Single Molecule Tracking and Localization of Mitochondrial Protein Complexes in Live Cells

2017 ◽  
pp. 273-291 ◽  
Author(s):  
Timo Appelhans ◽  
Karin Busch
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Mitochondrial Protein ◽  
Live Cells ◽  
Single Molecule Tracking

Assembly and Dynamics of the Bacterial Flagellum

2020 ◽  
Vol 74 (1) ◽  
pp. 181-200 ◽  
Author(s):  
Judith P. Armitage ◽  
Richard M. Berry
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Complex Structure ◽  
Live Cells ◽  
Flagellar Motor ◽  
Interacting Protein ◽  
Bacterial Flagellum ◽  
Bacterial Flagellar Motor ◽  
The Stability

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45–50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.

scholarly journals Bacterial Type 3 Secretion Systems: High-Throughput 3D Single-Molecule Tracking of Sorting Platform Proteins in Live Cells

2017 ◽  
Vol 112 (3) ◽  
pp. 149a
Author(s):  
Julian Rocha ◽  
Andreas Diepold ◽  
Judith P. Armitage ◽  
Andreas Gahlmann
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Live Cells ◽  
Secretion Systems ◽  
Single Molecule Tracking ◽  
Type 3 ◽  
Bacterial Type

scholarly journals Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

2014 ◽  
Vol 107 (4) ◽  
pp. 803-814 ◽  
Author(s):  
Peter J. Bosch ◽  
Ivan R. Corrêa ◽  
Michael H. Sonntag ◽  
Jenny Ibach ◽  
Luc Brunsveld ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Tracking

scholarly journals Author response: Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus

2014 ◽  
Author(s):  
Ignacio Izeddin ◽  
Vincent Récamier ◽  
Lana Bosanac ◽  
Ibrahim I Cissé ◽  
Lydia Boudarene ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Molecule ◽  
Search Strategies ◽  
Author Response ◽  
Live Cells ◽  
Target Search ◽  
Single Molecule Tracking

scholarly journals Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Sandra Kunz ◽  
Anke Tribensky ◽  
Wieland Steinchen ◽  
Luis Oviedo-Bocanegra ◽  
Patricia Bedrunka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Live Cells ◽  
Independent Manner ◽  
Single Molecule Tracking ◽  
Content Type ◽  
Diguanylate Cyclases

ABSTRACT Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro, which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro, showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient. IMPORTANCE Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis. Thus, local-pool signal transduction operates extremely efficiently and specifically.

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

2019 ◽  
Vol 141 (40) ◽  
pp. 15747-15750 ◽  
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

scholarly journals Bound2Learn: A Machine Learning Approach for Classification of DNA-Bound Proteins from Single-Molecule Tracking Experiments

2020 ◽  
Author(s):  
Nitin Kapadia ◽  
Ziad W. El-Hajj ◽  
Rodrigo Reyes-Lamothe
Keyword(s):  
Machine Learning ◽  
Single Molecule ◽  
Protein Complexes ◽  
Essential Elements ◽  
Single Particle Tracking ◽  
Learning Approach ◽  
Single Molecule Tracking ◽  
Slow Kinetics ◽  
Machine Learning Approach

AbstractDNA-bound proteins are essential elements for the maintenance, regulation, and use of the genome. The time they spend bound to DNA provides useful information on their stability within protein complexes and insight into the understanding of biological processes. Single-particle tracking allows for direct visualization of protein-DNA kinetics, however, identifying whether a molecule is bound to DNA can be non-trivial. Further complications arise when tracking molecules for extended durations in processes with slow kinetics. We developed a machine learning approach, termed Bound2Learn, using output from a widely used tracking software, to robustly classify tracks in order to accurately estimate residence times. We validated our approach in silico, and in live-cell data from Escherichia coli and Saccharomyces cerevisiae. Our method has the potential for broad utility and is applicable to other organisms.

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