Defective assembly or function of primary cilia causes multiple diseases and developmental disorders (Sharma et al.
). This recent association between cilia and human disease has increased research and clinical interest into the mechanisms by which cilia are assembled and maintained and how defects in cilia function may influence cellular physiology. Although the length of primary cilia can vary between different tissues or even between cell types within a tissue, it appears that length is under regulatory control as cilia are relatively consistent in size within a specific cell type. This is also observed in the flagella of Chlamydomonas
, in which multiple genes involved in length control have now been indentified (Wemmer and Marshall, 2007
). Whether similar mechanisms exist in mammalian cells is not currently known; however, it seems likely as cells that extended multiple primary cilia in this study were found to have similar length under the different treatment regiments. In addition, it is not known whether signaling activities of the cilium alter its length and morphology as seen in Caenorhabditis elegans
(Blacque et al.
; Mukhopadhyay et al.
) or whether increased cilia length can influence a cilium’s ability to propagate a signal. It should be noted that multiple cystic kidney disease models involving cilia proteins have been reported and among these are models with either excessively long or short cilia (Taulman et al.
; Mokrzan et al.
; Tammachote et al.
), supporting the idea that abnormal cilia length does have physiological consequences. Furthermore, cilia length in the kidney is markedly increased by acute injury, raising the possibility that cilia are important in regulating or sensing cellular responses to external stresses (Verghese et al.
The data in this report indicate that there is an important link between the cytoskeleton and regulation of cilia length. Similar to the results reported by Kim et al.
) when the actin network is destabilized with CD, there was marked increase in cilia length. Here we extend this observation and show that actin stabilization also results in cilia elongation and that there is a concomitant increase in the level of soluble tubulin under these conditions. Importantly, taxol inhibited the effect of actin depolymerization and stabilization, and cilia were also elongated when cells were treated with subtle amounts of nocodazole. These data demonstrate a connection between the levels of soluble tubulin in a cell and the length of its cilium. However, it must be noted that treatment with potent factors, such as nocodazole, taxol, or CD, have wide spanning consequences that may not be directly related to the level of soluble tubulin. For example, changes in cytoskeleton have major effects on membrane and vesicular transport, cell division, cell spreading and migration, cell stress, and responses to mechanical stimuli. Thus at this point it cannot be unequivocally stated that there is a direct connection between the cilia length as observed in our analysis and the levels of soluble tubulin; however, the data do show a strong correlation between these events.
Data shown here, as well as those reported by Kim et al.
), indicate that actin disruption results in the elongation of cilia. Intriguingly, Kim et al.
used smoothened-GFP (Smo-GFP) to show that this was associated with the formation of a vesicular structure around the PCC. The PCC is thought to be a site of vesicle docking and a temporary reservoir for ciliary membrane and associated proteins. They conclude in their study that the effect of actin destabilization on cilia length was a result of increased membrane transport into the cilium resulting from the destabilization of the PCC. Although we did not assess the PCC, our data suggest that these effects are also mediated through the microtubule cytoskeleton and increased levels of soluble tubulin, keeping in mind the caveats of the pharmacological approach mentioned above. Further support for a soluble tubulin model in cilia length control comes from recent Chlamydomonas
studies in which the activity of CrKinesin-13, a cytosolic microtubule-depolymerizing kinesin (mammalian homologue; MCAK), was tightly coordinated with flagella regeneration and establishment of normal flagella length (Piao et al.
). Importantly, cells lacking kinesin-13 exhibit a much slower rate of flagella regeneration that is thought to be caused by stabilized cell body microtubules.
Unexpectedly, we found here that both actin stabilization and destabilization caused similar effects with regard to the length of the cilium. In nonmammalian cells in which actin localizes to cilia, disruption of actin results in cilia or flagella shortening (Boisvieux-Ulrich et al.
; Dentler and Adams, 1992
). Interestingly, CD can induce complete depolymerization of flagellar MTs in Chlamydomonas
(Dentler and Adams, 1992
). Given the actin localization to flagella, it is possible that MT depolymerization might be mediated through actin disruption and thus flagellar shortening in Chlamydomonas
Several studies have identified molecular candidates for mediating structural interactions between actin filament and microtubules in mammalian cells (Rodriguez et al.
). For instance, p150 subunit of microtubules motor dynein/dynactin complex colocalizes to F-actin cortical spots and sites of cell–cell contact (Busson et al.
) and is involved in centrosome reorientation during cell migration (Levy and Holzbaur, 2008
). BBS4, one of the Bardet-Biedl syndrome proteins, localizes to the centrosome/basal body of primary cilia and acts as an adaptor of p150 dynactin to recruit PCM1 (pericentriolar material protein 1) to the centriolar satellites (Kim et al.
). Kim et al.
also showed that silencing of BBS4
induces mislocalization of PCM1 and disorganized microtubules within the cell body. It should be noted that, later, another study showed that BBS4
null mice possess longer primary cilia in kidneys and in isolated renal tubules (Mokrzan et al.
