The mammalian target of rapamycin (mTOR) pathway has a crucial role in metabolism and cell growth1
. mTOR signalling is executed by two multi-protein complexes, mTORC1 and mTORC2. mTORC1 is activated by the GTPase Rheb to phosphorylate p70S6 kinase (S6K) and 4E-BP1 and thereby stimulate protein synthesis, but is effectively inhibited by treatment of cells with rapamycin. mTORC1 activity is regulated by diverse signals2–4
. Growth factors and amino acids activate mTORC1, whereas energy stress and the tumour suppressor Lkb1 inhibit mTORC1-mediated signalling through the energy sensor, AMP-activated protein kinase (AMPK)9
. Although knowledge of intracellular signal-transduction events is rapidly increasing, little information exists on where different external signals are processed to regulate mTOR signalling4
We hypothesized that cilia have a role in mTOR signalling. Cilia are signalling platforms that protrude as filiform organelles from the plasma membrane, and rely on kinesin-driven intraflagellar transport (IFT) for their form and function10,11
. They function as mechanosensors, which generates calcium currents12
, have a pivotal role in the hedgehog pathway13,14
and are involved in Wnt signalling15
. Mutations of ciliary proteins result in developmental defects, including situs-inversus and polydactyly, and postnatal diseases, such as retinal degeneration, obesity and polycystic kidney disease (PKD)8
. In PKD the tubular geometry of kidneys is distorted and fluid-filled cysts replace renal parenchyma16
. One hypothesis of why tubular cells are unable to maintain the tubular diameter is that there is a failure by the cilia to sense urine flow17
; however, the downstream effects of flow sensing are unknown. Cyst epithelia have increased mTORC1 activity7
. Interestingly, mTOR inhibitors markedly reduce cyst formation in PKD animal models5–7
, and are currently being tested in clinical trials18,19
. However, the mechanism of mTOR deregulation in PKD is not established. Polycystin-1, the most commonly mutated protein in autosomal dominant PKD (ADPKD), interacts with mTOR7
and reduces mTORC1 activity20
, but the role of cilia in mTOR regulation has not been investigated.
In polycystic kidneys, cells lining the cysts are larger than normal tubular cells21
, raising the possibility that cilia have a role in cell-size control. To determine whether the loss of primary cilia affects cell size in vivo
, we measured cell volumes in renal collecting ducts of mouse kidneys with a mutation in the kinesin subunit, Kif3a22
. These mice lose cilia by postnatal day 10 (P10) and start to form cysts at P14 (ref. 22
). The collecting duct cells of Kif3a
mutants were larger than cells in control animals () and the size distribution of Kif3a
-mutant cells was shifted to the right (), suggesting that primary cilia have a role in the regulation of cell volume.
Figure 1 Cilia regulate cell size under flow conditions. (a) 3D reconstruction of collecting ducts in kidney sections from control mice (left) and kidney-specific Kif3a-mutant-mice (right). Apical membrane stained with DBA; green, basolateral membrane stained (more ...)
To facilitate the study of how cilia regulate cell size, we generated MDCK cells with inducible knockdown of Kif3a (Kif3a-i). On incubation of these cells with tetracycline, Kif3a is knocked down and GFP (green fluorescent protein) is expressed (). Immunostaining for acetylated tubulin, a cilia marker, confirmed that GFP-positive knockdown cells lack cilia ( and Supplementary Information, Fig. S1a
). However, in contrast to the in vivo
findings, no size difference was seen in Kif3a-depleted cells, compared with non-induced control cells (). This discrepancy suggested that physiological requirements for cell-size control were missing in the in vitro
experiment. In renal tubules, cilia function as flow sensors12,23
, so we hypothesized that bending of the cilia by fluid flow might be the physiological stimulus that regulates cell size. To test this hypothesis, we analysed ciliated MDCK cells in a flow chamber that allows cultivation of cells for several days under permanent fluid flow, mimicking the physiological conditions in renal tubules24
. Interestingly, after 6 days under flow conditions the average cell size appeared smaller than in cells grown without flow (). To further validate this finding, we performed a time-course analysis and found that cell size decreases between day 1 and 4, but with no further decrease from day 4 to 8 ( and Supplementary Information, Fig. S1b
). Cells in stationary medium also reached a plateau after 4 days, but remained significantly larger, despite a similarly low mitotic index24
(Supplementary Information, Fig. S1c
). Further analysis of cross-sections was performed to ensure that differences in the x
plane are not offset by different cell heights, but no difference was found in z
-axis measurements (Supplementary Information, Fig. S1d, e
). Similarly, three-dimensional (3D) volume estimation of membrane-labelled cells demonstrated that cells under flow were significantly smaller than controls (). These findings raised the possibility that cilia require flow to regulate cell size. Indeed, when Kif3a-i cells were analysed in the flow chamber the cell size of Kif3a-depleted cells was significantly larger than non-induced cells ( and Supplementary Information, Fig. S1f
). Thus, our in vitro
findings in the flow chamber mimic the deregulation of cell size in Kif3a
-mutant mice and demonstrate that bending of cilia by flow is critical for cell size regulation.
We tested our hypothesis in a second cilia-deficient cell line, as Kif3a has known extra-ciliary functions25
. We constructed MDCK cells for the inducible depletion of the IFT-molecule Ift88 (Supplementary Information, Fig. S1g–i
). Mutation of Ift88 leads to loss of cilia and causes PKD in mice26,27
. Knockdown of Ift88 effectively suppressed cilia formation (Supplementary Information, Fig. S1j
). Similarly to Kif3a-depleted cells, unciliated Ift88-i cells under flow were significantly larger when compared with non-induced cells (Supplementary Information, Fig. S2a–d
), but no size difference was observed in two independent control cell-lines (Supplementary Information, Fig. S2e–g
). These results confirm that cell-size regulation under flow requires cilia.
