Time-lapse imaging of primary cilium behavior with physiological expression of fluorescent ciliary proteins.

Abstract

Almost all cell types of mammals have a small protrusion named a primary cilium on their surface. Primary cilia are enriched by cilia-specific ion channels and G-protein-coupled receptors. They are known to regulate various cellular functions that contribute to the development and homeostasis of living organisms by receiving extracellular signals and transfusing them to the cell body. All functions are performed when the structure of the primary cilia is maintained properly. Abnormalities in primary cilia or their signaling can lead to a collection of diseases in various organs called ciliopathies. The primary cilium is dynamic, static, or fixed. The length of primary cilia varies as the cell cycle progresses and is also altered by extracellular stimuli. Ligand binding to cilia-specific receptors is also known to alter the length. Thus, there is a need for a method to study the morphological changes of the primary cilium in a time-dependent manner, especially under stimuli or mechanical shocks. Time-lapse imaging of primary cilia is one of the most powerful methods to capture the time-dependent behavior of primary cilia. Overexpression of ciliary proteins fused to fluorescent proteins is commonly used for the time-lapse imaging of primary cilia. However, overexpression has drawbacks in terms of artifacts. In addition, the time-lapse imaging of the tiny primary cilia requires some technical tricks. Here, we present a detailed description of the methods for time-lapse imaging of primary cilium, from the generation of cell lines that stably express fluorescent protein-labeled cilia-localized proteins at the physiological level to image analysis, including quantification through image acquisition.