scholarly journals Strain-Dependent Kinetic Properties of KIF3A and KIF3C Tune the Mechanochemistry of the KIF3AC Heterodimer

2019 ◽  
Author(s):  
Brandon M. Bensel ◽  
Michael S. Woody ◽  
Serapion Pyrpassopoulos ◽  
Yale E. Goldman ◽  
Susan P. Gilbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Load ◽  
Optical Trap ◽  
Slow Step ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Cargo Transport ◽  
Mammalian Neuron ◽  
Kinetics Of ◽  
Strain Dependent

AbstractKIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo-and heterodimeric constructs, and to determine their transport potential under mechanical load, we assayed motor activity using interferometric scattering (iSCAT) microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1 = 11 s−1) without apparent kinetic asymmetry in stepping. Asymmetry was also not apparent under hindering or assisting mechanical loads of 1 pN in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA, yet is more capable of stepping against mechanical load than KIF3CC. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, indicating the homodimers mechanically impact each other’s motility to reproduce the behavior of the heterodimer. We conclude that the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner which is similar to application of an assisting load, and the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. These results suggest that KIF3AC-based cargo transport likely requires multiple motors, and its mechanochemical properties arise due to the strain-dependences of KIF3A and KIF3C.Significance StatementKinesins are important long-range intracellular transporters in neurons required by the extended length of the axon and dendrites and selective cargo transport to each. The mammalian kinesin-2, KIF3AC, is a neuronal heterodimer of fast and slow motor polypeptides. Our results show that KIF3AC has a single observed stepping rate in the presence and absence of load and detaches from the microtubule rapidly under load. Interestingly, both KIF3A and assisting loads accelerate the kinetics of KIF3C. These results suggest that KIF3AC is an unconventional cargo transporter and its motile properties do not represent a combination of alternating fast and slow step kinetics. We demonstrate that the motile properties of KIF3AC represent a mechanochemistry that is specific to KIF3AC and may provide functional advantages in neurons.

scholarly journals The mechanochemistry of the kinesin-2 KIF3AC heterodimer is related to strain-dependent kinetic properties of KIF3A and KIF3C

2020 ◽  
Vol 117 (27) ◽  
pp. 15632-15641
Author(s):  
Brandon M. Bensel ◽  
Michael S. Woody ◽  
Serapion Pyrpassopoulos ◽  
Yale E. Goldman ◽  
Susan P. Gilbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Load ◽  
Optical Trap ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Force Sensitivity ◽  
Mammalian Neuron ◽  
Kinetics Of ◽  
Strain Dependent ◽  
Transport Potential

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo- and heterodimeric constructs and determine their transport potential under load, we assayed motor activity using interferometric scattering microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1= 11 s−1) without apparent kinetic asymmetry. Asymmetry was also not apparent under hindering or assisting mechanical loads in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA yet is more capable of stepping against mechanical load than KIF3CC. Interestingly, the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, suggesting the homodimers mechanically impact each other’s motility to reproduce the behavior of the heterodimer. Our observations are consistent with a mechanism in which the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner, similar to application of an assisting load. These results suggest that the mechanochemical properties of KIF3AC can be explained by the strain-dependent kinetics of KIF3A and KIF3C.

scholarly journals The C-terminal actin binding domain of talin forms an asymmetric catch bond with F-actin

2020 ◽  
Author(s):  
Leanna M. Owen ◽  
Nick A. Bax ◽  
William I. Weis ◽  
Alexander R. Dunn
Keyword(s):  
Single Molecule ◽  
Optical Trap ◽  
Focal Adhesions ◽  
Actin Binding ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Dimerization Domain ◽  
Catch Bond ◽  
Cellular Cytoskeleton

AbstractFocal adhesions (FAs) are large, integrin-based adhesion complexes that link cells to the extracellular matrix (ECM). Previous work demonstrates that FAs form only when and where they are necessary to transmit force between the cellular cytoskeleton and the ECM, but how this occurs remains poorly understood. Talin is a 270 kDa adapter protein that links integrins to filamentous (F)-actin and recruits additional components during FA assembly in a force-dependent manner. Cell biological and developmental data demonstrate that the third, and C-terminal, F-actin binding site (ABS3) of talin is required for normal FA formation. However, ABS3 binds F-actin only weakly in in vitro, biochemical assays. We used a single-molecule optical trap assay to examine how and whether ABS3 binds F-actin under physiologically relevant, pN mechanical loads. We find that ABS3 forms a directional catch bond with F-actin when force is applied towards the pointed end of the actin filament, with binding lifetimes more than 100-fold longer than when force is applied towards the barbed end. Long-lived bonds to F-actin under load require the ABS3 C-terminal dimerization domain, whose cleavage is known to regulate focal adhesion turnover. Our results support a mechanism in which talin ABS3 preferentially binds and orients actin filaments with barbed ends facing the cell periphery, thus nucleating long-range order in the actin cytoskeleton. We suggest that talin ABS3 may function as a molecular AND gate that allows FA growth only when sufficient integrin density, F-actin polarization, and mechanical tension are simultaneously present.

