scholarly journals Single-particle tracking of Nucleotide Excision Repair proteins inside living bacteria

2018 ◽  
Author(s):  
Alicja Piotrowska ◽  
Mathew Stracy ◽  
Pawel Zawadzki
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Excision Repair ◽  
Ccd Camera ◽  
Single Particle Tracking ◽  
Point Of View ◽  
Photoactivated Localization Microscopy ◽  
Nucleotide Excision ◽  
Multiple Frames

Single-particle tracking (SPT) combined with Photoactivated Localization Microscopy (sptPALM) provides an opportunity to perform complex molecular biology experiments inside living cells. By tracking the motion of DNA repair proteins in vivo, information can be extracted not only about their diffusion, but also about the kinetics and spatial distribution of DNA binding1–3. From a methodological point of view, a Total Internal Reflection Microscope equipped with a sensitive detector (usually an EM-CCD camera4) is commonly used, allowing detection of individual fluorophores. The signal from individual emitters can be analysed and the position of a given fluorophore established with high accuracy (up to a single nm) by Gaussian fitting. To determine the mobility of each fluorophore, the positions of individual molecules are linked into trajectories over multiple frames using a tracking algorithm5.

scholarly journals Cega: A Single Particle Segmentation Algorithm to Identify Moving Particles in a Noisy System

2020 ◽  
Author(s):  
Erin M. Masucci ◽  
Peter K. Relich ◽  
E. Michael Ostap ◽  
Erika L. F. Holzbaur ◽  
Melike Lakadamyali
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Molecular Motors ◽  
Single Particle Tracking ◽  
Particle Detection ◽  
Identification Rate ◽  
Detection Techniques ◽  
Noticeable Improvement

ABSTRACTImprovements to particle tracking algorithms are required to effectively analyze the motility of biological molecules in complex or noisy systems. A typical single particle tracking (SPT) algorithm detects particle coordinates for trajectory assembly. However, particle detection filters fail for datasets with low signal-to-noise levels. When tracking molecular motors in complex systems, standard techniques often fail to separate the fluorescent signatures of moving particles from background noise. We developed an approach to analyze the motility of kinesin motor proteins moving along the microtubule cytoskeleton of extracted neurons using the Kullback-Leibler (KL) divergence to identify regions where there are significant differences between models of moving particles and background signal. We tested our software on both simulated and experimental data and found a noticeable improvement in SPT capability and a higher identification rate of motors as compared to current methods. This algorithm, called Cega, for ‘find the object’, produces data amenable to conventional blob detection techniques that can then be used to obtain coordinates for downstream SPT processing. We anticipate that this algorithm will be useful for those interested in tracking moving particles in complex in vitro or in vivo environments.

scholarly journals Probing Membrane Nanodomain Organization with Single Particle Tracking via Photoactivated Localization Microscopy (spt-PALM)

2019 ◽  
Vol 25 (S2) ◽  
pp. 1248-1249
Author(s):  
Yerim Lee ◽  
Carey Phelps ◽  
Tao Huang ◽  
Barmak Mostofian ◽  
Daniel Zuckerman ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy

scholarly journals Extracting transition rates in single-particle tracking using analytical diffusion distribution analysis

2020 ◽  
Author(s):  
Jochem N.A. Vink ◽  
Stan J.J. Brouns ◽  
Johannes Hohlbein
Keyword(s):  
Dna Polymerase ◽  
Particle Tracking ◽  
Single Particle ◽  
Diffusion Coefficients ◽  
Dna Interaction ◽  
Single Particle Tracking ◽  
Distribution Analysis ◽  
Transition Rates ◽  
Chromosomal Dna

AbstractSingle-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.

scholarly journals Silver Ions Caused Faster Diffusive Dynamics of Histone-Like Nucleoid-Structuring Proteins in Live Bacteria

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Asmaa A. Sadoon ◽  
Prabhat Khadka ◽  
Jack Freeland ◽  
Ravi Kumar Gundampati ◽  
Ryan H. Manso ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Antimicrobial Agents ◽  
Silver Ions ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy ◽  
Diffusive Dynamics ◽  
Live Bacteria

ABSTRACT The antimicrobial activity and mechanism of silver ions (Ag+) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag+ ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag+ ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag+ ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag+ ions: Ag+-induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria. IMPORTANCE As so-called “superbug” bacteria resistant to commonly prescribed antibiotics have become a global threat to public health in recent years, noble metals, such as silver, in various forms have been attracting broad attention due to their antimicrobial activities. However, most of the studies in the existing literature have relied on the traditional bioassays for studying the antimicrobial mechanism of silver; in addition, temporal resolution is largely missing for understanding the effects of silver on the molecular dynamics inside bacteria. Here, we report our study of the antimicrobial effect of silver ions at the nanoscale on the diffusive dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy. This work highlights the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Sign in / Sign up

Export Citation Format

Share Document