Calcium–axonemal microtubuli interactions underlie mechanism(s) of primary cilia morphological changes
We have used cell culture of astrocytes aligned within microchannels to investigate calcium effects on primary cilia morphology. In the absence of calcium and in the presence of flow of media (10 μL.s−1) the majority (90%) of primary cilia showed reversible bending with an average curvature of 2.1 ± 0.9 × 10−4 nm−1. When 1.0 mM calcium was present, 90% of cilia underwent bending. Forty percent of these cilia demonstrated strong irreversible bending, resulting in a final average curvature of 3.9 ± 1 × 10−4 nm−1, while 50% of cilia underwent bending similar to that observed during calcium-free flow. The average length of cilia was shifted toward shorter values (3.67 ± 0.34 μm) when exposed to excess calcium (1.0 mM), compared to media devoid of calcium (3.96 ± 0.26 μm). The number of primary cilia that became curved after calcium application was reduced when the cell culture was pre-incubated with 15 μM of the microtubule stabilizer, taxol, for 60 min prior to calcium application. Calcium caused single microtubules to curve at a concentration ≈1.0 mM in vitro, but at higher concentration (≈1.5 mM) multiple microtubule curving occurred. Additionally, calcium causes microtubule-associated protein-2 conformational changes and its dislocation from the microtubule wall at the location of microtubule curvature. A very small amount of calcium, that is 1.45 × 1011 times lower than the maximal capacity of TRPPs calcium channels, may cause gross morphological changes (curving) of primary cilia, while global cytosol calcium levels are expected to remain unchanged. These findings reflect the non-linear manner in which primary cilia may respond to calcium signaling, which in turn may influence the course of development of ciliopathies and cancer.
KeywordsPrimary cilia Calcium Axonemal microtubules Interactions Morphology Nonlinear dynamics
We would like to express our sincere gratitude to Professor Maxwell Bennett, AO (Professor of Neuroscience, University Chair, Founder and Scientific Director of The Brain and Mind Research Institute, The University of Sydney) for his genuine interest in this work and continued support. We are especially grateful to Professor Bennett for allowing us to use his lab’s equipment and cell culture to produce the data shown in Fig. 1, Fig. 2 and Fig. 3.
We cordially thank to Professor Boris Martinac, AO (Professor of Biophysics, Head of Mechanobiology Laboratory, Victor Chang Cardiac Research Institute, University of New South Wales) for critical reading of the initial version of this manuscript and his highly valuable comments.
We also kindly thank Dr. Ellie Cable, Senior Microscopist and Laboratory Manager at the Australian Centre for Microscopy and Microanalysis, The University of Sydney, for her friendly and highly professional help.
The authors dedicate this paper to the memory of Dr Vlado A. Buljan, the lead author, who unexpectedly passed away in March 2017 when this manuscript was under review. Dr Buljan was a dedicated scientist who pursued his research into the biophysics of tubulin with exemplary vigour. He will be greatly missed by his colleagues.
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Conflict of interest
The authors declare that they have no conflicts of interest.
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