Abstract
The primary monocilium, or cilium, is a single antenna-like organelle that protrudes from the surface of most mammalian cell types, and serves as a signaling hub. Mutations of cilia-associated genes result in severe genetic disorders termed ciliopathies. Among these, the most common is autosomal dominant polycystic kidney disease (ADPKD); less common genetic diseases include Bardet–Biedl syndrome, Joubert syndrome, nephronophthisis, and others. Important signaling cascades with receptor systems localized exclusively or in part at cilia include Sonic Hedgehog (SHH), platelet derived growth factor alpha (PDGFRα), WNTs, polycystins, and others. Changes in ciliation during development or in pathological conditions such as cancer impacts signaling by these proteins. Notably, ciliation status of cells is coupled closely to the cell cycle, with cilia protruding in quiescent (G0) or early G1 cells, declining in S/G2, and absent in M phase, and has been proposed to contribute to cell cycle regulation. Because of this complex biology, the elaborate machinery regulating ciliary assembly and disassembly receives input from many cellular proteins relevant to cell cycle control, development, and oncogenic transformation, making study of genetic factors and drugs influencing ciliation of high interest. One of the most effective tools to investigate the dynamics of the cilia under different conditions is the imaging of live cells. However, developing assays to observe the primary cilium in real time can be challenging, and requires a consideration of multiple details related to the cilia biology. With the dual goals of identifying small molecules that may have beneficial activity through action on human diseases, and of identifying ciliary activities of existing agents that are in common use or development, we here describe creation and evaluation of three autofluorescent cell lines derived from the immortalized retinal pigmented epithelium parental cell line hTERT-RPE1. These cell lines stably express the ciliary-targeted fluorescent proteins L13-Arl13bGFP, pEGFP-mSmo, and tdTomato-MCHR1-N-10. We then describe methods for use of these cell lines in high throughput screening of libraries of small molecule compounds to identify positive and negative regulators of ciliary disassembly.
Highlights
Primary cilia, known as monocilia, are evolutionarily conserved, antenna-like organelles that protrude from the cell surface of almost all types of mammalian cell types, with limited exceptions
In a response to growth factors stimuli, the disassembly of the primary cilium is associated with the de-polymerization of the axoneme microtubules, which is partially controlled by an activation of kinesin superfamily protein 2A (KIF2A) and polo-like kinase-1 (PLK1) (Seeger-Nukpezah et al, 2012; Miyamoto et al, 2015)
We present data comparing the efficiency of visualization of cilia using targeting moieties provided by fusion of these fluorescent moieties to ADP-ribosylation factorlike protein 13b (ARL13b), SMO, and melanin-concentrating hormone receptor 1 (MCHR1) in the hTERT1-RPE1 cell line
Summary
Known as monocilia, are evolutionarily conserved, antenna-like organelles that protrude from the cell surface of almost all types of mammalian cell types, with limited exceptions (e.g., lymphocytes). In a response to growth factors stimuli, the disassembly of the primary cilium is associated with the de-polymerization of the axoneme microtubules, which is partially controlled by an activation of kinesin superfamily protein 2A (KIF2A) and polo-like kinase-1 (PLK1) (Seeger-Nukpezah et al, 2012; Miyamoto et al, 2015). Cascade dependent on the GLI family of transcription factors; following SHH-PTCH1 interactions at cilia, PTCH1 translocates from the cilium, and Smoothened (SMO) enters, where it interacts with and activates GLI transcription factors (Bangs and Anderson, 2017) Because of this and other ciliary signaling receptor systems, control of ciliation is relevant to a number of clinically significant human pathological conditions (Pan et al, 2013). We discuss relevant issues for optimizing analysis, and present an approach using an automated imaging system to visualize, quantify, and establish significance of ciliation data, using alisertib and ganetespib as examples of small molecules that influence ciliation
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