Interferometric Scattering Microscopy for the Study of Molecular Motors

Andrecka, Joanna; Takagi, Yasuharu; Mickolajczyk, Keith J.; Lippert, Lisa G.; Sellers, James R.; Hancock, William O.; Goldman, Yale E.; Kukura, Philipp

doi:10.1016/bs.mie.2016.08.016

Cited by 22 publications

(21 citation statements)

References 69 publications

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“…1B). MTs were detected label-free, as expected (35)(36)(37), and tubulin-bound gold nanoparticles (tubulin-gold) appeared as points of high contrast (Fig. 1C).…”

Section: Monitoring Individual Tubulins Within Growing Microtubules Wsupporting

confidence: 68%

“…We combined two recently introduced technologies, recombinant tubulin(32) and interferometric scattering microscopy (iSCAT) (33)(34)(35)(36), to make single-molecule observations of individual tubulin dimers binding to growing MT tips. We first generated a homogenous population of S. cerevisiae tubulin biotinylated through a C-terminal KCK epitope (tubulin-KCK) on the β-subunit (Tub2) ( Fig.…”

Section: Monitoring Individual Tubulins Within Growing Microtubules Wmentioning

confidence: 99%

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Direct observation of individual tubulin dimers binding to growing microtubules

Mickolajczyk¹,

Geyer²,

Kim³

et al. 2018

Preprint

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The biochemical basis of microtubule growth has remained elusive for over thirty years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make the first direct, single-molecule measurements of tubulin on-and off-rates. We detect two populations of transient dwell times, and determine via binding-interface mutants that they are separated by the formation of inter-protofilament bonds. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data, and furthermore predict plus-end tapering. Overall, we provide the most direct and complete quantification of how microtubules grow to date. SIGNIFICANCEMicrotubule polymerization dynamics are fundamental to cell migration and cell division, where they are targets for chemotherapy drugs. Despite significant progress, the precise structural and biochemical events occurring at growing microtubule tips are not well defined, and better understanding is necessary for discriminating mechanisms of microtubule dynamics regulation in cells. Here, we visualize individual tubulin subunits reversibly and irreversibly interacting with dynamic microtubule tips, and thereby directly measure tubulin on-and off-rates. By analyzing plus-tip residence times of wild-type and mutant tubulin, we characterize the relative contributions of longitudinal (along protofilaments) and lateral (between protofilaments) bond energies to microtubule growth. This work provides insights into microtubule tip structure and potential modes of microtubule dynamics regulation.

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“…1B). MTs were detected label-free, as expected (35)(36)(37), and tubulin-bound gold nanoparticles (tubulin-gold) appeared as points of high contrast (Fig. 1C).…”

Section: Monitoring Individual Tubulins Within Growing Microtubules Wsupporting

confidence: 68%

Section: Monitoring Individual Tubulins Within Growing Microtubules Wmentioning

confidence: 99%

Direct observation of individual tubulin dimers binding to growing microtubules

Mickolajczyk¹,

Geyer²,

Kim³

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…Monitoring Individual Tubulins Within Growing MTs by iSCAT. We combined two recently introduced technologies, recombinant tubulin (32) and iSCAT (36)(37)(38)(39), to make single-molecule observations of individual tubulin dimers binding to growing MT tips. We first generated a homogenous population of Saccharomyces cerevisiae tubulin biotinylated through a C-terminal KCK epitope (tubulin-KCK) on the β-subunit (Tub2) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the present work, we overcome the longstanding challenge of single-molecule tubulin measurements by applying two recently developed technologies: recombinant tubulin (32)(33)(34)(35) and interferometric scattering microscopy (iSCAT) (36)(37)(38)(39). We first created biochemically homogeneous populations of tubulin subunits sparsely labeled with 20-nm gold nanoparticles.…”

Section: Significancementioning

confidence: 99%

Direct observation of individual tubulin dimers binding to growing microtubules

Mickolajczyk

Geyer

Kim

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.

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“…We sought to resolve the stepping kinetics of individual KIF3A and KIF3C motor domains in homo-and heterodimeric motors and to determine the transport potential of the dimers under mechanical load. To accomplish this, we assayed motor activity using interferometric scattering (iSCAT) microscopy, a recent advance in light microscopy which has sufficient spatiotemporal precision to resolve single steps of a kinesin motor during processive runs (19)(20)(21)(22)(23), and optical trapping which reveals the motor performance under forces hindering and assisting plus-end directed motility (24)(25)(26)(27)(28)(29). We observed that the step dwell time distribution of KIF3AC is not a combination of the dwell time distributions observed for KIF3AA and KIF3CC and is adequately fit by a single exponential rate.…”

Section: Introductionmentioning

confidence: 99%

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

Bensel

Woody

Pyrpassopoulos

et al. 2019

Preprint

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250 max) 249 words 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 homoand 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.Key Words: kinesin  molecular motors single-molecule motility high-speed optical trapping iSCAT microscopy  neuron  microtubule  intracellular transport  cytoskeleton Significance Statement -(120 max) 120 words Kinesins 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.

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Interferometric Scattering Microscopy for the Study of Molecular Motors

Cited by 22 publications

References 69 publications

Direct observation of individual tubulin dimers binding to growing microtubules

Direct observation of individual tubulin dimers binding to growing microtubules

Direct observation of individual tubulin dimers binding to growing microtubules

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

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