Confocal Analysis of Primary Cilia Structure and Colocalization With the Golgi Apparatus in Chondrocytes and Aortic Smooth Muscle Cells

Abstract

Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

c-Met and NF-κB–Dependent Overexpression of Wnt7a and -7b and Pax2 Promotes Cystogenesis in Polycystic Kidney Disease

“…14 The fact that abnormal Wnt7a and -7b expression may result from both abnormal protein trafficking and ciliary dysfunction suggests that either events in the cilia are affecting protein trafficking in the cilia or conversely, abnormal trafficking in the Golgi affects the cilia. Because IFT20 is also found in the Golgi, our studies and others 52,[54][55][56] suggest a functional link between the Golgi and primary cilia that may be broadly involved in cyst formation.…”

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma

“…The sections were incubated with testicular hyaluronidase (2 mg/ml in 0.1 M Tris saline, pH 5.0; Sigma-Aldrich, St Louis, MO, USA) at 37 1C for 2 h and with 0.5% Triton X-100 in PHEM buffer (PIPES 0.05 M, HEPES 0.025 M, EGTA 0.01 M and MgCl 2 0.01 M) at room temperature for 5 min as previously described. 7,26 In addition, to increase antibody penetration, proteinase K (5 ml/ml in 0.1 M Tris-buffered saline, pH 5.0; DakoCytomation, Carpinteria, CA, USA) was applied at room temperature for 3 min. The sections were stained with primary monoclonal antibodies against acetylated a-tubulin (clone 6-11b-1, 1:1000; Sigma-Aldrich, Steinheim, Germany), g-tubulin (clone GTU-88, 1:1000; Sigma-Aldrich, USA) and/ or Ki-67 (1:100) and/or a polyclonal antibody against KIF3A (1:100; Abcam, Cambridge, UK) at 4 1C overnight.…”

Assembly of primary cilia

“…However, in some cases the centriole that becomes the basal body moves peripherically towards the apical cell membrane, where the whole ciliary assembly process takes place. Either way, the cilium can increase considerably in length, (e.g., kidney epithelial cells; Wheatley and Bowser, 2000), and may arise directly from the cell surface (e.g., chrondrocytes; Poole et al, 1997), or from deeply inside the cell close to the nucleus (e.g., adrenal zona glomerulosa cells; Wheatley, 1967).…”