The cooperative effect of multiple affinity binding interactions creating a stable bond, known as avidity, is particularly important in assessing the potency of potential drugs such as monoclonal antibodies, CAR T, or NK cells to treat cancer. However, predicting avidity based on in vitro single affinity interactions has limitations and often fails to describe the avidity effects observed in vivo. Acoustic force- based assays have recently emerged as a reliable method for direct avidity measurements, expressed as adhesion forces, which positively correlate with drug efficacy. However, to better understand avidity, in particular for cell-cell interactions and correlate it with affinity, a cell model system with controlled avidity-related properties is needed. This study presents a method for producing a cell model system using effector beads that can be used in acoustic force spectroscopy-based avidity assays or any other bead-based avidity assay. The protein of interest is biotinylated in vivo in E.coli, purified and subsequently mixed with streptavidin coated beads to create effector beads. The results demonstrate the dependency of rupture force on the receptor surface density and force loading rate, thus providing valuable information for designing future effector bead assays as well as cell avidity measurements for screening and characterization purposes.

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 fromPhyscomitrella patensand PttCesA8 fromPopulus tremula x tremuloidesthat 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 µM, 108 µM and Vmax = 7.88x10-5µmol/min, 4.31x10-5µmol/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.

Carbohydrate binding modules (CBMs) are non-catalytic domains associated with cell wall degrading enzymes that are often present in nature tethered to distinct catalytic domains (CD). CBMs initiate the association of CDs to polysaccharides by binding to the specific substrate. This protein-carbohydrate interaction has been used beneficially to enhance enzymatic deconstruction of biomass as well as the recognition of carbohydrates through immunocytochemistry. Several CBMs have also been used to visualize the presence of various polysaccharides present in the cell wall of plant cells and tissues. However, most of these previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of optimal CBM designs for recognizing specific plant cell wall glycans and also have not been used to study cell wall regeneration in plant protoplasts. Here, 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 reversible binding affinity towards cellulose-I using equilibrium binding assays. To compute the adsorption (kon) and desorption (koff) rate constants of single versus tandem CBM designs towards nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation (QCM-D). Our results indicate that tandem CBM3a exhibits a five-fold increased adsorption to cellulose compared to single CBM3a, making tandem CBM3a suitable for live-cell imaging applications. However, single CBM3a showed a higher binding affinity, albeit with poor binding reversibility, compared to all other CBM designs. Finally, we used these engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy (CLSM) and fluorescence microscopy. Our studies demonstrate how CBMs could be used to target growing cellulose chains in real-time and not perturb the dynamic movement and cross-linking of cellulose chains during cell wall regeneration in living Arabidopsis protoplasts.

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk on ) and desorption (k off ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

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