Cynthia G. Jensen

The resorption of primary cilia during mitosis in a vertebrate (PtK1) cell line

A high-voltage electron microscope (HVEM) was used to investigate the fate of primary cilia during late interphase and mitosis in a ciliated subline of PtK1 (Potorous tridactylis). The results provide direct evidence that PtK1 cells which possess primary cilia resorb their cilia during the early stages of spindle formation and subsequently complete a normal mitotic division. During late interphase the single diplosome with its attached cilium replicates to form two diplosomes, only one of which has an associated cilium. It was found that during the early stages of spindle formation (i.e., during prophase to prometaphase) the primary cilium is progressively resorbed so that by metaphase each spindle pole contains two normal centrioles. During resorption the axoneme with its attached centriole is incorporated into the cytoplasm where it is gradually disorganized from its distal end. The resorption of cilia during the early stages of mitosis suggests that this organelle is part of the cytoplasmic microtubule complex (CMTC). This report constitutes the first documentation of ciliary resorption during mitosis in a vertebrate cell.

Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ

Hyaline cartilage chondrocytes express one primary cilium per cell, but its function remains unknown. We examined the ultrastructure of chick embryo sternal chondrocyte cilia and their interaction with extracellular matrix molecules by transmission electron microscopy (TEM) and, for the first time, double‐tilt electron tomography. Ciliary bending was also examined by confocal immunohistochemistry. Tomography and TEM showed the ciliary axoneme to interdigitate amongst collagen fibres and condensed proteoglycans. TEM also revealed the presence of electron‐opaque particles in the proximal axoneme which may represent intraciliary‐transport (ICT) particles. We observed a wide range of ciliary bending patterns. Some conformed to a heavy elastica model associated with shear stress. Others were acutely deformed, suggesting ciliary deflection by collagen fibres and proteoglycans with which the cilia make contact. We conclude that mechanical forces transmitted through these matrix macromolecules bend the primary cilium, identifying it as a potential mechanosensor involved in skeletal patterning and growth.

Localization of Extracellular Matrix Receptors on the Chondrocyte Primary Cilium

A single primary cilium is found in chondrocytes and other connective tissue cells. We have previously shown that extracellular matrix (ECM) macromolecules such as collagen fibers closely associate with chondrocyte primary cilia, and their points of contact are characterized by electron-opaque plaques suggesting a direct link between the ECM and the cilium. This study examines the expression of receptors for ECM molecules on chondrocyte primary cilia. Embryonic chick sterna were fluorescently labeled with antibodies against α and β integrins, NG2, CD44, and annexin V. Primary cilia were labeled using acetylated α-tubulin antibody. Expression of ECM receptors was examined on chondrocyte plasma membranes and their primary cilia using immunofluorescence and confocal microscopy. All receptors examined showed a punctate distribution on the plasma membrane. α2, α3, and β1 integrins and NG2 were also present on primary cilia, whereas annexin V and CD44 were excluded. The number of receptor-positive cilia varied from 8/50 for NG2 to 43/50 for β1 integrin. This is the first study to demonstrate the expression of integrins and NG2 on chondrocyte primary cilia. The data strongly suggest that chondrocyte primary cilia have the necessary machinery to act as mechanosensors, linking the ECM to cytoplasmic organelles responsible for matrix production and secretion.

Primary cilia mediate mechanotransduction through control of ATP-induced Ca2+ signaling in compressed chondrocytes

