Published: Single-molecule localization microscopy as an emerging tool to probe multiscale food structures

J. Hohlbein, Food Structure, 30, 100236, 2021, [link]

Optical microscopy is an indispensable tool to characterize the microstructure of foods at ambient conditions. Depending on both the wavelength of light used to illuminate the sample and the opening angle of the microscope objective, the achievable resolution is limited to around 200 nm. This so-called classical diffraction limit implies that smaller structural features cannot be resolved or separated from each other. As many food structures are ultimately defined by the molecular interactions of single proteins or single molecules, the classical resolution is insufficient to reveal structural details in the (tens of) nanometer range. Intriguingly, recent advancements in imaging techniques originating mostly in the (biomedical) life sciences have been closing the gap, pushing the resolution towards true molecular resolution. In this perspective, we want to highlight some of these emerging techniques and provide an outlook on potential future applications.

Published: Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework

K.J.A. Martens, A. Jabermoradi, S. Yang, and J. Hohlbein, Methods, 193, 2021, [link], previously on bioRxiv [link]

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub- pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.

Published: Probing DNA – transcription factor interactions using single-molecule fluorescence detection in nanofluidic devices

M. Fontana, Š. Ivanovaite , S. Lindhoud, E. van der Wijk, K. Mathwig, W. van den Berg, D. Weijers, and J. Hohlbein, Advanced Biology, 2100953, 2021, [link], preprint: bioRxiv, 2021, [link]

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements, and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element

Published: Quantitative spatiotemporal mapping of lipid and protein oxidation in mayonnaise

S. Yang, A.A. Verhoef, D.W.H Merkx, J.P.M. van Duynhoven, and J. Hohlbein, Antioxidants, 9, 1278, 2020, [link]

Lipid oxidation in food emulsions is mediated by emulsifiers in the water phase and at the oil-water interface. To unravel the physico-chemical mechanisms and to obtain local lipid and protein oxidation rates, we used confocal laser scanning microscopy (CLSM), thereby monitoring changes in both the fluorescence emission of a lipophilic dye BODIPY 665/676 and protein auto-fluorescence. Our data show that the removal of lipid-soluble antioxidants from mayonnaises promotes lipid oxidation within oil droplets as well as protein oxidation at the oil-water interface. Furthermore, we demonstrate that ascorbic acid acts as either a lipid antioxidant or pro-oxidant depending on the presence of lipid-soluble antioxidants. The effects of antioxidant formulation on local lipid and protein oxidation rates were all statistically significant (p < 0.0001). The observed protein oxidation at the oil-water interface was spatially heterogeneous, which is in line with the heterogeneous distribution of lipoprotein granules from the egg yolk used for emulsification. The impact of the droplet size on local lipid and protein oxidation rates was significant (p < 0.0001) but minor compared to the effects of ascorbic acid addition and lipid-soluble antioxidant depletion. The presented results demonstrate that CLSM can be applied for unraveling the roles of colloidal structure and transport in mediating lipid oxidation in complex food emulsions.

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Published: Extracting transition rates in particle tracking using analytical diffusion distribution analysis

J. Vink, S.J.J. Brouns, and J. Hohlbein, Biophysical Journal, 119, 1970–83, 2020, [link], preprint: bioRxiv, 2020, [link]

Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models, especially for molecules that rapidly switch between different diffusional states. Here, we present analytical diffusion distribution analysis (anaDDA), a framework that allows for extracting transition rates from distributions of apparent diffusion coefficients calculated from short trajectories that feature less than 10 localizations per track. Under the assumption that the system is Markovian and diffusion is purely Brownian, we show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics, especially in the fast regime of 0.1–10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalyzed experimental data of DNA polymerase I diffusing in live Escherichia coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behavior of biomolecules in living cells.

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Published: Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis

A. Freire-Rios, K. Tanaka, I. Crespo, E. van der Wijk, Y. Sizentsova, V. Levitsky, S. Lindhoud, M. Fontana, J. Hohlbein, D.R. Boer, V. Mironova, and D. Weijers, Proceedings of the National Academy of Science of the United States of America, 117 (39), 24557, 2020, [link]

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this “caliper” model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF–DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

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Published: Design principles of a minimal auxin response system

H. Kato, S.K. Mutte, H. Suzuki, I. Crespo, S. Das, T. Radoeva, M. Fontana, Y. Yoshitake, E. Hainiwa, W. van den Berg, S. Lindhoud, L. Ishizaki, J. Hohlbein, J.W. Borst, D.R. Boer, R. Nishihama, T. Kohchi, D. Weijers, Nature Plants, 6, 472, 2020, [link], bioRxiv, 2019, [link]

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF tran-scription factor activity. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

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Published: Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of Pol β-DNA complexes

C. Fijen, M. Kronenberg, R. Kaup, M. Fontana, J. Towle-Weicksel, J. Sweasy, J. Hohlbein, Journal of Biological Chemistry, 295, 9012–20, 2020, [link], previous bioRxiv preprint: [link]

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA 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 its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET (smFRET) to measure distance changes associated with DNA binding and pre-chemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase–DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and, thus, repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of pre-chemistry nucleotide selection.

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Published: Spatiotemporal heterogeneity of κ‐carrageenan gels investigated via single-particle-tracking fluorescence microscopy

K.J.A. Martens, J. van Duynhoven, and J. Hohlbein, Langmuir, 36, 5502, 2020, [link]

Hydrogels made of the polysaccharide κ-carrageenan are widely used in the food and personal care industry as thickeners or gelling agents. These hydrogels feature dense regions embedded in a coarser bulk network, but the characteristic size and behavior of these regions has remained elusive. Here, we use single-particle-tracking fluorescence microscopy (sptFM) to quantitatively describe κ-carrageenan gels. Infusing fluorescent probes into fully gelated κ-carrageenan hydrogels resulted in two distinct diffusional behaviors. Obstructed self-diffusion of the probes revealed that the coarse network consists of κ-carrageenan strands with a typical diameter of 3.2 ± 0.3 nm leading to a nanoprobe diffusion coefficient of ~1-5∙10^-12 m2/s. In the dense network regions, we found a fraction with a largely decreased diffusion coefficient of ~1∙10^-13 m2/s. We also observed dynamic exchange between these states. The computation of spatial mobility maps from diffusional data indicated that the dense network regions have a characteristic diameter of ~1 µm and are itself mobile on the seconds-to-minutes timescale. sptFM provides an unprecedented view on spatiotemporal heterogeneity of hydrogel networks, which we believe bears general relevance for understanding transport and release of both low- and high molecular weight solutes.

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Publication: Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism

J.N.A. Vink, K.J.A. Martens, M. Vlot, R.E. McKenzie, C. Almendros, B. Estrada Bonilla, D.J.W. Brocken, J. Hohlbein, S.J.J. Brouns, Molecular Cell, 77, 39-50.e10, 2020, [link], preprint here [link]

CRISPR-Cas systems encode RNA-guided surveil-lance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism andkinetics of target search and its impact on CRISPRprotection levels have remained unknown. Here, wevisualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunitCas8e, Cascade spends half its search time rapidly probing DNA (30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.

Graphical abstract