Comparison of in-vitro and in-vivo DNA Hybridization Kinetics using 3D Single-Molecule Tracking Method

ABSTRACT Although DNA-compacting proteins have been extensively characterized in vitro, knowledge of their DNA binding dynamics in vivo is greatly lacking. We have employed single-molecule tracking to characterize the motion of the three major chromosome compaction factors in Bacillus subtilis, Smc (structural maintenance of chromosomes) proteins, topoisomerase DNA gyrase, and histone-like protein HBsu. We show that these three proteins display strikingly different patterns of interaction with DNA; while Smc displays two mobility fractions, one static and one moving through the chromosome in a constrained manner, gyrase operates as a single slow-mobility fraction, suggesting that all gyrase molecules are catalytically actively engaged in DNA binding. Conversely, bacterial histone-like protein HBsu moves through the nucleoid as a larger, slow-mobility fraction and a smaller, high-mobility fraction, with both fractions having relatively short dwell times. Turnover within the SMC complex that makes up the static fraction is shown to be important for its function in chromosome compaction. Our report reveals that chromosome compaction in bacteria can occur via fast, transient interactions in vivo, avoiding clashes with RNA and DNA polymerases. IMPORTANCE All types of cells need to compact their chromosomes containing their genomic information several-thousand-fold in order to fit into the cell. In eukaryotes, histones achieve a major degree of compaction and bind very tightly to DNA such that they need to be actively removed to allow access of polymerases to the DNA. Bacteria have evolved a basic, highly dynamic system of DNA compaction, accommodating rapid adaptability to changes in environmental conditions. We show that the Bacillus subtilis histone-like protein HBsu exchanges on DNA on a millisecond scale and moves through the entire nucleoid containing the genome as a slow-mobility fraction and a dynamic fraction, both having short dwell times. Thus, HBsu achieves compaction via short and transient DNA binding, thereby allowing rapid access of DNA replication or transcription factors to DNA. Topoisomerase gyrase and B. subtilis Smc show different interactions with DNA in vivo, displaying continuous loading or unloading from DNA, or using two fractions, one moving through the genome and one statically bound on a time scale of minutes, respectively, revealing three different modes of DNA compaction in vivo.

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Measuring DNA Hybridization Kinetics in Live Cells Using a Time-Resolved 3D Single-Molecule Tracking Method

Journal of the American Chemical Society ◽

10.1021/jacs.9b08036 ◽

2019 ◽

Vol 141 (40) ◽

pp. 15747-15750 ◽

Cited By ~ 1

Author(s):

Yuan-I Chen ◽

Yin-Jui Chang ◽

Trung Duc Nguyen ◽

Cong Liu ◽

Stephanie Phillion ◽

...

Keyword(s):

Single Molecule ◽

Dna Hybridization ◽

Live Cells ◽

Time Resolved ◽

Single Molecule Tracking ◽

Tracking Method

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The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽

10.3390/biom11030360 ◽

2021 ◽

Vol 11 (3) ◽

pp. 360

Author(s):

Pieterjan Debie ◽

Noemi B. Declerck ◽

Danny van Willigen ◽

Celine M. Huygen ◽

Bieke De Sloovere ◽

...

Keyword(s):

Single Molecule ◽

Gamma Ray ◽

Nuclear Imaging ◽

Primary Amines ◽

Resection Margins ◽

Fluorescent Light ◽

Single Structure ◽

Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

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Single molecule tracking of Ace1p in Saccharomyces cerevisiae defines a characteristic residence time for non-specific interactions of transcription factors with chromatin

Nucleic Acids Research ◽

10.1093/nar/gkw744 ◽

2016 ◽

Vol 44 (21) ◽

pp. e160-e160 ◽

Cited By ~ 23

Author(s):

David A Ball ◽

Gunjan D Mehta ◽

Ronit Salomon-Kent ◽

Davide Mazza ◽

Tatsuya Morisaki ◽

...

Keyword(s):

Transcription Factors ◽

Residence Time ◽

Single Molecule ◽

Specific Binding ◽

Underlying Mechanism ◽

Specific Interactions ◽

Single Molecule Tracking ◽

Reliable Performance ◽

Transient Interactions

Abstract In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12–0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.

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Synthesis runs counter to directional folding of a nascent protein domain

Nature Communications ◽

10.1038/s41467-020-18921-8 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xiuqi Chen ◽

Nandakumar Rajasekaran ◽

Kaixian Liu ◽

Christian M. Kaiser

Keyword(s):

Single Molecule ◽

Molten Globule ◽

Elongation Factor ◽

Protein Domain ◽

Folding Pathway ◽

Native Structure ◽

Folding Intermediates ◽

C Terminus

Abstract Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

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Rational design of protein-specific folding modifiers

10.1101/2020.04.28.064113 ◽

2020 ◽

Author(s):

Anirban Das ◽

Anju Yadav ◽

Mona Gupta ◽

R Purushotham ◽

Vishram L. Terse ◽

...

Keyword(s):

Single Molecule ◽

Rational Design ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Force Measurements ◽

Folding Nucleus ◽

Dynamics Simulations ◽

General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

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C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

10.1101/835082 ◽

2019 ◽

Cited By ~ 4

Author(s):

Laura Fumagalli ◽

Florence L. Young ◽

Steven Boeynaems ◽

Mathias De Decker ◽

Arpan R. Mehta ◽

...

Keyword(s):

Axonal Transport ◽

Motor Neurons ◽

Single Molecule ◽

Induced Pluripotent Stem Cell ◽

Cytoplasmic Dynein ◽

Inhibitory Interaction ◽

Hexanucleotide Repeat ◽

Repeat Expansions

ABSTRACTHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

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Synthesis runs counter to directional folding of a nascent protein domain

10.1101/2020.04.29.068593 ◽

2020 ◽

Cited By ~ 1

Author(s):

Xiuqi Chen ◽

Nandakumar Rajasekaran ◽

Kaixian Liu ◽

Christian M. Kaiser

Keyword(s):

Single Molecule ◽

Molten Globule ◽

Elongation Factor ◽

Protein Domain ◽

Folding Pathway ◽

Native Structure ◽

Folding Intermediates ◽

C Terminus

AbstractFolding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

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Transcription factor clusters regulate genes in eukaryotic cells

eLife ◽

10.7554/elife.27451 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 38

Author(s):

Adam JM Wollman ◽

Sviatlana Shashkova ◽

Erik G Hedlund ◽

Rosmarie Friemann ◽

Stefan Hohmann ◽

...

Keyword(s):

Single Molecule ◽

Functional Unit ◽

Yeast Genome ◽

Gene Promoters ◽

Intrinsically Disordered ◽

Extracellular Signal ◽

Genome Model ◽

Nuclear Targets

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

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Vesicle Size Regulates Nanotube Formation in the Cell

Scientific Reports ◽

10.1038/srep24002 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 20

Author(s):

Qian Peter Su ◽

Wanqing Du ◽

Qinghua Ji ◽

Boxin Xue ◽

Dong Jiang ◽

...

Keyword(s):

Single Molecule ◽

Model Systems ◽

Organelle Biogenesis ◽

Membrane Deformation ◽

Vesicle Size ◽

Cellular Processes ◽

Vesicle Recycling ◽

Physiological Relevance

Abstract Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling.

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