Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes

Abstract

The sodium dodecyl sulfate (SDS)-solubilized freeze fracture replica labeling (SDS-FRL) technique has been successfully applied to a vascular plant, Vigna angularis [Kimura et al. 1999, Plant Cell 11: 2075–2085] and a bacterium, Acetobacter xylinum [Kimura et al. 2001, J. Bacteriol. 183: 5668–5674] for understanding cellulose biosynthesis. However, we do not know yet how many gold particles can be bound with antibodies that label a single rosette or linear TC. We analyzed these techniques from a theoretical background by considering the size of gold particle, primary and secondary antibody, and the rosette and linear TC. The 10 nm gold particles coated with primary and secondary antibodies result in a 30 nm diameter for the gold-conjugated antibody that binds to a rosette TC. Therefore, a rosette TC theoretically could be labeled with, at most, four gold particles and commonly with 1–3 gold particles after the topological consideration of the immunogold labeling of the rosette TC. We interpreted an actual image of freeze-fracture labeling of TCs by comparison with a scale model of the antibody-labeling to a rosette TC. The labeling of linear TCs with the gold-conjugated antibody of c-di-GMP binding protein was quite stable, and 75% of the 277 gold particles were found within 20 nm of the linear row. Labeled TCs with depressions and indistinct particle arrays and non-labeled TCs with distinct particle arrays were observed on the P-fracture face of the outer membrane, suggesting that TCs in A. xylinum are composed of two types of subunits.