A play on cilia beating

Abstract

Motile cilia, hairlike structures present on the cell surface, have a well-appreciated role in human physiology, including sweeping mucus, dirt and debris out of the respiratory tract. However, we are only beginning to understand the mechanisms governing cilia growth, maintenance and function. In this issue, Arora et al. reveal new details about the control of cilia growth. They identify a previously unrecognized connection between adenylate cyclase 6 (AC6), a cilia signaling mediator, and the autophagy-mediated regulation of motile cilia length via kinesin Kif19a, a regulator of cilia length. These findings provide new insights into motile cilia biology and may lead to novel ciliopathy treatments.

Cilia are small, microtubule-based organelles protruding from the surface of eukaryotic cells, and the number and function of cellular cilia can vary across cell types and organisms. For instance, many unicellular organisms use one or more cilia for locomotion and sensation. In vertebrates, most cells make a single nonmotile primary cilium, which regulates signal transduction during development (1). However, cells of various specialized vertebrate epithelial tissues (i.e. airway epithelium) have tufts of several dozens of cilia, which beat in a coordinated manner to drive directional flow of fluids (2). Dysfunctions of cilia manifest as complex multisystem disorders termed ciliopathies. In the context of airway epithelium, cilia defects lead to respiratory problems due to inefficient mucociliary clearance (3). Thus, understanding the biological mechanisms regulating the formation and function of cilia has important basic science and clinically relevant implications.

Cilia are specialized signaling compartments with a distinct composition of receptors and second messengers, including cAMP. Adenylate cyclase (AC) has been shown to be a major supplier of cAMP in nonmotile cilia and is implicated in the orchestration of compartmental signaling and regulation of ciliary beating (1, 2). However, how AC and cAMP may regulate growth and dynamics of motile cilia has not been determined.

It is well-established that cilia growth depends on the microtubules contained within them. Kif19a, a motor protein that binds to tubulin in an ATP-dependent manner, was shown to be a key regulator of cilia length by causing the depolymerization of microtubules at cilium tips (4, 5). Additionally, autophagy, one of the main paths for protein and organelle degradation, can selectively clear cilia-associated proteins and regulate cilia growth (6, 7). These findings prompted Arora et al. (8) to explore whether AC6 regulates cilia growth via the autophagic degradation of Kif19a.

The authors started by using a clever combination of in vitro primary cell experiments and in vivo approaches using conditional deletion of the AC6 gene (Adcy6) in tracheal epithelial cells (Adcy6^Δ/Δ mice). The authors found that movement of cilia in tracheal epithelial cells was impaired in Adcy6^Δ/Δ trachea, which is in agreement with the previously described role of cAMP in the regulation of airway epithelium ciliary beating and mucociliary clearance (9, 10). Interestingly, the authors also found that cilia were significantly longer in both Adcy6^Δ/Δ mice and AC6 RNAi-treated primary human bronchial epithelial cells. Based on these data, they concluded that loss of AC6 results in elongated cilia, which may contribute to defective ciliary beating in airway epithelium, but how?

It turned out that the effects of AC6 depletion on cilia phenotype resembled previously reported Kif19a-related defects (5), so the authors decided to test possible functional connections between AC6 and Kif19a. In doing so, they not only confirmed previous data linking Kif19a loss and cilium length, but also observed that protein levels of Kif19a were dramatically reduced in isolated Adcy6^Δ/Δ mice tracheae (8). Given the reported links between cilia formation, protein degradation, and autophagy (6, 7), these findings provided the authors with a potential missing link that would explain how AC6 regulates cilia growth: by modulating Kif19a levels via autophagy! Thus, they investigated whether their data could be explained by deregulated autophagy in AC6-depleted cells. Indeed, they found elevated levels of autophagy markers (i.e. phosphorylated AMPK) in Adcy6^Δ/Δ tracheae, indicating that AC6 inhibits AMPK activity and prompting them to test the involvement of AMPK and autophagy in the regulation of Kif19a turnover. Remarkably, they observed that inhibition of autophagy by either direct ablation of AMPK activity or treatment with chloroquine, an autophagy inhibitor, resulted in elevated levels of Kif19a, whereas activation of autophagy/AMPK caused Kif19a down-regulation. These data not only established Kif19a as an autophagy target, but importantly provided support for a model where AC6 deficiency leads to increased activation of AMPK, enhanced autophagy, and in turn diminished protein levels of Kif19a.

Finally, the authors sought to answer how modulation of this novel pathway translates into changes in cilia growth. They designed elegant functional rescue experiments using primary tracheal cells in combination with pharmacological inhibition or activation of AMPK activity. This approach allowed them to correlate changes in Kif19a steady-state levels with the effects on cilia length and beating, respectively. In their experiments, activation of AMPK caused down-regulation of Kif19a and, in turn, longer cilia in control, but not in Adcy6^Δ/Δ cells, suggesting that AMPK activation and its effect on Kif19a degradation in Adcy6^Δ/Δ had already reached saturation. Conversely, inhibition of AMPK in Adcy6^Δ/Δ increased Kif19a levels and improved cilia beating, and the degree of improvement correlated with cilia length. AMPK inhibitor treatment of control cells did not affect cilia length or beating.

As has already been mentioned, cilia play an instrumental role in human physiology, but our knowledge of the molecular mechanisms governing their formation and function is still far from complete. The current work of Arora et al. (8) demonstrates that AC6 negatively regulates cilium length via mechanisms involving Kif19a and regulation of its turnover by autophagy (Fig. 1). Their work represents an important step on the way toward our better understanding of motile cilia biology. It has several pertinent implications, as it directly connects regulations of cilia dynamics (length) and function (cilia beating) to a single controlling mechanism, the AC6/AMPK/Kif19a pathway. Adenylate cyclase and cAMP are involved in regulation of cilia beating and dynamics in various systems. Thus, it is tempting to speculate that the AMPK/Kif19a module might provide a means for how activation of ciliary signaling fine-tunes ciliary dynamics with direct consequences for the motile cilia function. It seems quite plausible that in a situation where a cell is undergoing stress and conservation of energy is vital, lack of appropriate stimuli translates into increased autophagy, thus slowing ciliary beating and conserving necessary energy resources. From this perspective, it will be interesting to see whether this pathway is specific to the AC6/airway epithelium or if it applies to other AC isoforms in different multiciliated cell types. In addition, it should also be rewarding to explore in more detail the mechanism of autophagy dysregulation in the context of AC6/cAMP. Last but not least, a very attractive clinical implication of the work of Arora et al. is a potential to treat motile ciliopathies by pharmacological modulation of the AC6/AMPK/Kif19a pathway, which should be closely examined in future studies.

Figure 1.

Role of AC6 in regulation of cilia length. Lack of AC6 leads to increased phosphorylation of AMPK (p-AMPK) and activation of autophagy, which mediates degradation of microtubule-depolymerizing kinesin Kif19a. This translates to increased cilia length and impaired beating. See the main text for more details.