Biology subjects

Jayachandran, D.

Publications and source records attributed to Jayachandran, D..

5 recordsLinked to original sources

bioRxiv2024

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

Significance StatementCellulose is a major extracellular matrix component of cells that is critical for plant development and has applications to bioenergy, agricultural food/feed, textile, and wood production. Cellulose is thought to be assembled by the closely coordinated motion of plasma membrane-embedded cellulose synthase enzyme complexes. To date, however, it has not been possible to visualize de novo plant cell wall synthesis at the single cell level with the necessary spatiotemporal resolution to derive a data-driven model of how plant cells can resynthesize and assemble cell wall after its removal. Based on our time-resolved data, we propose a new model for cellulose biosynthesis after successfully performing live protoplast time-lapse imaging to visualize for the first time the complex dynamics of de novo cellulose biosynthesis and assembly into an intertwined microfibril network. Plant cell walls are composed of polysaccharides among which cellulose is the most abundant component. Cellulose is processively synthesized as bundles of linear {beta}-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.

plant biology↗

bioRxiv2024

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Polysaccharides are a major class of natural polymers found abundantly across all major life forms and play a critical role as structural, metabolic, or functional components in biomolecular processes. Some polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs). Despite the fact that the GT-2 family has the maximum number of deposited sequences, the biochemistry of GT-2 family enzymes is still poorly understood due to difficulties associated with GT membrane protein expression, purification, and reconstitution in lipid carriers. Here, we chose Populus tremula x tremuloides cellulose synthase 8 (PttCesA8) and Streptococcus equisimilis hyaluronan synthase (SeHas) as putative family-2-GTs to be expressed in a wheat-germ-based cell-free expression (CFE) system as proteoliposomes. The cell-free products were obtained as reconstituted liposomes directly from CFE reactions at high yields and short processing times compared to other approaches. GT enzymes expression was confirmed using SDS-PAGE and immunoblotting, and the integration of GTs in lipid layers was observed using cryogenic electron microscopy. Both GTs tested were catalytically active when incubated with their respective substrates and cofactors. The Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 {micro}M, and SeHas was 321.51 {micro}M (toward UDP N-Acetyl Glucosamine) and 207.88 {micro}M (toward UDP Glucuronic Acid), respectively. UDP was found to actively inhibit both these GTs with apparent inhibition constants of 10.08 {micro}M and 24.38 {micro}M. Mutation of specific conserved residues in structure-deficit SeHas confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, wheat-germ-based CFE can be used to express functionally active and liposome-reconstituted family-2 GTs at high yields with relative ease to enable classical enzymology assays and will also enable more detailed structural studies in the near future.

bioengineering↗

bioRxiv2023

Plant cellulose synthase membrane protein isolation directly from Pichia pastoris protoplasts, liposome reconstitution, and its enzymatic characterization

The most abundant renewable biopolymer on earth, viz., cellulose, acts as carbon storage reserve in plant and microbial cell walls that could potentially be converted into biofuels or other valuable bioproducts. Cellulose is synthesized by a plant cell membrane-integrated processive glycosyltransferase (GT) called cellulose synthase (CesA). Since only a few of these plant CesAs have been purified and characterized to date, there are huge gaps in our mechanistic understanding of these enzymes. Furthermore, the coordination between different CesAs involved in primary and secondary cell wall formation is yet to be unveiled. The biochemistry and structural biology studies of CesAs are currently hampered by challenges associated with their expression and extraction at high yields. To aid in understanding CesA reaction mechanisms and to provide a more efficient CesA extraction method, two putative plant CesAs - PpCesA5 from Physcomitrella patens and PttCesA8 from Populus tremula x tremuloides that are involved in primary and secondary cell wall formation in plants were expressed using Pichia pastoris as an expression host. We developed a protoplast-based membrane protein extraction approach to directly isolate both these membrane-bound enzymes for purification, as detected by immunoblotting and mass spectrometry-based analyses. Our method results in a higher purified protein yield by 3-4-fold than the standard cell homogenization protocol. Our purified CesAs were reconstituted into liposomes to yield active enzymes that gave similar biochemical characteristics (e.g., substrate utilization and cofactor requirements, no primer needed to initiate polymerization reaction) as enzymes isolated using the standard protocol. This method resulted in reconstituted CesA5 and CesA8 with similar Michaelis-Menten kinetic constants, Km = 167 M, 108 M and Vmax = 7.88x10-5 mol/min, 4.31x10-5 mol/min, respectively, in concurrence with the previous studies. Taken together, these results suggest that CesAs involved in primary and secondary cell wall formation can be expressed and purified using a simple and more efficient extraction method. This could potentially help unravel the mechanism of native and engineered cellulose synthase complexes involved in plant cell wall biosynthesis.

