scholarly journals Motor reattachment kinetics play a dominant role in multimotor-driven cargo transport

2017 ◽  
Author(s):  
Qingzhou Feng ◽  
Keith J. Mickolajczyk ◽  
Geng-Yuan Chen ◽  
William O. Hancock
Keyword(s):  
Single Molecule ◽  
Motor Coordination ◽  
Transport Model ◽  
Dominant Role ◽  
Coordination Mechanisms ◽  
Stopped Flow ◽  
Single Molecule Level ◽  
Cargo Transport ◽  
Run Lengths

ABSTRACTKinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multi-motor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be 4-fold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of gold-nanoparticle-labeled cargo with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than do kinesin-1. Finally, cargo transported by kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multi-motor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads while kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

Enhancing the generating and collecting efficiency of single particle upconverting luminescence at low power excitation

Nanophotonics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1993-2000 ◽  
Author(s):  
Chenshuo Ma ◽  
Chunyan Shan ◽  
Kevin Park ◽  
Aaron T. Mok ◽  
Paul J. Antonick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Particle ◽  
Excitation Power ◽  
Single Particle Tracking ◽  
Rare Earth Ions ◽  
High Excitation ◽  
Single Molecule Level ◽  
Collecting Efficiency ◽  
High Luminescence Intensity

AbstractUpconverting luminescent nanoparticles are photostable, nonblinking, and low chemically toxic fluorophores that are emerging as promising fluorescent probes at the single molecule level. High luminescence intensity upconversion nanoparticles (UCNPs) have previously been achieved by doping with high amounts of rare-earth ions using high excitation power (>2.5 MW/cm2). However, such particles are inadequate for in vitro live-cell imaging and single-particle tracking, as high excitation power can cause photodamage. Here, we compared UCNP luminescence intensities with different dopant concentrations and presented more efficient (about seven times) UCNPs at low excitation power by increasing the concentrations of Yb3+ and Tm3+ dopants (NaYF4: 60% Yb3+, 8% Tm3+) and adding a core-shell structure.

scholarly journals Intracellular cargo transport by single-headed kinesin motors

2019 ◽  
Vol 116 (13) ◽  
pp. 6152-6161 ◽  
Author(s):  
Kristin I. Schimert ◽  
Breane G. Budaitis ◽  
Dana N. Reinemann ◽  
Matthew J. Lang ◽  
Kristen J. Verhey
Keyword(s):  
Intracellular Transport ◽  
Motor Coordination ◽  
Optical Trap ◽  
Coiled Coil ◽  
Force Output ◽  
Cellular Assays ◽  
Cellular Events ◽  
Cargo Transport ◽  
In Cells

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Applied Nano ◽  
2022 ◽  
Vol 3 (1) ◽  
pp. 16-41
Author(s):  
Aurimas Kopūstas ◽  
Mindaugas Zaremba ◽  
Marijonas Tutkus
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Dna Interaction ◽  
Dna Interactions ◽  
Ensemble Averaging ◽  
Target Search ◽  
Single Molecule Level ◽  
Protein Dna Interactions ◽  
Long Time

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

scholarly journals Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks

2019 ◽  
Vol 47 (16) ◽  
pp. 8521-8536 ◽  
Author(s):  
Rogelio Hernández-Tamayo ◽  
Luis M Oviedo-Bocanegra ◽  
Georg Fritz ◽  
Peter L Graumann
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Bacillus Subtilis ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Replication Forks ◽  
Single Molecule Level ◽  
Time Dynamics ◽  
Bacterium Bacillus

AbstractDNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

scholarly journals Molecular motors: forty years of interdisciplinary research

2011 ◽  
Vol 22 (21) ◽  
pp. 3936-3939 ◽  
Author(s):  
James A. Spudich
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Single Molecule Level ◽  
In Vitro Motility ◽  
Nonmuscle Cell ◽  
Nonmuscle Cells ◽  
City Plan

A mere forty years ago it was unclear what motor molecules exist in cells that could be responsible for the variety of nonmuscle cell movements, including the “saltatory cytoplasmic particle movements” apparent by light microscopy. One wondered whether nonmuscle cells might have a myosin-like molecule, well known to investigators of muscle. Now we know that there are more than a hundred different molecular motors in eukaryotic cells that drive numerous biological processes and organize the cell's dynamic city plan. Furthermore, in vitro motility assays, taken to the single-molecule level using techniques of physics, have allowed detailed characterization of the processes by which motor molecules transduce the chemical energy of ATP hydrolysis into mechanical movement. Molecular motor research is now at an exciting threshold of being able to enter into the realm of clinical applications.