We investigated the role of the chondrocyte primary cilium in mechanotransduction events related to cartilage extracellular matrix synthesis. We generated conditionally immortalized wild‐type (WT) and IFT88orpk (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca2+ signaling, extracellular matrix synthesis, and ATP release in response to physiologically relevant compressive strains in a 3‐dimensional chondrocyte culture system. All conditions were compared to unloaded controls. We found that cilia were required for compression‐induced Ca2+ signaling mediated by ATP release, and an associated up‐regulation of aggrecan mRNA and sulfated glycosaminosglycan secretion. However, chondrocyte cilia were not the initial mechanoreceptors, since both WT and ORPK cells showed mechanically induced ATP release. Rather, we found that primary cilia were required for downstream ATP reception, since ORPK cells did not elicit a Ca2+ response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca2+ signaling may be regulated by polycystin‐1, since ORPK cells only expressed the C‐terminal tail. This is the first study to demonstrate that primary cilia are essential organelles for cartilage mechanotransduction, as well as identifying a novel role for primary cilia not previously reported in any other cell type, namely cilia‐mediated control of ATP reception.—Wann, A. K. T., Zuo, N., Haycraft, C. J., Jensen, C. G., Poole, C. A., McGlashan, S. R., Knight, M. M. Primary cilia mediate mechanotransduction through control of ATP‐induced Ca2+ signaling in compressed chondrocytes. FASEB J. 26, 1663‐1671 (2012). www.fasebj.org

Articular chondrocytes express connexin 43 hemichannels and P2 receptors – a putative mechanoreceptor complex involving the primary cilium?

Mechanical loading is essential for the health and homeostasis of articular cartilage, although the fundamental mechanotransduction pathways are unclear. Previous studies have demonstrated that cyclic compression up‐regulates proteoglycan synthesis via an intracellular Ca2+ signalling pathway, mediated by the release of ATP. However, the mechanism(s) of ATP release has not been elucidated. The present study examines expression of the putative mechanosensitive ATP‐release channel, connexin 43 and whether it is expressed on the chondrocyte primary cilium, which acts as a mechanosensor in a variety of other cell types. In addition the study characterized the expression of a range of purine receptors through which ATP may activate downstream signalling events controlling cell function. Bovine articular chondrocytes were isolated by sequential enzyme digestion and seeded in agarose constructs. To verify the presence of functional hemichannels, Lucifer yellow (LY) uptake into viable cells was quantified following treatment with a hemichannel agonist (EGTA) and antagonist (flufenamic acid). LY uptake was observed in 45% of chondrocytes, increasing to 83% following EGTA treatment (P < 0.001). Treatment with the hemichannel blocker, flufenamic acid, significantly decreased LY uptake to less than 5% with and without EGTA. Immunofluorescence and confocal microscopy confirmed the presence of primary cilia and the expression of connexin 43. Approximately 50% of bovine chondrocyte primary cilia were decorated with connexin 43. Human chondrocytes in situ within cartilage explants also expressed connexin 43 hemichannels. However, expression was confined to the upper 200 µm of the tissue closest to the articular surface. Immunofluorescence revealed the expression of a range of P2X and P2Y receptor subtypes within human articular cartilage. In conclusion, the expression of functional hemichannels by articular chondrocytes may represent the mechanism through which mechanical loading activates ATP release as part of a purinergic mechanotransduction pathway. Furthermore, the expression of connexin 43 on the chondrocyte primary cilium suggests the possible involvement of the cilium in this pathway.

Primary cilia in osteoarthritic chondrocytes: from chondrons to clusters.

Osteoarthritis (OA) is a common joint disease characterized by articular cartilage degeneration. The etiology of OA is unknown. Because several previous studies have shown that primary cilia play critical roles in joint development, this study examined the incidence and morphology of primary cilia in chondrocytes during joint degeneration in a naturally occurring bovine model of OA. Primary cilia were detected using antibodies to acetylated α‐tubulin in normal cartilage as well as in mild and severe OA tissue. In normal cartilage, cilia number and length were lowest in the superficial zone and increased with distance from the articular surface. In OA tissue, the incidence and length of cilia increased at the eroding articulating surface, resulting in an overall increased proportion of cilia. This is the first study to show that primary cilia are present on chondrocytes throughout OA progression and that the overall percentage of ciliated cells within the degenerating cartilage increases with OA severity. Developmental Dynamics 237:2013–2020, 2008.

