Inhibition of Ca2+ Transport Triggers Changes in Ciliary Intraflagellar Transport Rate and Particle Composition

Abstract

The primary cilium is a specialized microtubule-based organelle that extends from the apical surface of many cell types. It has been found to play important roles in chemo- and mechano-sensation acting as a signal transducer of extracellular stimuli into intracellular signaling. The cilium is assembled and maintained by the transport of biomolecules in and out of this cellular compartment. Defects in the proteins that make cilia, such as those mutations found in so-called human ciliopathy genes can cause defects in cellular signaling that result in severe phenotypes including retinal degeneration, kidney cysts, and tissue homeostasis disruption. Primary cilia were found to be unique calcium compartments regulated by the polycystin (PKD) family of transient receptor potential channels (PKD2, PKD2L1) in mice and humans. The effect of ciliary Ca2+ regulation on intraflagellar transport (IFT), bidirectional transport system required for establishing, maintaining and disassembling of cilia, and the relation of these to developmental patterning and renal disease, is currently under study. Gadolinium known to be a powerful inhibitor of the Ca2+ transport was utilized to perturb the ciliary calcium balance. A genetically encoded calcium sensor GCaMP3 targeted to the primary cilium was used to monitor the level of the ciliary calcium. Changes in IFT motility in the primary cilia of mammalian kidney epithelial cells were measured via live cell TIRF microscopy. Fluorescence image moment analysis together with dSTORM super-resolution techniques were hired to investigate the stoichiometry of IFT particles. The biophysical measurements performed in the current study helped to understand the connection between ciliary Ca2+ balance and IFT transport in mammalian primary cilia.