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The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Kidney cysts are characterized by an abnormal tubular geometry that may result from loss of orientation and random cell divisions during renal development. Since cystic kidney disease is caused by mutations of ciliary proteins and cilia act as flow sensors in the kidney, we examined three polarized events in Madin Darby Canine Kidney cells under flow: cell division, cell migration, and centriole movement. We found that the mitotic orientation of dividing cells was not affected by flow and was randomly distributed in relation to the direction of the flow. Flow did not alter the direction and speed of cell migration in a wound-healing assay. However, flow resulted in increased motility of centrioles and biased centrioles to move along the axis of the flow. This effect was lost after flow-induced calcium signaling was abolished by a mutant polycystin 2. Our findings suggest that the cilium may translate fluid flow into altered centriole movements to provide tubular epithelial cells with the spatial orientation required to establish and/or maintain a normal tubular geometry.

Objective: Inherited Polycystic kidney disease has been linked to defects in cilial function of renal epithelial cells. Cilial bending in vitro through mechanical stimulation or flow has been shown to result in an increase in intracellular calcium, which is polycystin-1 and polycystin-2 dependent. The present experiments aimed at imaging downstream signalling events in MDCK cells by time lapse imaging. Methods: A parallel plate chamber was designed for life cell imaging under flow conditions. Ciliated MDCK cells and nonciliated control cells were imaged under different flow conditions and increase of intracellular calcium was determined by fura-2 ratio imaging. Virally transduced MDCK cells expressing a Protein Kinase C (PKC) gamma-GFP fusion construct, or a deletion construct with the C2 domain fused to GFP, were examined under flow conditions and the subcellular localization of PKC was tracked by time lapse imaging. Results: Intracellular calcium increased in wild type MDCK cells in response to flow in cilated cells only, when compared with non ciliated control cells. Ciliated MDCK cells expressing PKC-GFP or C2-GFP showed translocation of PKC to the plasma membrane in response to flow. Translocation of C2-GFP was enhanced, when compared to full length GFP, as would be expected in the absence of the C1 domains. Conclusions: The increase in intracellular calcium in renal epithelial cells in response to cilial bending is associated with the translocation of Protein Kinase C to the plasma membrane. Further studies will attempt to identify targets of downstream signalling of PKC. [ABSTRACT FROM AUTHOR]