scholarly journals Single-molecule transport kinetics of a glutamate transporter homolog shows static disorder

2020 ◽  
Vol 6 (22) ◽  
pp. eaaz1949 ◽  
Author(s):  
Didar Ciftci ◽  
Gerard H. M. Huysmans ◽  
Xiaoyu Wang ◽  
Changhao He ◽  
Daniel Terry ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Glutamate Transporter ◽  
Resonance Energy ◽  
Fold Increase ◽  
Transport Kinetics ◽  
Kinetic Properties ◽  
Molecule Transport ◽  
Kinetics Of

Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer–based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting “molecular memory.”

scholarly journals Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells

2020 ◽  
Author(s):  
Amanda Koch ◽  
Luis Aguilera ◽  
Tatsuya Morisaki ◽  
Brian Munsky ◽  
Timothy J. Stasevich
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Host Cells ◽  
Spatial Distributions ◽  
Dependent Manner ◽  
Single Molecule Tracking ◽  
Single Molecule Level ◽  
The One ◽  
Kinetics Of ◽  
Relative Kinetics

ABSTRACTViruses use IRES sequences within their RNA to hijack translation machinery and thereby rapidly replicate in host cells. While this process has been extensively studied in bulk assays, the dynamics of hijacking at the single-molecule level remain unexplored in living cells. To achieve this, we developed a bicistronic biosensor encoding complementary repeat epitopes in two ORFs, one translated in a Cap-dependent manner and the other translated in an IRES-mediated manner. Using a pair of complementary probes that bind the epitopes co-translationally, our biosensor lights up in different colors depending on which ORF is being translated. In combination with single-molecule tracking and computational modeling, we measured the relative kinetics of Cap versus IRES translation and show: (1) Two non-overlapping ORFs can be simultaneously translated within a single mRNA; (2) EMCV IRES-mediated translation sites recruit ribosomes less efficiently than Cap-dependent translation sites but are otherwise nearly indistinguishable, having similar mobilities, sizes, spatial distributions, and ribosomal initiation and elongation rates; (3) Both Cap-dependent and IRES-mediated ribosomes tend to stretch out translation sites; (4) Although the IRES recruits two to three times fewer ribosomes than the Cap in normal conditions, the balance shifts dramatically in favor of the IRES during oxidative and ER stresses that mimic viral infection; and (5) Translation of the IRES is enhanced by translation of the Cap, demonstrating upstream translation can positively impact the downstream translation of a non-overlapping ORF. With the ability to simultaneously quantify two distinct translation mechanisms in physiologically relevant live-cell environments, we anticipate bicistronic biosensors like the one we developed here will become powerful new tools to dissect both canonical and non-canonical translation dynamics with single-molecule precision.Graphical Abstract

Multiplex single-molecule kinetics of nanopore-coupled polymerases

2020 ◽  
Author(s):  
Mirkό Palla ◽  
Sukanya Punthambaker ◽  
P. Benjamin Stranges ◽  
Frederic Vigneault ◽  
Jeff Nivala ◽  
...  
Keyword(s):  
Single Molecule ◽  
Screening Method ◽  
Rapid Screening ◽  
Kinetic Properties ◽  
Nanopore Sequencing ◽  
Sequencing Platform ◽  
Dna Strands ◽  
Potential Applications ◽  
Polymerase Mutants ◽  
Kinetics Of

AbstractDNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for unique properties gives the opportunity to identify polymerases with novel features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to a α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ~100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with unique or altered physical properties.

scholarly journals KIF3A accelerates KIF3C within the kinesin-2 heterodimer to generate symmetrical phosphate release rates for each processive step

