Published: Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis

S.P.B. van Beljouw, S. van der Els, K.J. A. Martens, M. Kleerebezem, P.A. Bron, J. Hohlbein, Physical Biology, 16, 035001, 2019, [link]

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays.

Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.

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News: Welcome to Suyeon Yang and George Vogelaar

Suyeon joined the group in November 2018 for her PhD thesis. She will study lipid oxidation at oil/water interfaces using super-resolution microscopy. George just started his MSc thesis to study diffusion of dCas9 in Lactococcus lactis using single-particle tracking PALM.

Published: High-throughput, non-equilibrium studies of single biomolecules using glass made nanofluidic devices

M. Fontana, C. Fijen, S. G. Lemay, K. Mathwig and J. Hohlbein, Lab on a Chip, 19, 79, 2019. [link]

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observing DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level.

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Pre-print: An open microscopy framework suited for tracking dCas9 in live bacteria

K.J.A. Martens, S. van Beljouw, S. van der Els, S. Baas, J.N.A. Vink, S.J.J. Brouns, P. van Baarlen, M. Kleerebezem, J. Hohlbein, bioRxiv, 2018 [link]

Super-resolution microscopy is frequently employedin the life sciences, but the number of freely accessible and affordable microscopy frameworks, especially for single particle tracking photo-activation localization microscopy (sptPALM), remains limited. To this end, we designed the miCube: a versatile super-resolution capable fluorescence microscope, which combines high spatiotemporal resolution, good adaptability, low price, and easy installation. By providing all details, we hope to enable interested researchers to build an identical or derivative instrument. The capabilities of the miCube are assessed with a novel sptPALM assay relying on the heterogeneous expression of target genes. Here, we elucidate mechanistic details of catalytically inactive Cas9 (dead Cas9) in live Lactococcus lactis. We show that, lacking specific DNA target sites, the binding and unbinding of dCas9 to DNA can be described using simplified rate constants of kbound_free = 30 – 80 s-1 and kfree_bound = 15 – 40 s-1. Moreover, after providing specific DNA target sites via DNA plasmids, the plasmid-bound dCas9 population size decreases with increasing dCas9 copy number via a mono-exponential decay, indicative of simple disassociation kinetics.

Published: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

B. Hellenkamp, S. Schmid, O. Doroshenko, O. Opanasyuk, R. Kühnemuth, S. Rezaei Adariani, B. Ambrose, M. Aznauryan, A. Barth, V. Birkedal, M.E. Bowen, H. Chen, T. Cordes, T. Eilert, C. Fijen, C. Gebhardt, M. Götz, G. Gouridis, E. Gratton, T. Ha, P. Hao, C.A. Hanke, A. Hartmann, J. Hendrix, L.L. Hildebrandt, V. Hirschfeld, J. Hohlbein, B.g Hua, C.G. Hübner, E. Kallis, A.N. Kapanidis, J.Y. Kim, G. Krainer, D.C. Lamb, N.K. Lee, E.A. Lemke, B. Levesque, M. Levitus, J.J. McCann, N. Naredi-Rainer, D. Nettels, T. Ngo, R. Qiu, N.C. Robb, C. Röcker, H. Sanabria, M. Schlierf, T. Schröder, B. Schuler, H. Seidel, L. Streit, J. Thurn, P. Tinnefeld, S. Tyagi, N. Vandenberk, A. Manuel Vera, K.R. Weninger, B. Wünsch, I.S. Yanez-Orozco, J. Michaelis, C.A.M. Seidel, T.D. Craggs, T. Hugel, Nature Methods, 15, 669, 2018, [link], preprint on arXiv: [link]

Single-molecule Förster resonance energytransfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ± 0.02 and ± 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

News: The miCube moved to Github

Our open (single-molecule) microscopy project, the #miCube, is now on Github [link]!  The page shows a detailed and updated overview of the components, some information on phasor-based SMLM, and many links to similar open hardware projects.

 

Pre-print: DNA polymerase β fingers movement revealed by single-molecule FRET suggests a partially closed conformation as a fidelity checkpoint

C. Fijen, M. Mahmoud, R. Kaup, J. Towle-Weicksel, J. Sweasy, and J. Hohlbein, bioRxiv, 2018 [link]

The eukaryotic DNA polymerase βplays an important role in cellular DNA repair as it fills gaps in single nucleotide gapped DNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially substrate binding and a fidelity-related conformational change called fingers closing. Here, we applied single-molecule Förster resonance energy transfer to study the conformational dynamics of Pol β. Using an acceptor labelled polymerase and a donor labelled DNA substrate, we measured distance changes associated with DNA binding and fingers movement. Our findings suggest that Pol β does not bend its gapped DNA substrate to the extent related crystal structures indicate: instead, bending seems to be significantly less profound. Furthermore, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. Additionally, we provide evidence that the fingers close only partially when an incorrect nucleotide is bound. This ajar conformation found in Pol β, a polymerase of the X-family, suggests the existence of an additional fidelity checkpoint similar to what has been previously proposed for a member of the A-family, the bacterial DNA polymerase I.

2018_PolB_overview

News: Welcome to Meike Kronenberg and farewell to Carel Fijen

Meike joined the group for her BSc thesis and will work on DNA polymerase beta. Carel successfully defended his PhD thesis in March and decided to continue working on DNA polymerase beta by joining the Sweasy lab at Yale as a postdoctoral research assistent. All the best and we hope receiving new stocks soon!

News: Welcome to Andreas Hentrich and Hans Dekker…

…who joined the group for an 6 week internship (Andreas) and a short capita selecta (Hans). Andreas will try unifying some fragmented software packages for in vivo single particle tracking. Hans will push the limits of our ultrafast pSMLM algorithm for super-resolution microscopy.

Pre-print: Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

T.D. Craggs, M. Sustarsic, A. Plochowietz, M. Mosayebi, H. Kaju, A. Cuthbert, J. Hohlbein, L. Domicevica, P.C. Biggin, J.P. K. Doye, A.N. Kapanidis, bioRxiv, 2018 [link]

DNA-binding proteins utilisedifferent recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarse-grained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy ( = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.

 

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