Super-resolution imaging of voltage-gated calcium channel Ca_V1.3 in primary cilia of retinal pigment epithelium

Abstract

Purpose : Cells of retinal pigment epithelium (RPE) need primary cilia (PC) for proper development. We recently showed that L-type Ca²⁺ channel Ca_V1.3 localizes to the vicinity of PC during development of human embryonic stem cell derived RPE (hESC-RPE). Based on earlier knowledge in literature on the role of Ca²⁺ signaling in PC, we hypothesize that Ca_V1.3 participates in ciliogenesis during hESC-RPE maturation or that its expression is coupled to the functioning of primary cilia in RPE maturation. As a first step to approach these hypotheses, we studied the detailed localization of Ca_V1.3 near PC.

Methods : We cultured hESC-RPE on inserts and evaluated their maturity based on expression and polarization of functionally relevant proteins, transepithelial resistance, growth factor secretion and phagocytosis. PC were visualized with ARL13B immunostaining and the base of PC with pericentrin (PCNT). Imaging was conducted with Nikon N-SIM super-resolution structured imaging microscope (SR-SIM), which allows sub-diffraction limited resolution of approximately 120 nm. The SR-SIM images were reconstructed in Nikon NIS-elements software and further processed in imageJ. Standard Zeiss LSM 780 laser scanning confocal microscope was used for imaging larger fields.

Results : Confocal images revealed the localization of Ca_V1.3 near the base of the PC in maturing hESC-RPE. This localization was observed in the monolayers of confluent, cobblestone hESC-RPE until they reached sufficient maturation and Ca_V1.3 gained a more homogeneous labelling pattern at the RPE cell membrane. Further analysis with SR-SIM during hESC-RPE development showed that Ca_V1.3 appeared as ring-like foci in the close proximity of PCNT. Interestingly, the images indicated the localization of Ca_V1.3 right above the PCNT in the transition zone region.

Conclusions : SR-SIM is a powerful tool for imaging small cellular structures and can be used to visualize the structural organization of the primary cilia in RPE. In this study, SR-SIM images reveal the localization of Ca_V1.3 above the basal body but below the axoneme in maturing hESC-RPE. This positioning may provide important insight into the role of Ca_V1.3 on RPE maturation and PC functionality since PCNT is suggested to recruit protein complexes involved in cilia assembly and calcium signalling to the base of the PC.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.