Excited about this collaborative preprint that had critical contributions from my previous lab members Dharanidaran Jayachandran and Mohammed Irfan, who alongside the Lee and Lam lab members at Rutgers have developed a novel methodology to visualize cellulose microfibrils synthesized in real-time by live plant cells for the very first time to discover some amazing insights into the process of cellulose biosynthesis and assembly as microfibrils in regenerated plant protoplast cell walls. Now need to get this work peer reviewed and published soon. Check it out the preprint if you are interested. Preprint is under peer review now! Briefly, 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.
Our work should prove valuable to others in the glycoscience community exploring mechanistic aspects of cell wall polymers synthesis by visualizing polysaccharides biosynthesis and assembly in higher order structures in real-time using high resolution imaging instruments. As this manuscript undergoes peer review over the coming month/s, we look forward to feedback from the scientific research community at large.
Special thanks to the U.S. Department of Energy (DOE) for primarily supporting this work as part of the BER Bioimaging program. Also thanks to National Science Foundation (NSF), Rutgers University, Great Lakes Bioenergy Research Center (GLBRC) for support on various aspects of this work.