). These various observations suggest a connection between actin–microtubule structural interactions within the cell body and regulation of primary cilia length. Although we do not yet understand the absolute cellular connection between the actin network and tubulin dynamics, the effect on cilia length correlated directly with soluble tubulin levels and was impaired by treatment with taxol.
In addition to actin depolymerization, several studies have indicated that forskolin can increase cilia length (Low et al.
; Besschetnova et al.
). This occurred through adenylyl cyclase producing cAMP that in turn activates PKA. The effects of cAMP/PKA on cilia length in Besschetnova et al.
) were attributed to an increase in the rate of anterograde IFT. Data from our study, as well as from Prasain et al.
), indicated that cAMP also causes changes in the state of MT polymerization with a concomitant increase in the level of soluble tubulin. Thus it was interesting that cilia elongation in response to forskolin in our analysis was attenuated by taxol, which would prevent the increase in soluble tubulin. The fact that taxol did not completely inhibit cAMP-induced effects supports the idea that cAMP may have multiple influences on cilia length control. As shown by Besschetnova et al.
), this would include increasing anterograde IFT rates and, as shown here, increased levels of soluble tubulin, the basic building blocks of the cilia axoneme. Taxol would be able to inhibit the liberation of free tubulin induced by cAMP but not the increase in IFT, thus leading to the intermediate cilia length observed.
In contrast to what was observed with forskolin/cAMP, the increase in cilia length caused by actin disruption was not blocked by inhibition of PKA activity. These findings suggest either that PKA functions upstream of actin depolymerization or that there are independent pathways, both of which influence levels of soluble tubulin. The possibility of functioning upstream seems unlikely based on the data indicating that taxol can completely inhibit the cilia length increase caused by actin depolymerization, whereas it does not completely inhibit the cAMP-mediated increase in cilia length. These data again support the possibility that cAMP/PKA has multiple roles in regulation of cilia length.
In our studies, we used taxol to inhibit formation of soluble tubulin. However, Reed et al.
) have demonstrated that taxol, in addition to stabilizing MTs, also results in enrichment of MT posttranslational modifications (Hammond et al.
). This was found to alter kinesin-1 trafficking in the cell. Thus we investigated the possibility that the effects of taxol on cilia length in our assays may involve increased PTMs that could subsequently alter the activity of motor proteins, such as the IFT kinesin. This was accomplished by globally increasing tubulin acetylation using inhibitor of HDAC6. Despite a massive increase in the levels of MT acetylation, cilia assembly and length were unaffected. Furthermore, data have shown that over expression of α-tubulin acetyltransferase, mec-17 in Tetrahymena thermophila
does not affect cilia assembly (Akella et al.
). Thus we do not believe that our findings are related to PTMs; however, further studies are needed to evaluate whether other PTMs of tubulin, such as detyrosination, glycylation, or glutamylation, have the ability to alter cilia assembly or length.
Findings reported recently by Besschetnova et al.
), as well as by us here, indicate that forskolin increases cilia length; however, another study has determined that cilia length can be increased by treatment with lithium chloride (LiCl), but not by forskolin (Ou et al.
). Although LiCl is a potent inhibitor of GSK3β, which is known to affect tubulin dynamics (Hong et al.
), the proposed mechanism for LiCl by Ou et al.
) was through inhibition of adenylate cyclase III (AC3). The cause of this discrepancy is not known and may be related to the different cells used in the analyses. Ou et al.
) analyzed LiCl in fibroblast-like synoviocytes, NIH3T3 cells, astrocytes, and PC12 cells, whereas both our analyses and those by Besschetnova et al.
) used renal or retinal-pigmented epithelia. In support of this possibility, another recent study has demonstrated that LiCl treatment can cause cilia elongation in some regions of the brain but not others, thus indicating that some cilia respond differently to LiCl than others (Miyoshi et al.
Here we also show that the stunted cilia observed on cells with reduced IFT (IFT88orpk
renal cells) can be rescued by forskolin. IFT88orpk
is a hypomorphic allele where anterograde IFT is impaired; thus, based on data from Besschetnova et al.
) it might be expected that, by increasing the rate of anterograde IFT by cAMP, it would increase cilia length. As shown previously by Kim et al.
(2010), we also found that CD (as well as nocodazole) increased cilia length in the IFT88orpk
mutant cells. In these cases, it is possible that the increase in soluble tubulin precursors resulting from these treatments makes them more readily available for IFT mediated transport into the cilium under the impaired anterograde IFT conditions in the IFT88orpk
In summary, our data show that cilia length can be markedly increased using multiple pharmacological approaches to increase cAMP or to disrupt the actin and microtubule networks. In all of these cases, the increase in cilia length was directly correlated with the levels of soluble tubulin. On the basis of these findings, we propose that one aspect of cilia length control involves regulation of soluble tubulin levels. Further studies will be needed to dissect the mechanisms and signals that the cell uses to balance polymerization/depolymerization state of microtubules and how this influences the length and signaling or sensory capabilities of the cilium.