Left-handed DNA-PAINT for improved super-resolution imaging in the nucleus

Geertsema, Hylkje; Aimola, Giulia; Fabricius, Valentin; Fuerste, J.P.; Kaufer, Benedikt B.; Ewers, Helge

doi:10.1038/s41587-020-00753-y

Cited by 27 publications

(29 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To provide a definitive understanding of the events that took place and led to the observed phenotypes, one needs to be able to visualize the presence of chromosomal and plasmid DNA from the two parent cells in single cells during the selection process of Figure 1. This will require the development of new bioimaging technology not currently available for prokaryotic cells, such as the DNA PAINT technology (20)(21)(22).…”

Section: Discussionmentioning

confidence: 99%

Novel mechanism of plasmid-DNA transfer mediated by heterologous cell fusion in syntrophic coculture of Clostridium organisms

Charubin

Gregory

Papoutsakis

2021

Preprint

View full text Add to dashboard Cite

The evolution of bacteria is driven by random genetic mutations and horizontal gene transfer (HGT) of genetic material from other bacteria. HGT can occur via transformation, transduction, and conjugation. Here, we present a potential new mechanism of HGT which occurs in a syntrophic Clostridium coculture. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, the two organisms undergo heterologous cell fusion, which includes fusion of the peptidoglycan cell walls and membranes. Heterologous cell fusion facilitated a large-scale exchange of cytoplasmic protein and RNA between the two organisms, leading to the formation of hybrid bacterial cells containing cytoplasmic material of the two parent organisms. Here we present new evidence that cell fusion events also facilitate the exchange of plasmid DNA between the two organisms of the syntrophic coculture. Through the use of a selective subculturing process, we successfully isolated wild-type C. acetobutylicum clones which have acquired a portion of the plasmid DNA – containing the antibiotic resistance marker – from a recombinant strain of C. ljungdahlii. Fusion events led to formation of persistent aberrant hybrid cells with distinct morphogenetic characteristics. Furthermore, our data support the concept of a novel, interspecies, mechanism of acquiring antibiotic resistance. Since neither organism contains any known conjugation machinery or mechanism, these findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.IMPORTANCEInvestigations of natural multispecies microbiomes and the field of synthetic and syntrophic microbial cocultures are attracting renewed interest based on their potential application in biotechnology, ecology, and medical fields. A variety of synthetic and natural cocultures have been examined in terms of their metabolic output, but relatively few systems have been interrogated at the cellular and molecular level. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms, and leads to the formation of hybrid bacterial cells. Continuing this line of investigation, we now show that heterologous cell fusion between the two Clostridium organisms can also facilitate the exchange of DNA between the two organisms. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Fusion led to persistent hybrid cells containing DNA from both parents but with distinct properties and morphologies. Moreover, we provide evidence for a novel mechanism of acquiring antibiotic resistance mediated by the syntrophic interactions of this system. This is a major finding that may shed light on a new mechanism of bacteria’s ability to acquire antibiotic resistance.

show abstract

Section: Discussionmentioning

confidence: 99%

Novel mechanism of plasmid-DNA transfer mediated by heterologous cell fusion in syntrophic coculture of Clostridium organisms

Charubin

Gregory

Papoutsakis

2021

Preprint

View full text Add to dashboard Cite

show abstract

“…24,26,[28][29][30] Furthermore, the use of left-handed DNA has improved specificity in DNA containing samples. 31 When compared directly, resPAINT offers some advantages over DNA-PAINT in that: 1) it is fully compatible with DNA containing samples; 2) conjugation with DNA can be experimentally complex and 3) DNA-PAINT can suffer from binding-site depletion, although appropriate buffers can somewhat mitigate this. 32 Indeed, a potential application of resPAINT would be imaging DNA in whole cell nuclei with Hoechst.…”

Section: Discussionmentioning

confidence: 99%

“…Instead, methods have been developed where the probe ‘lights up’ or is concentrated on target versus in solution by way of Förster resonance energy transfer (FRET), 26 fluorogenic probes, 27 repeat PAINT, 28 quenching, 29 photoactivation 30 and many others. While these strategies greatly improve volumetric imaging with DNA PAINT, they retain inherent drawbacks including: unspecific binding 31 , photocleavage 32 , the necessity of immunolabeling and an inability to observe direct binding events such as peptide/receptor interactions. Application of similar lightup strategies to Fab or small molecule binders would greatly increase the range of biological interactions that can be observed using PAINT.…”

Section: Introductionmentioning

confidence: 99%

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Sanders

Carr

Bruggeman

et al. 2022

Preprint

View full text Add to dashboard Cite

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a "reservoir" of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix point-spread function, the tetrapod point-spread function and single-molecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

show abstract

“…Thus, live-cell applications of DNA-PAINT have so far been very limited, and only employed for imaging molecules on cell surfaces ( Strauss et al, 2018 ). A very recent study surmounted the latter challenge by designing “docker” and ‘imager’ probes with left-handed DNA (L-DNA) which do not hybridize with endogenous nucleic acids ( Geertsema et al, 2021 ). Still, it seems clear that continued refinement of these techniques will be necessary to gain further experimental insight into dynamic changes in CRU structure and function at the length scale of single RyRs.…”

Section: The Changing Interaction Of Experiments and Simulation In Th...mentioning

confidence: 99%

Image-Driven Modeling of Nanoscopic Cardiac Function: Where Have We Come From, and Where Are We Going?

2022

View full text Add to dashboard Cite

Complementary developments in microscopy and mathematical modeling have been critical to our understanding of cardiac excitation–contraction coupling. Historically, limitations imposed by the spatial or temporal resolution of imaging methods have been addressed through careful mathematical interrogation. Similarly, limitations imposed by computational power have been addressed by imaging macroscopic function in large subcellular domains or in whole myocytes. As both imaging resolution and computational tractability have improved, the two approaches have nearly merged in terms of the scales that they can each be used to interrogate. With this review we will provide an overview of these advances and their contribution to understanding ventricular myocyte function, including exciting developments over the last decade. We specifically focus on experimental methods that have pushed back limits of either spatial or temporal resolution of nanoscale imaging (e.g., DNA-PAINT), or have permitted high resolution imaging on large cellular volumes (e.g., serial scanning electron microscopy). We also review the progression of computational approaches used to integrate and interrogate these new experimental data sources, and comment on near-term advances that may unify understanding of the underlying biology. Finally, we comment on several outstanding questions in cardiac physiology that stand to benefit from a concerted and complementary application of these new experimental and computational methods.

show abstract

Left-handed DNA-PAINT for improved super-resolution imaging in the nucleus

Cited by 27 publications

References 29 publications

Novel mechanism of plasmid-DNA transfer mediated by heterologous cell fusion in syntrophic coculture of Clostridium organisms

Novel mechanism of plasmid-DNA transfer mediated by heterologous cell fusion in syntrophic coculture of Clostridium organisms

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Image-Driven Modeling of Nanoscopic Cardiac Function: Where Have We Come From, and Where Are We Going?

Contact Info

Product

Resources

About