluorescent protein distribution across the short axis of rod-shaped bacteria. To demonstrate the strength of our approach in extracting spatial distributions and visualizing dynamic intracellular processes, we analyzed sparse fluorescence signals from single-molecule time-lapse images of individual Escherichia coli cells. In principle, our tool can be used to provide information on the distribution of signal intensity across the short axis of any rod-shaped object.
Van Zijp, HM, Barendrecht, AD, Riegman, J, Goudsmits, JMH, De Jong, AM, Kress, H & Prins, MWJ 2014, 'Quantification of platelet-surface interactions in real-time using intracellular calcium signaling', Biomedical Microdevices, vol. 16, no. 2, pp. 217-227.View/Download from: Publisher's site
Platelets get easily activated when in contact with a surface. Therefore in the design of microfluidic blood analysis devices surface activation effects have to be taken into account. So far, platelet-surface interactions have been quantified by morphology changes, membrane marker expression or secretion marker release. In this paper we present a simple and effective method that allows quantification of platelet-surface interactions in real-time. A calcium indicator was used to visualize intracellular calcium variations during platelet adhesion. We designated cells that showed a significant increase in cytosolic calcium as responding cells. The fraction of responding cells upon binding was analyzed for different types of surfaces. Thereafter, the immobilized platelets were chemically stimulated and the fraction of responding cells was analyzed. Furthermore, the time between the binding or chemical stimulation and the increased cytosolic calcium level (i.e. the response delay time) was measured. We used surface coatings relevant for platelet-function testing including Poly-L-lysine (PLL), anti-GPIb and collagen as well as control coatings such as Bovine Serum Albumin (BSA) and mouse immunoglobulin (IgG). We found that a lower percentage of responding cells upon binding, results in a higher percentage of responding cells upon chemical stimulation after binding. The measured delay time between platelet binding under sedimentation and calcium response was the lowest on a PLL-coated surface, followed by an anti-GPIb and collagen-coated surface and IgG-coated surface. The presented method provides real-time information of platelet-surface interactions on a single cell as well as on a cell ensemble level. For future in-vitro diagnostic tests, this real-time single-cell function analysis can reveal heterogeneities in the biological processes of a cell population. © 2013 Springer Science+Business Media.
Erkens, GB, Hänelt, I, Goudsmits, JMH, Slotboom, DJ & Van Oijen, AM 2013, 'Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters', Nature, vol. 502, no. 7469, pp. 119-123.View/Download from: Publisher's site
Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter Glt Ph. Each subunit in the homotrimeric Glt Ph supports the coupled translocation of one aspartate molecule and three Na + ions as well as an uncoupled flux of Cl - ions. Recent crystal structures of Glt Ph revealed three possible conformations for the subunits, but it is unclear whether the motions of individual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in individual Glt Ph trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three Glt Ph subunits undergo conformational changes stochastically and independently of each other. © 2013 Macmillan Publishers Limited. All rights reserved.
Jergic, S, Horan, NP, Elshenawy, MM, Mason, CE, Urathamakul, T, Ozawa, K, Robinson, A, Goudsmits, JMH, Wang, Y, Pan, X, Beck, JL, Van Oijen, AM, Huber, T, Hamdan, SM & Dixon, NE 2013, 'A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode', EMBO Journal, vol. 32, no. 9, pp. 1322-1333.View/Download from: Publisher's site
Processive DNA synthesis by the αÉ"θ core of the Escherichia coli Pol III replicase requires it to be bound to the β 2 clamp via a site in the α polymerase subunit. How the É" proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of É". Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in É" that, in conjunction with the site in α, maintains a closed state of the αÉ"θ-β 2 replicase in the polymerization mode of DNA synthesis. The É"-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states. © 2013 European Molecular Biology Organization.