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Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli

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Abstract

Primary cilia with a diameter of ~200 nm have been implicated in development and disease. Calcium signaling within a primary cilium has never been directly visualized and has therefore remained a speculation. Fluid-shear stress and dopamine receptor type-5 (DR5) agonist are among the few stimuli that require cilia for intracellular calcium signal transduction. However, it is not known if these stimuli initiate calcium signaling within the cilium or if the calcium signal originates in the cytoplasm. Using an integrated single-cell imaging technique, we demonstrate for the first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor. Importantly, this flow-induced calcium signaling depends on the ciliary polycystin-2 calcium channel. While DR5-specific agonist induces calcium signaling mainly in the cilioplasm via ciliary CaV1.2, thrombin specifically induces cytosolic calcium signaling through the IP3 receptor. Furthermore, a non-specific calcium ionophore triggers both ciliary and cytosolic calcium responses. We suggest that cilia not only act as sensory organelles but also function as calcium signaling compartments. Cilium-dependent signaling can spread to the cytoplasm or be contained within the cilioplasm. Our study thus provides the first model to understand signaling within the cilioplasm of a living cell.

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Acknowledgments

The authors thank Charisse Montgomery for comments regarding this manuscript. X. Jin’s work partially fulfilled the requirements for a PhD degree in Pharmacology. This work was supported by National Institute of Health, R01DK080640 (SMN) and R01GM083120 (KM). AMM is supported by F31DK096870.

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Authors and Affiliations

  1. Department of Medicine, College of Medicine, The University of Toledo, Toledo, OH, 43606, USA

    Xingjian Jin & Surya M. Nauli

  2. Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH, 43606, USA

    Ashraf M. Mohieldin, Brian S. Muntean & Surya M. Nauli

  3. Department of Pharmacology, College of Medicine, The Ohio State University, Columbus, OH, 43210, USA

    Jill A. Green & Kirk Mykytyn

  4. Department of Systems Biology, Harvard Medical School and Renal Division, Brigham and Women’s Hospital, Boston, MA, 02115, USA

    Jagesh V. Shah

  5. Department of Pharmacology, MS 1015, Health Education Building, Room 282D, The University of Toledo, 3000 Arlington Ave, Toledo, OH, 43614, USA

    Surya M. Nauli

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  1. Xingjian Jin
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  2. Ashraf M. Mohieldin
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  3. Brian S. Muntean
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  4. Jill A. Green
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Correspondence to Surya M. Nauli.

Electronic supplementary material

Supplemental Materials (Movies)

Movie 1. Fluid-shear stress induces cilium bending

The movie was taken with a high-resolution, high-speed differential interference contrast microscope. The cell was challenged with fluid-shear stress (flow). Number represents time in seconds.

Movie 2. Fluid-shear stress induces calcium signaling in the cilioplasm and cytoplasm.

Using high-speed excitation wavelength exchanger for the DG4/DG5 system, a movie of fluorescence was captured simultaneously with Movie 1. Color bar indicates calcium level, where black-purple and yellow–red colors represent low and high calcium levels, respectively. Number represents time in seconds.

Movie 3. Fluid-shear stress induces calcium signaling in the cilioplasm followed by the cytoplasm.

A movie of fluorescence changes in an experiment independent from Movie 2. Color bar indicates calcium level, where black-purple and yellow–red colors represent low and high calcium levels, respectively. Number represents time in seconds.

Movie 4. The cilium and cell body remain in focus in a cell treated with fenoldopam.

The movie was taken with a high-resolution, high-speed differential interference contrast microscope. The cell was challenged with fenoldopam (FD). Number represents time in seconds.

Movie 5. Fenoldopam induces calcium signaling specifically in the cilioplasm.

Using high-speed excitation wavelength exchanger for DG4/DG5 system, a movie of fluorescence changes was captured simultaneously with Movie 4. Color bar indicates calcium level, where black-purple and yellow–red colors represent low and high calcium levels, respectively. Number represents time in seconds.

Movie 6. The primary cilium and cell body remain in focus in a cell treated with thrombin.

The movie was taken with a high-resolution, high-speed differential interference contrast microscope. The cell was challenged with thrombin (TH). Number represents time in seconds.

Movie 7. Thrombin induces calcium signaling specifically in the cytoplasm.

Using high-speed excitation wavelength exchanger for DG4/DG5 system, a movie of fluorescence changes was captured simultaneously with Movie 6. Color bar indicates calcium level, where black-purple and yellow–red colors represent low and high calcium levels, respectively. Number represents time in seconds.

Movie 8. The primary cilium and cell body remain in focus in a cell treated with ionomycin.

The movie was taken with a high-resolution, high-speed differential interference contrast microscope. The cell was challenged with ionomycin (IO). Number represents time in seconds.

Movie 9. Ionomycin induces calcium signaling in both the cilioplasm and cytoplasm.

Using high-speed excitation wavelength exchanger for DG4/DG5 system, a movie of fluorescence changes was captured simultaneously with Movie 8. Color bar indicates calcium level, where black-purple and yellow–red colors represent low and high calcium levels, respectively. Number represents time in seconds.

Below is the link to the electronic supplementary material.

Supplementary material 1 (MOV 5825 kb)

Supplementary material 2 (MOV 608 kb)

Supplementary material 3 (MOV 1006 kb)

Supplementary material 4 (MOV 5966 kb)

Supplementary material 5 (MOV 1210 kb)

Supplementary material 6 (MOV 5263 kb)

Supplementary material 7 (MOV 1928 kb)

Supplementary material 8 (MOV 5129 kb)

Supplementary material 9 (MOV 1367 kb)

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Jin, X., Mohieldin, A.M., Muntean, B.S. et al. Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli. Cell. Mol. Life Sci. 71, 2165–2178 (2014). https://doi.org/10.1007/s00018-013-1483-1

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  • DOI: https://doi.org/10.1007/s00018-013-1483-1

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