Theory of Measuring the Selfsame Single Fluorescent Molecule in Solution Suited for Studying Individual Molecular Interactions by SPSM-FCS

Pteridines ◽  
2002 ◽  
Vol 13 (3) ◽  
pp. 73-82 ◽  
Author(s):  
Zeno Földes-Papp
Keyword(s):  
Single Molecule ◽  
Molecular Interactions ◽  
Spatial Separation ◽  
Absolute Number ◽  
Theoretical Concept ◽  
Correlation Spectroscopy ◽  
Single Individual ◽  
Fluorescent Molecule ◽  
Single Molecule Level

Abstract Three exact criteria are first derived for the probabilities that determine single molecule sensitivity in single-phases, for example in solutions or membranes. It is shown how the criteria can be used to decide whether single molecule sensitivity is obtained. Most straightforwardly, all we require is that we experimentally determine the Poisson probability for the absolute number of fluorescent molecules in the volume of observation. This is achieved by fluorescence correlation spectroscopy and allows identifying the selfsame, one single fluorescent molecule in single-phases. Further, we first provide the new and powerful analytical capability to fluorescencetagged inidividual molecules and reactions in in-vitro and living systems to follow their molecular interactions and function by means of spatial stochastic behavior. This fully stochastic modeling is derived which describes the influence of spatial separation on the reaction coordinate of one single individual molecule in single-phases within the observation volume. The theoretical concept is applied to experimental data 'at the single-molecule level' of photosensitive riboflavin and riboflavin-containing blue fluorescence protein. The data were taken from the literature. As simulation results of the elucidative examples, experiments are suggested that would measure individual, single molecules of flavins and flavoproteins m solution. They are of importance for experimentalists.

VISUALIZATION EX SITU OF SINGLE DNA MOLECULES INCUBATION: A FIRST STEP FOR QUANTITATIVE ANALYSIS ON MULTI-SITE DEGRADATION AND ENZYMATIC KINETICS

2009 ◽  
Vol 16 (01) ◽  
pp. 79-85
Author(s):  
XINYAN WANG ◽  
HAIJUN YANG ◽  
HUABIN WANG ◽  
PENG WANG ◽  
HAI LI
Keyword(s):  
Single Molecule ◽  
Ex Situ ◽  
In Vitro Tests ◽  
Dna Molecules ◽  
Single Molecule Level ◽  
Single Dna Molecules ◽  
Enzymatic Kinetics ◽  
Afm Imaging ◽  
Molecular Combing

Herein, we showed a different approach to directly single-molecule level visualization of the degradation of DNA in vitro tests using DNase I incubation based on high-resolution AFM imaging ex situ with fine relocation nanotechnology. A method of nanomanipulation termed as "modified dynamic molecular combing" (MDMC) was used to pattern DNA samples for further degradation and enzymatic kinetics. This strategy is potentially able to quantitatively address the mechanical-induced kinetic profiles of multi-site degradation of individual DNA molecules with very stable tension and strong immobilization on a surface and discover the mechanochemistry.

scholarly journals Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

2013 ◽  
Vol 14 (2) ◽  
pp. 3961-3992 ◽  
Author(s):  
Carina Monico ◽  
Marco Capitanio ◽  
Gionata Belcastro ◽  
Francesco Vanzi ◽  
Francesco Pavone
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Optical Methods ◽  
Dna Interactions ◽  
Single Molecule Level ◽  
Protein Dna Interactions
Sign in / Sign up

Export Citation Format

Share Document