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.

A Novel Dynein Light Intermediate Chain Colocalizes with the Retrograde Motor for Intraflagellar Transport at Sites of Axoneme Assembly inChlamydomonasand Mammalian Cells

“…The assembly of primary cilia can also vary with culture conditions and the stage of the cell cycle (Tucker et al, 1979;Alieva et al, 1999). However, when the primary cilium is analyzed directly, this organelle colocalizes with the centrosome, and it is often in proximity to the Golgi apparatus (Poole et al, 1997(Poole et al, , 2001Aughsteen, 2001). Previous studies have shown that the DHC2/D2LIC complex localizes to the centrosomal region in mammalian tissue culture cells, where it overlaps with the position of the Golgi apparatus (Vaisberg et al, 1996;Grissom et al, 2002).…”

“…Perrone et al the primary function of the cDHC1b isoform is its role in flagellar assembly (Pazour et al, 1999;Porter et al, 1999;Wicks et al, 2000). Because the centrosomal region is also the site of assembly of the primary cilium (Wheatley et al, 1996;Poole et al, 1997Poole et al, , 2001, we reexamined the distribution of the complex in mammalian cells relative to sites of cilia assembly. Preliminary experiments with polarized cultures of MDCK cells indicated that D2LIC is present in a diffuse pattern throughout the apical cytoplasm (Figure 7, A and D).…”

Failure to ubiquitinate c-Met leads to hyperactivation of mTOR signaling in a mouse model of autosomal dominant polycystic kidney disease

“…Polycystins-1 and -2 and many other proteins involved in cystogenesis are localized to the cilia, among other subcellular compartments (58). Several recent observations have begun to draw physical links between the cilia and the Golgi apparatus (47,59). For example, several proteins including IFT20, dyenin2, and kinesin-2 are found in both the Golgi apparatus and cilia (60).…”

Proteomic Surveillance of Autoantigens in Relapsing Polychondritis

“…One of the possible mechanisms would be modification of tubulin alpha chains. It was reported that chondrocyte primary cilium, structurally supported by microtubular cytoskeleton, plays important roles in obtaining environmental information from the extracellulr matrix of cartilage (28). Further, structural changes of cytoskeleton affect the viscoelastic properties of chondrocytes (34).…”