2020 ◽  
pp. jbc.RA120.015272
Author(s):  
Sean M. Quinn ◽  
Troy Vargason ◽  
Nilisha Pokhrel ◽  
Edwin Antony ◽  
Juergen Hahn ◽  
...  
Keyword(s):  
Single Molecule ◽  
Slow Step ◽  
Phosphate Release ◽  
Intrinsic Kinetics ◽  
The Central Nervous System ◽  
Atp Binding And Hydrolysis ◽  
Future Work ◽  
Rate Limiting ◽  
Kinetics Of

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in the central nervous system and is associated with vesicles in neurons. KIF3AC is an intriguing member of the kinesin-2 family because the intrinsic kinetics of KIF3A and KIF3C when expressed as homodimers and analyzed in vitro are distinctively different from each other. For example, the single-molecule velocities of the engineered homodimers KIF3AA and KIF3CC are 293 nm/s and 7.5 nm/s, respectively, whereas KIF3AC has a velocity of 186 nm/s. These results led us to hypothesize that heterodimerization alters the intrinsic catalytic properties of the two heads, and an earlier computational analysis predicted that processive steps would alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping. To test this hypothesis directly, we measured the presteady-state kinetics of phosphate release for KIF3AC, KIF3AA, and KIF3CC followed by computational modeling of the KIF3AC phosphate release transients. The results reveal that KIF3A and KIF3C retain their intrinsic ATP binding and hydrolysis kinetics. Yet within KIF3AC, KIF3A activates the rate of phosphate release for KIF3C such that the coupled steps of phosphate release and dissociation from the microtubule become more similar for KIF3A and KIF3C. These coupled steps are the rate-limiting transition for the ATPase cycle suggesting that within KIF3AC, the stepping kinetics are similar for each head during the processive run. Future work will be directed to define how these properties enable KIF3AC to achieve its physiological functions.

scholarly journals Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

2012 ◽  
Vol 287 (42) ◽  
pp. 35275-35285 ◽  
Author(s):  
Rustem I. Litvinov ◽  
Andrey Mekler ◽  
Henry Shuman ◽  
Joel S. Bennett ◽  
Valeri Barsegov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Transition ◽  
Dissociation Constants ◽  
Optical Trap ◽  
Free Association ◽  
Correlation Spectroscopy ◽  
Two Dimensional ◽  
Integrin Αiibβ3 ◽  
High Affinity ◽  
Kinetics Of

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (kon1 = 1.4 × 10−4 and kon2 = 2.3 × 10−4 μm2/s), off-rates (koff1 = 2.42 and koff2 = 0.60 s−1), and dissociation constants (Kd1 = 1.7 × 104 and Kd2 = 2.6 × 103 μm−2). The integrin activator Mn2+ changed the on-rates and affinities (Kd1 = 5 × 104 and Kd2 = 0.3 × 103 μm−2) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn2+-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.

Mouse paternal-RNAs initiate pattern of metabolic disorders in a strain dependent manner

2016 ◽  
Author(s):  
Guzide Satir Basaran ◽  
Yagut Akbarova ◽  
Kezban Korkmaz ◽  
Kursad Unluhizarci ◽  
Francois Cuzin ◽  
...  
Keyword(s):  
Metabolic Disorders ◽  
Dependent Manner ◽  
Strain Dependent

Atomistic Simulation Study of Controlled Nanostructure Patterning

2003 ◽  
Vol 775 ◽  
Author(s):  
Byeongchan Lee ◽  
Kyeongjae Cho
Keyword(s):  
Diffusion Barrier ◽  
Atomistic Simulation ◽  
Degrees Of Freedom ◽  
Embedded Atom Method ◽  
Surface Kinetics ◽  
Bulk Properties ◽  
Embedded Atom ◽  
Kinetics Of ◽  
Dissociation Barrier ◽  
Strain Dependent

AbstractWe investigate the surface kinetics of Pt using the extended embedded-atom method, an extension of the embedded-atom method with additional degrees of freedom to include the nonbulk data from lower-coordinated systems as well as the bulk properties. The surface energies of the clean Pt (111) and Pt (100) surfaces are found to be 0.13 eV and 0.147 eV respectively, in excellent agreement with experiment. The Pt on Pt (111) adatom diffusion barrier is found to be 0.38 eV and predicted to be strongly strain-dependent, indicating that, in the compressive domain, adatoms are unstable and the diffusion barrier is lower; the nucleation occurs in the tensile domain. In addition, the dissociation barrier from the dimer configuration is found to be 0.82 eV. Therefore, we expect that atoms, once coalesced, are unlikely to dissociate into single adatoms. This essentially tells that by changing the applied strain, we can control the patterning of nanostructures on the metal surface.

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