The differential distribution of acetylated and detyrosinated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage chondrocytes

Abstract

The primary cilium is a ubiquitous cytoplasmic organelle of unknown function. Ultrastructural evidence of primary cilia in chondrocytes, and their colocalisation with the Golgi apparatus, has led to speculation that these structures are functionally linked. To investigate the relationship between these organelles, we examined the molecular anatomy of the microtubular cytoskeleton in the chondrocytes of chick embryo sterna. Thick cryosections were immunolabelled with antibodies directed against acetylated α-tubulin (C3B9), detyrosinated α-tubulin (ID5) and total α-tubulin (TAT), and imaged at high magnification using confocal laser scanning microscopy. Transmission electron microscopy confirmed the ultrastructure of the chondrocyte primary cilium and its structural relationship to the Golgi apparatus. Detyrosinated and acetylated α-tubulins were concentrated in the centrioles, centrosome and microtubule organising centre adjacent to the nucleus, with total α-tubulin distributed throughout the cytoplasm. ID5 stained the primary cilium at an incidence of 1 per cell, its colocalisation with C3B9 identifying the primary cilium as one of the most stable features of the microtubular cytoskeleton. Primary cilia varied from 1 to 4 μm in length, and 3 patterns of projection into the extracellular matrix were identified; (1) full extension and matrix contact, with minor undulations along the length; (2) partial extension and matrix contact, with a range of bending deflections; (3) cilium reclined against the cell surface with minimal matrix contact. Ultrastructural studies identified direct connections between extracellular collagen fibres and the proteins which decorate ciliary microtubules, suggesting a matrix–cilium–Golgi continuum in hyaline chondrocytes. These results strengthen the hypothesis that the primary cilium acts as a ‘cellular cybernetic probe' capable of transducing environmental information from the extracellular matrix, communicating this information to the centrosome, and regulating the exocytosis of Golgi-derived secretory vesicles.