A cell‐based screen for inhibitors of flagella‐driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow

Abstract

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound "ciliabrevin" due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

“…The library's current format of arrayed colonies on solid medium is compatible with high-throughput robotic screens using colony size as a powerful and versatile phenotype, as illustrated in yeast (Costanzo et al, 2010). The library is also compatible with a range of other quantitative phenotyping methods, including measurement of pigment levels, chlorophyll fluorescence analysis of arrays on agar (Johnson et al, 2009), and motility screens in 96-well plates (Engel et al, 2011).…”

Regulation of Cilium Length and Intraflagellar Transport by the RCK-Kinases ICK and MOK in Renal Epithelial Cells

“…The most plausible explanation is provided by the balance point model, in which cilium length is determined by a balance between cilium assembly and disassembly rates [8], [9]. The assembly rate is dependent on the availability of axonemal tubulin and other structural components, supplemented by anterograde IFT and probably the pool of these proteins at the base of the cilium [10], [11]. Indeed, changes in both anterograde and retrograde IFT are accompanied by changes in cilium length [8], [12]–[15].…”

A systems-biology approach to understanding the ciliopathy disorders

“…Application of genetic, chemical or even genome-wide small interfering RNA libraries to ciliated cells followed by assessment of signaling or ciliary structure provides an important complementary approach to understanding ciliary-specific genetic requirements [41-44]. These cellular analyses have uncovered many important signaling mechanisms, some of which are pharmacologically tractable and may serve as a basis for the development of future therapy, in particular because the assays can be targeted to bypass specific patient-related cellular defects.…”

“…For instance, we found that positive regulators of actin polymerization functioned as negative regulators of ciliogenesis [44], and vice versa, prompting us to question whether actin destabilizers might promote ciliogenesis in cellular models of disease. We found that low dose application of the actin polymerization inhibitor cytochalasin D partially rescued cilum elongation defects caused by the hypomorphic mouse mutation Ift88 [41-44]. Further genomic screening focusing on a specific cellular process, such as ciliation linked to cell cycle regulation, post-translational modification of microtubules and regulation of ciliary transport and signaling, will lead to a greater understanding of ciliary structure/function relationships, their relevance to disease and potential treatments.…”