Integrated multi-wavelength microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes

Nong, Daguan; Haviland, Zachary K.; Kuntz, Kate L. Vasquez; Anderson, Charles T.; Hancock, William O.

doi:10.1364/boe.423798

Cited by 14 publications

(21 citation statements)

References 21 publications

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“…The Qdot-labeled Cel7A was imaged by TIRFM using a 488-nm laser ( Fig. 2 A ) on a custom-build microscope ( 28 ). Tetraspeck beads (Thermo Scientific) (brighter objects in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2 A ) were imaged simultaneously as fiduciary markers to compensate for stage drift ( 29 ). Immobilized cellulose was imaged by interference reflectance microscopy (IRM) ( 22 , 23 , 28 ) ( Fig. 2 B ).…”

Section: Resultsmentioning

confidence: 99%

“…Changes in the positions of the Tetraspeck particles were subtracted to compensate for stage drift in the x to y plane, and drift in the z -direction was minimized by an active feedback system on the microscope. A custom analysis program written in Matlab was used to analyze particle trajectories ( 28 ). The tracking precision of the system was determined by immobilizing a Qdot525 on a coverslip, moving the piezoelectric stage in a stepwise pattern, and determining particle positions by the tracking software.…”

Section: Resultsmentioning

confidence: 99%

“…The tracking precision of the system was determined by immobilizing a Qdot525 on a coverslip, moving the piezoelectric stage in a stepwise pattern, and determining particle positions by the tracking software. The particle positions accurately reflected the step displacements, and the SD of position at each step was 1.5 nm ( 28 ). Using this system, we analyzed the binding dynamics and movement of 11,116 Cel7A molecules across two independent experiments.…”

Section: Resultsmentioning

confidence: 99%

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Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Haviland

Nong²,

Kuntz³

et al. 2021

Journal of Biological Chemistry

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Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled Tr Cel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which Tr Cel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.

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“…The Qdot-labeled Cel7A was imaged by TIRFM using a 488-nm laser ( Fig. 2 A ) on a custom-build microscope ( 28 ). Tetraspeck beads (Thermo Scientific) (brighter objects in Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Haviland

Nong²,

Kuntz³

et al. 2021

Journal of Biological Chemistry

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“…Active Motor Active Motor+Unbound Motor+Rigor Motor TIRF assays and analysis Single-molecule tracking was performed on a custom built micromirror TIRF microscope with a dual-view for two-channel imaging (Nong et al, 2021). All experiments were performed at 21 °C.…”

Section: Mt Pelleting Assaymentioning

confidence: 99%

Kinesin-1, -2 and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport

Gicking

Feng

et al. 2022

Preprint

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Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2 and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2 and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.

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Measuring PETase enzyme kinetics by single-molecule microscopy

Zhang,

Hancock

2024

Biophysical Journal

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Integrated multi-wavelength microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes

Cited by 14 publications

References 21 publications

Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Kinesin-1, -2 and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport

Measuring PETase enzyme kinetics by single-molecule microscopy

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