Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; Hancock, William O.

doi:10.1091/mbc.e14-06-1146

Cited by 66 publications

(92 citation statements)

References 32 publications

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“…Given that the CesA complexes appear to contain equimolar heterotrimeric CesAs, i.e. 1:1:1 of CesA1, 3 and 6-like CesAs and of CesA4, 7 and 8 in primary and secondary wall CesA complexes, respectively [43,44] the VAEM photo-bleach study [42] supports 36 CesA subunits per complex. However, it remains to be seen how these data will compare against EM-based work, if all CesA subunits in the complex are active, and if there are cell, tissue and species dependent differences of CesA complex size (Fig.…”

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confidence: 83%

“…A recent elegant study used variable-angle epifluorescence microscopy (VAEM) in combination with quantitative photo-bleaching to enable optical assessment of the CesA3 stoichiometry in CesA complexes in epidermal hypocotyl cells of Arabidopsis [42]. By using a step-detection algorithm this study estimated the number of GFP-CesA3 molecules to be at least 10 in one CesA complex [42,4]. Still, these estimates are likely low because the imaged plants were only in a partial-loss-of-function mutant (i.e.…”

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confidence: 99%

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Cellulose and callose synthesis and organization in focus, what's new?

Schneider

Hanak

Persson

et al. 2016

Current Opinion in Plant Biology

104

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Article available under the terms of the CC-BY-NC-ND licence (https://creativecommons.org/licenses/by-nc-nd/4.0/) eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. Abstract Plant growth and development are supported by plastic but strong cell walls. These walls consist largely of polysaccharides that vary in content and structure. Most of the polysaccharides are produced in the Golgi apparatus and are then secreted to the apoplast and built into the growing walls. However, the two glucan polymers cellulose and callose are synthesized at the plasma membrane by cellulose or callose synthase complexes, respectively. Cellulose is the most common cell wall polymer in land plants and provides strength to the walls to support directed cell expansion. In contrast, callose is integral to specialized cell walls, such as the cell plate that separates dividing cells and growing pollen tube walls, and maintains important functions during abiotic and biotic stress responses. The last years have seen a dramatic increase in our understanding of how cellulose and callose are manufactured, and new factors that regulate the synthases have been identified. Much of this knowledge has been amassed via various microscopy-based techniques, including various confocal techniques and super-resolution imaging. Here, we summarize and synthesize recent findings in the fields of cellulose and callose synthesis in plant biology. Title

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confidence: 83%

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confidence: 99%

Cellulose and callose synthesis and organization in focus, what's new?

Schneider

Hanak

Persson

et al. 2016

Current Opinion in Plant Biology

104

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“…Step finding was performed on the y-displacement versus time traces using the tDetector algorithm (55), which returned step sizes and durations. Randomness measurements were calculated using the formula (56,57),…”

Section: Methodsmentioning

confidence: 99%

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

Chen

Mickolajczyk

Hancock

2016

Journal of Biological Chemistry

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Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATPtriggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rearhead-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.

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“…S2G). To extract steps, the motor position along the microtubule axis was analyzed using a model-free t test-based algorithm (29), and 982 total steps were detected. The plateau SD, calculated through pairwise differences with outliers removed (Materials and Methods and Fig.…”

Section: Significancementioning

confidence: 99%

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.

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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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Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

Cited by 66 publications

References 32 publications

Cellulose and callose synthesis and organization in focus, what's new?

Cellulose and callose synthesis and organization in focus, what's new?

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

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

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