bioengineering↗

bioRxiv2023

Site-specific Effector Protein Functionalization to Create Bead-based Avidity Model Systems

The cooperative effect of multiple affinity binding interactions creating a stable bond, known as avidity, is a universal biological phenomenon seen in diverse systems. For example, avidity based biomolecular interactions are particularly important in assessing the potency of potential drugs such as monoclonal antibodies, chimeric antigen receptor (CAR) T-cell, or Natural Killer, cells to treat cancer or engineering microbes with cell surface immobilized enzyme complexes for consolidated bioprocessing (CBP) of cellulosic biomass to fuels and chemicals. However, predicting or measuring avidity based on in vitro single affinity interactions with non-complexed protein-ligand binding model systems has limitations and often fails to describe the avidity effects observed in vivo with cell surface complexed proteins interacting with multivalent ligands at solid interfaces. Acoustic force spectroscopy (AFS) based assays have recently emerged as a reliable method for direct avidity measurements, expressed as adhesion or rupture forces, which positively correlate with in vivo avidity interactions. However, to better understand and model avidity, in particular for cell-cell interactions and to correlate it with classical binding affinity, a cell mimetic model system with controlled avidity-related properties is needed. Here, we present a method for producing such a cell mimetic model system using "effector beads" that can be used in AFS-based avidity assays or any other bead-based avidity assay. The protein of interest is heterologously expressed and biotinylated in vivo in E. coli, purified, and subsequently tethered with streptavidin coated micron-sized beads to create effector beads. Our experimental results, combined with simulations of the multivalent binding phenomena, demonstrate the dependency of bead rupture force on its receptor protein surface density and force loading rate as well as the intrinsic kinetic binding parameters of the protein-ligand system of interest. These insights provide valuable information for designing future effector bead assays and cell avidity measurements for screening and characterization purposes for diverse applications.

bioengineering↗

bioRxiv2023

Engineering and characterization of carbohydrate-binding modules to enable real-time imaging of cellulose fibrils biosynthesis in plant protoplasts

O_LICarbohydrate binding modules (CBMs) are non-catalytic domains associated with cell wall degrading carbohydrate-active enzymes (CAZymes) that are often present in nature tethered to distinct catalytic domains (CD). Fluorescently labeled CBMs have been also used to visualize the presence of specific polysaccharides present in the cell wall of plant cells and tissues. C_LIO_LIPrevious studies have provided a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of optimal CBM designs for recognizing specific plant cell wall glycans. Furthermore, CBMs also have not been used to study cell wall regeneration in plant protoplasts. C_LIO_LIHere, we examine the dynamic interactions of engineered type-A CBMs (from families 3a and 64) with crystalline cellulose-I and phosphoric acid swollen cellulose (PASC). We generated tandem CBM designs to determine their binding parameters and reversibility towards cellulose-I using equilibrium binding assays. Kinetic parameters - adsorption (kon) and desorption (koff) rate constants-for CBMs towards nanocrystalline cellulose were determined using quartz crystal microbalance with dissipation (QCM-D). Our results indicate that tandem CBM3a exhibits a five-fold increased adsorption rate to cellulose compared to single CBM3a, making tandem CBM3a suitable for live-cell imaging applications. We next used engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using wide-field fluorescence and confocal laser scanning microscopy (CLSM). C_LIO_LIIn summary, tandem CBMs offer a novel polysaccharide labeling probe for real-time visualization of growing cellulose chains in living Arabidopsis protoplasts. C_LI

plant biology↗