Mechanical loading modulates chondrocyte primary cilia incidence and length

The pathways by which chondrocytes of articular cartilage sense their mechanical environment are unclear. Compelling structural evidence suggests that chondrocyte primary cilia are mechanosensory organelles. This study used a 3D agarose culture model to examine the effect of compressive strain on chondrocyte cilia. Chondrocyte/agarose constructs were subjected to cyclic compression (0–15%; 1 Hz) for 0.5–48 h. Additional constructs were compressed for 48 h and allowed to recover for 72 h in uncompressed free‐swelling conditions. Incidence and length of cilia labelled with anti‐acetylated α‐tubulin were examined using confocal microscopy. In free‐swelling chondrocytes, these parameters increased progressively, but showed a significant decrease following 24 or 48 h compression. A 72 h recovery partially reversed this effect. The reduced cilia incidence and length were not due to increased cell division. We therefore propose that control of primary cilia length is an adaptive signalling mechanism in response to varying levels and duration of mechanical loads during joint activity.

The binding of MAP-2 and tau on brain microtubules in vitro: implications for microtubule structure.

We have presented data that indicate that MAP-2 associates with brain microtubules at nonrandomly distributed sites, whose distribution on the microtubule polymer can best be described by the 12-dimer MAP superlattice originally described by Amos; because of the additional spacings, however, between MAP-2 projections observed on MAP-2-saturated microtubules, we suggest that the 6-dimer MAP superlattice, or what we will call the double Amos superlattice, more completely specifies the total set of MAP-binding sites on cytoplasmic microtubules. Second, we have shown that brain microtubules reassembled in vitro contain a heterogeneous population of MAP-binding sites, which differ in their affinities for the two MAPs, MAP-2 and tau. Third, we have shown that microtubule populations that differ in their MAP content have subtle, but detectable differences in their tubulin isotype composition. Based on all the data presented here, we have presented the idea of a nonrandom distribution of tubulin isotypes within a microtubule as a means by which a cell could specify both the identity and the distribution of MAP-binding sites.

The occurrence and structure of primary cilia in a subline of Potorous tridactylus.

Abstract The paper describes a subline of rat kangaroo Potorous tridactylus kidney (PtK1) cells capable of producing a single primary (9+0) cilium during interphase. Antitubulin immunofluorescence, scanning electron microscopy (SEM) and high voltage electron microscopy (HVEM) were used to demonstrate the occurrence and structural features of the cilia. During repeated subculturing the frequency of cilia ranged from approx. 60% of the cells in a growing population to almost 100% in confluent cultures. We believe that the subline may provide excellent material for high resolution correlative light- and electron microscopic studies on the development, function and subsequent fate of primary cilia during the cell cycle.

INHIBITION OF CYTOKINESIS BY ASBESTOS AND SYNTHETIC FIBRES

Using high‐resolution timelapse microscopy, we have followed individual phagocytized fibres through the later stages of division in MeT‐5A human mesothelial cells and LLC‐MK2monkey epithelial cells. The fibres used were crocidolite and chrysotile asbestos, fibrous glass (MMVF), and refractory ceramic fibres (RCF). Long fibres (15–80μm) trapped within the cleavage furrow can partially or completely block cytokinesis. Cells proceed in one of three ways: (1) eventual completion of cytokinesis; (2) incomplete cytokinesis, resulting in two cells joined by a fibre‐containing intercellular channel; or (3) failure of cytokinesis, resulting in a binucleate or trinucleate cell. Two factors associated with fibre‐induced bi/trinucleation are: (1) an initial association between the fibre and the forming daughter nuclei, which is sometimes lost over time, and (2) disintegration of the midbody. The studies suggest that delay of cytokinesis by interzonal fibres can result in bi/trinucleation through the loss of midbody/intercellular bridge proteins that are required for completion of cytokinesis.