Testing the ion-current model for flagellar length sensing and IFT regulation

Abstract

Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca2+ channels along the flagellum and that the Ca2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca2+ spikes. Thus, Ca2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.

Chlamydomonas as a model system to study cilia and flagella using genetics, biochemistry, and microscopy

“…There are a large number of potential mechanisms by which the length of the flagellum could be sensed, many of which have been shown to be capable, in theory, of fitting available measurements of flagellar regeneration ( Ludington et al, 2015 ). Testing these models has employed a combination of specific mutations with quantitative imaging, which has allowed us to rule out three models so far: (a) an “initial bolus” model where flagella are loaded with a fixed quantity of IFT particles that circulate back and forth, ruled out by photobleaching experiments ( Ludington et al, 2015 ); (b) a “time of flight” model where length is sensed by a molecular timer attached to the IFT particle, ruled out using mutants that change the speed of retrograde IFT ( Ishikawa and Marshall, 2017 ); and (c) an “ion current” model where length is sensed by the magnitude of a calcium current flowing across the flagellar membrane, ruled out using mutations and chemical perturbations that alter flagellar calcium transport ( Ishikawa et al, 2023 ).…”

Ciliary length regulation by intraflagellar transport in zebrafish

“…Moreover, the rate at which IFT trains enter the flagella, known as the IFT injection rate, is negatively correlated with flagellar length during regeneration (Engel et al, 2009; Ludington et al, 2013). Researchers have proposed and tested several theoretical models to explain the regulatory mechanism of the IFT injection rate (Ishikawa et al, 2023; Marshall, 2023; Wemmer et al, 2020). Intriguingly, recent studies in the parasitic protist Giardia, which possesses eight flagella of varying lengths, revealed that the ciliary tip localization of microtubule-depolymerizing kinesin-13 is inversely correlated with flagellar length, similar to its role in flagellar length regulation (McInally et al, 2019; Piao et al, 2009; Wang et al, 2013).…”

Role of intraflagellar transport in transcriptional control during flagellar regeneration in Chlamydomonas

“…Flagellar assembly and length maintenance involves a kinesin-based transport mechanism known as intraflagellar transport or IFT ( Kozminski et al ., 1993 ; Cole et al ., 1998 ; Rosenbaum and Witman, 2002 ; Bhogaraju et al ., 2014 ) that actively transports tubulin and other building blocks to the site of assembly at the tip of the growing flagellum ( Qin et al ., 2004 ; Hao et al ., 2011 ; Bhogaraju et al ., 2013 ; Craft et al ., 2015 ). The activity of the IFT pathway is a function of flagellar length, such that the rate of IFT decreases according to 1/ L ( Engel et al ., 2009 ; Ludington et al ., 2013 ), but the mechanism by which length regulates IFT remains unclear ( Ludington et al., 2015 ; Ishikawa and Marshall, 2017 ; Hendel et al ., 2018 ; Ishikawa et al ., 2022 ). The transport of tubulin and other cargoes by IFT also varies as a function of length ( Wren et al ., 2013 ; Craft et al ., 2015 ), but at least in the case of tubulin it remains unclear whether this represents regulation of a binding interaction or a length dependence of the number of binding sites ( Wemmer et al., 2020 ).…”