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.
After recently defending his PhD thesis titled “Pinpointing macromolecular motion. Single-particle tracking fluorescence microscopy advances and applications” with distinction (cum laude!), Koen Martens left the group for a postdoc position in the lab of Dr. Ulrike Endesfelder (CMU, Pittsburgh). All the best, Koen, you will be missed!
A big welcome to Martijn Gobes and Cleo Bagchus who both joined the group for their MSc thesis. Martijn will improve our computational workflow for super-resolution microscopy (his first ImageJ plugin for fast temporal median filtering is available here). Cleo will continue our work on single-particle tracking in live cells together with Simon van de Els, who joined the lab as a (semi) post-doctoral research assistant.
Last but not least, we welcome Niels Zijlstra as a guest scientist from the Cordes lab in Munich. Niels will work on combining the miCube platform with spectrally resolved super-resolution imaging.
Good news! Two proposals got granted!
For the first one (IPM-3, Innovation Program Microbiology, an initiative from the Laboratory of Microbiology at Wageningen University and Research), we will use single-particle tracking Photo-Activated Localisation Microscopy (sptPALM) to study CRISPR-Cas in live bacteria. Looking forward collaborating with Raymond Staals from the Laboratory of Microbiology. A one-year post-doc position will be available, please contact me (with CV) for further details.
For the second, the Road-to-Innovation business development grant, we will work on a neat idea for spectrally resolved single-molecule localisation microscopy.
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.
C. Fijen, M. Kronenberg, R. Kaup, M. Fontana, J. Towle-Weicksel, J. Sweasy, J. Hohlbein, Journal of Biological Chemistry, jbc.RA120.0130049, 2020, [link], previous bioRxiv preprint [link]
Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. Observed diffusion coefficients in vivo, for example, enable 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 interchange between different diffusional states. Here, we present analytic diffusion distribution analysis (anaDDA), a framework that allows extracting transition rates from distributions of observed diffusion coefficients. 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 reanalysed experimental data of DNA polymerase I diffusing in live E. 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 behaviour of biomolecules in living cells.
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.
The point spread function (PSF) of single molecule emitters can be engineered in the Fourier plane to encode three-dimensional localization information, creating double-helix, saddle-point or tetra-pod PSFs. Here, we describe and assess adaptations of the phasor-based single-molecule localization microscopy (pSMLM) algorithm to localize single molecules using these PSFs with sub-pixel accuracy. For double-helix, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations, while for saddle-point or tetra-pod, a novel phasor-based deconvolution approach is used. The 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. pSMLM substantially improves the localization rate by a factor of 2 – 4x on a standard CPU, with 1-1.5·104 (double-helix) or 2.5·105 (saddle-point/tetra-pod) localizations/second.
With Mariska, the third PhD student in our NWO funded LocalBioFood project on super-resolution based localisation of biomolecules at food-related interfaces has started. Mariska started her PhD thesis in the Voets lab in Eindhoven and will relocate to our lab in early 2022.
Dani started his BSc thesis in the lab during Covid-19 times. He will look into improving our computational workflow for single-particle tracking in live cells.
Ezra just started his BSc thesis in a new project together with the Laboratory of Microbiology (Wen Wu & Dr. Raymond Staals). He will work on visualising CRISPR-Cas interactions in live bacteria using sptPALM.