Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy

Schneider, René; Glaser, Tilman; Berndt, Michael; Diez, Stefan

doi:10.1364/oe.21.003523

Cited by 17 publications

(20 citation statements)

References 39 publications

(49 reference statements)

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“…Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7)(8)(9)(10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4,11,12), consistent with the motor walking in a handover-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7)(8)(9)(10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.…”

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confidence: 57%

“…Kinesin has served as an important model system for advancing single-molecule techniques (7)(8)(9)(10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.…”

mentioning

confidence: 99%

“…This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7,19,21,22) and single-molecule tracking studies (4,(8)(9)(10)(11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11,12,14,15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.…”

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confidence: 99%

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Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Mickolajczyk

Deffenbaugh

Arroyo

et al. 2015

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

119

222

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To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the onehead-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.kinesin | iSCAT | microscopy | structural kinetics | structure-function K inesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1-6).Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7-10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4,11,12), consistent with the motor walking in a handover-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7-10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head-bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads-bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4,11,(14)(15)(16)(17)(18)(19)(20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7,19,21,22) and sin...

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confidence: 57%

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confidence: 99%

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confidence: 99%

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Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Mickolajczyk

Deffenbaugh

Arroyo

et al. 2015

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

119

222

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“…Experiments to obtain the stepping velocity of single rKin430-SBP-GFP motors were performed in flow channels made of silanized coverslips, as described in ref. 44. Briefly, first a solution of β-tubulin antibodies (0.5% SAP.4G5; Thermo Fisher Scientific) diluted in H20S75 buffer was flushed into a flow channel and incubated for 5 min, followed by a washing step with H20S75 buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescent, Taxol-stabilized rhodamine microtubules (used in the stepping motility assays as well as for the data in Fig. 3 A and B) were prepared as described previously (44). Taxol-stabilized GMP-CPP microtubules (also referred to as "double-stabilized microtubules," used throughout our work, unless mentioned otherwise) were prepared by polymerizing 2.5 μM tubulin mix (1:3 rhodamine-labeled tubulin:unlabeled tubulin) in BRB80 buffer (80 mM Pipes, 1 mM MgCl 2 , 1 mM EGTA, pH 6.9) supplemented with 1.25 mM GMP-CPP and 1.25 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

Grover

Fischer

Schwarz

et al. 2016

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

Self Cite

111

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In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors' anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted "membrane-anchored" gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surfaceimmobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using singlemolecule fluorescence microscopy. Our results illustrate the importance of motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. molecular motors | lipid bilayers | transport efficiency | motor-cargo coupling | streptavidin-binding peptide I ntracellular transport of membrane-bound vesicles and organelles is a process fundamental to many cellular functions including morphogenesis, signaling, and growth (1-4). Active cargo transport inside eukaryotic cells is mediated by ensembles of motor proteins, such as kinesins and dynein, walking on microtubule tracks (5), and myosins walking on actin filaments (6). Gaining mechanistic insight into the functioning of these motors inside the complex environment of cells is challenging. Several studies have thus used in vitro approaches to investigate transport mediated by groups of same or different motors attached to cargos such as silica beads (7), quantum dots (8), glass coverslips (9, 10), or DNA scaffolds (11,12). Although these approaches provide us with knowledge about the collective dynamics of multimotor transport, a key anomaly in these in vitro systems is the use of rather nonphysiological rigid cargo. Vesicular cargo transport by molecular motors requires their attachment to a fluid lipid bilayer either directly or via different adaptor molecules. The anchoring of motors in a diffusive lipid environment induces loose intermotor coupling along with the motors diffusing within the lipid bilayer, thereby increasing the flexibility of the system....

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High-Resolution Single-Molecule Kinesin Assays at kHz Frame Rates

Mickolajczyk

Hancock

2018

Methods in Molecular Biology

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This chapter describes methods for high-speed, unloaded, in vitro single-molecule kinesin tracking experiments. Instructions are presented for constructing a total internal reflection dark-field microscope (TIRDFM) and labeling motors with gold nanoparticles. An AMP-PNP unlocking assay is introduced as a specialized means of capturing processive events in a reduced field of view. Finally, step-finding tools for analyzing high frame-rate tracking data are described.

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Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy

Cited by 17 publications

References 39 publications

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

High-Resolution Single-Molecule Kinesin Assays at kHz Frame Rates

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