223. Itoh, T., Kimura, S. and R.M. Brown, Jr. 2007. Immunogold Labeling of Cellulose-Synthesizing Terminal Complexes. In Cellulose: Molecular and Structural Biology. Springer pp 237-255.
Rosette TCs that are visualized exclusively by freeze-fracture electron microscopy have long been thought of as "putative" cellulose-synthesizing terminal complexes (TCs). We succeeded in directly demonstrating that the TCs contain cellulose synthases by the application of novel techniques combined with both freeze-fracture and immuno-gold labeling. Since the purification of cellulose syntheses has not succeeded in higher plant cells, we cloned GhCesA using cotton cDNA libraries and prepared polyclonal antibodies against the GhCesA protein. We were successful in applying SDS-FRL techniques to plant cells for the first time by using mixtures of cellulases and pectolyases to digest cell walls after freeze replication and immunogold labeling. Using SDS-FRL, the antibodies of cellulose synthases specifically labeled the rosette TCs in the plasma membrane of' higher plant cells. This provided direct evidence that the rosettes contain the catalytic subunit of the cellulose synthase. In this chapter, we have analyzed the mechanism of labeling compared with actual dimensions of rosettes, gold particles, and antibodies.
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