Detecting the orientation of newly-deposited crystalline cellulose with fluorescent CBM3

Pfaff, Sarah A.; Wang, Xuan; Wagner, Edward R.; Wilson, Liza; Kiemle, Sarah N.; Cosgrove, Daniel J.

doi:10.1016/j.tcsw.2022.100089

Cited by 5 publications

(3 citation statements)

References 75 publications

(97 reference statements)

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“…The immunolabeling of crystalline cellulose in living stomatal complexes required the succession of three molecular probes. In place of a primary antibody, a His-tagged CBM3 carbohydrate binding module was used (Pfaff et al 2022 ; Zhang et al 2018 ). The His-tag was recognized by a mouse anti-His-tag antibody, which was in turn targeted by a FITC-conjugated anti-mouse IgG (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Cell wall anisotropy plays a key role in Zea mays stomatal complex movement: the possible role of the cell wall matrix

Gkolemis,

Giannoutsou,

Adamakis

et al. 2023

Plant Mol Biol

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The opening of the stomatal pore in Zea mays is accomplished by the lateral displacement of the central canals of the dumbbell-shaped guard cells (GCs) towards their adjacent deflating subsidiary cells that retreat locally. During this process, the central canals swell, and their cell wall thickenings become thinner. The mechanical forces driving the outward displacement of the central canal are applied by the asymmetrically swollen bulbous ends of the GCs via the rigid terminal cell wall thickenings of the central canal and the polar ventral cell wall (VW) ends. During stomatal pore closure, the shrinking bulbous GC ends no longer exert the mechanical forces on the central canals, allowing them to be pushed back inwards, towards their initial position, by the now swelling subsidiary cells. During this process, the cell walls of the central canal thicken. Examination of immunolabeled specimens revealed that important cell wall matrix materials are differentially distributed across the walls of Z. mays stomatal complexes. The cell walls of the bulbous ends and of the central canal of the GCs, as well as the cell walls of the subsidiary cells were shown to be rich in methylesterified homogalacturonans (HGs) and hemicelluloses. Demethylesterified HGs were, in turn, mainly located at the terminal cell wall thickenings of the central canal, at the polar ends of the VW, at the lateral walls of the GCs and at the periclinal cell walls of the central canal. During stomatal function, a spatiotemporal change on the distribution of some of the cell wall matrix materials is observed. The participation of the above cell wall matrix polysaccharides in the well-orchestrated response of the cell wall during the reversible movements of the stomatal complexes is discussed.

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Section: Methodsmentioning

confidence: 99%

Cell wall anisotropy plays a key role in Zea mays stomatal complex movement: the possible role of the cell wall matrix

Gkolemis,

Giannoutsou,

Adamakis

et al. 2023

Plant Mol Biol

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“…CFW, although commonly used, is not optimal for visualizing cellulose in live cells because of its non-specific binding to various plant glycan epitopes and known cell toxicity (36)(37)(38). CBM-based proteinaceous probes were previously developed as a versatile, inert, and target-specific cell wall polysaccharide probe for enabling live-cell imaging as well as other immunolabeling applications (23,35,(47)(48)(49)(50)(51)(39)(40)(41)(42)(43)(44)(45)(46). In recent work, we developed a collection of engineered CBMs that have been extensively characterized by various biochemical and kinetic analyses to identify binding probes suitable for live-cell imaging.…”

Section: Arabidopsis Protoplasts Regenerate Cell Walls In the Presenc...mentioning

confidence: 99%

“…In contrast, carbohydrate-binding modules (CBMs) are non-catalytic protein domains associated with the glycosyl hydrolases that bind to cell wall polysaccharides in a target-specific manner: CBM3a for crystalline cellulose (39)(40)(41)(42); CBM17 and CBM28 for amorphous cellulose (42,43); CBM76 for xyloglucan (44); and CBM27 for mannans (45). CBMs have been used to label various cell wall polysaccharides via conjugation with fluorescent proteins (23,46,47) or chemical dyes (35,(48)(49)(50)(51). Such fluorescent probes can be imaged with widefield epifluorescence or scanning laser confocal microscopy (SLCM), but the diffraction-limited optical resolution often proves insufficient to resolve nanoscale fibril ultrastructures of cell wall polysaccharides.…”

Section: Introductionmentioning

confidence: 99%

Time-resolved tracking of cellulose biosynthesis and microfibril network assembly during cell wall regeneration in live Arabidopsis protoplasts

Huh,

Jayachandran,

Sun

et al. 2024

Preprint

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Plant cell walls are composed of polysaccharides among which cellulose is the most abundant component. Cellulose is processively synthesized as bundles of linear β-1,4-glucan homopolymer chains via the coordinated action of multiple enzymes in cellulose synthase complexes (CSCs) embedded within the plasma cell membrane. Plant cell walls are composed of multiple layers of cellulose fibrils that form highly intertwined extracellular matrix networks. However, it is not yet clear as to how cellulose fibrils synthesized by multiple CSCs are assembled into the intricate cellulose network deposited on plant cell surfaces. Herein, we have established an in vivo time-resolved imaging platform for visualizing cellulose during its biosynthesis and assembly into a complex fibrillar network on the surface of Arabidopsis thaliana mesophyll protoplasts as the primary cell wall regenerates. We performed total internal reflection fluorescence microscopy (TIRFM) with fluorophore-conjugated tandem carbohydrate binding modules (tdCBMs) that were engineered to specifically bind to nascent cellulose fibrils. Together with a well-controlled environment, it was possible to monitor in vivo cellulose fibril synthesis dynamics in a time-resolved manner for nearly one day of continuous cell wall regeneration on protoplast cell surfaces. Our observations provide the basis for a novel model of cellulose fibril network development in protoplasts driven by complex interplay of multi-scale dynamics that include: rapid diffusion and coalescence of short nascently synthesized cellulose fibrils; processive elongation of single fibrils; and cellulose fibrillar network rearrangement during cell wall maturation. This platform is valuable for exploring mechanistic aspects of cell wall synthesis while visualizing cellulose microfibrils assembly.

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Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

Nong,

Haviland,

Zexer

et al. 2024

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

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Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a K i of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme’s velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the “back door” product release site to slow activity and to the “front door” substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

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Detecting the orientation of newly-deposited crystalline cellulose with fluorescent CBM3

Cited by 5 publications

References 75 publications

Cell wall anisotropy plays a key role in Zea mays stomatal complex movement: the possible role of the cell wall matrix

Cell wall anisotropy plays a key role in Zea mays stomatal complex movement: the possible role of the cell wall matrix

Time-resolved tracking of cellulose biosynthesis and microfibril network assembly during cell wall regeneration in live Arabidopsis protoplasts

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

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