To test whether cilia regulate cell size through the mTOR pathway, we analysed cell lysates from ciliated MDCK cells for phosphorylation of mTOR and its target S6K, established markers of mTORC1 actvity28
. We found that cells under flow had less phosphorylated mTOR and S6K when compared with cells in stationary medium (), thus correlating with the decreased cell size. Conversely, the levels of phosphorylated mTOR and S6K increased after depletion of Kif3a in cells grown under flow (), which corresponds to the larger size of these cells, compared with control cells. We hypothesized that flow represses mTORC1 in ciliated cells, and that cells without cilia fail to downregulate mTOR. Indeed, in ciliated Kif3a-i cells after 5 days under flow, phosphorylated S6K was decreased, compared with baseline before flow, whereas in Kif3a-i cells induced with tetracycline and therefore unciliated, the downregulation of phosphorylated S6K was less pronounced (). Analogous results were obtained in Ift88-i cells (), but not in control lines (Supplementary Information, Fig. S2h, i
). Similarly to our in vitro
mutant kidneys contained more phosphorylated ribosomal-S6-protein (rS6), an S6K target, than kidneys from control animals () and epithelial phosphorylated rS6 staining was found in mutant mice but not in controls (). These results confirm a positive correlation between cell size and mTORC1 activity and suggested that the cell size changes are mediated through cilia-dependent downregulation of the mTOR pathway.
Figure 2 Cilia and flow modulate mTORC1 activity. (a) Western blot to assess mTOR phosphorylation in ciliated wild-type MDCK cells grown under flow and non-flow conditions. (b) Intensity of phosphorylated mTOR (p-mTOR) bands, relative to intensity of mTOR bands, (more ...)
To further test if cell-size regulation in vitro
occurs through mTOR we treated ciliated cells under flow with rapamycin29
. Compared with vehicle-treated cells, rapamycin had no effect on the size of ciliated Kif3a-i cells (Kif3a-i cells without tetracycline; ) suggesting that flow maximally suppresses mTORC1 activity in ciliated cells. However, in unciliated cells, rapamycin compensated for the absence of cilia and resulted in smaller cells (Kif3a-i cells with tetracycline; ). In a complementary approach, co-expression of dominant-negative Raptor, a scaffold protein of mTORC129,30
, or Raptor knockdown, in Kif3a-depleted cells, resulted in a similar size decrease under flow as in ciliated cells ( and Supplementary Information, Fig. S3a, b
) and thus demonstrated that the size defect with absent cilia is because of ineffective downregulation of mTORC1.
Figure 3 Flow-induced cell-size regulation is mTOR dependent and ablation of flow-induced calcium transients has no effect on cell size. (a) Size of Kif3a-i cells, in absence or presence of tetracycline and treated as indicated, under flow conditions. Uninduced (more ...)
Using an opposite approach, we reasoned that activation of mTOR would counteract the flow-mediated cell-size reduction. We transduced MDCK cells with a tetracycline-inducible gain-of-function mutant of the mTORC1 activator, Rheb31
; Supplementary Information, Fig. S3c
). Induction of this construct activated mTOR () and resulted in significantly larger cells under flow than the same cells without induction (). These results demonstrate that mechanical stimulation of cilia downregulates the mTOR pathway and cell size.
After establishing a link between cilia, mTOR and cell size, we were interested in the underlying mechanism. Protein kinase B (Akt) is a major activator of mTOR. Akt activates mTORC1 directly, as well as indirectly by releasing Rheb from inhibition by the Tsc complex2
. We hypothesized that if mTOR deregulation through lack of cilia involved Akt activation, loss of Akt function would rescue the phenotype. However, expression of a dominant-negative Akt mutant in Kif3a-depleted cells neither abrogated cell-size deregulation nor S6K phosphorylation under flow (Supplementary Information, Fig. S3d–f
), suggesting that a role for Akt in ciliary mTOR regulation is unlikely.
The mechanosensory function of cilia in the kidney involves the cation channel polycystin-2 (TRPP2), which is mutated in a subset of patients with ADPKD. TRPP2 translates fluid flow into calcium transients17
. However, it is unclear if deficient flow-mediated calcium transients have a role in cyst formation32
. We hypothesized that inhibition of the mTOR pathway through ciliary bending might be mediated by a calcium signal. Ciliated MDCK cells are a well-established model to analyse calcium signalling under flow12,23
. Consequently, we generated tetracycline-inducible TRPP2 knockdown cells. Tetracycline induction of these cells resulted in efficient depletion of TRPP2 (Supplementary Information, Fig. S3g
) and inhibited flow-induced calcium transients ( and Supplementary Information, Fig. S3h
). Surprisingly, in cells grown under flow conditions, no difference was found in cell size or levels of phosphorylated S6K after knockdown of TRPP2, compared with control cells ( and Supplementary Information, Fig. S3i, j
). This finding indicated that cell size and mTORC1 control by cilia occurs independently of flow-induced calcium signalling.
Next, we investigated the tumour suppressor, Lkb1, a negative regulator of mTOR9,33
. In fact, loss of Lkb1 increases cell mass in pancreatic β-cells through activation of mTOR34
. To test if Lkb1 has a role in the ciliary regulation of cell size, we generated MDCK cells with tetracycline-inducible knockdown of Lkb1 (). Similarly to previous observations35
, depletion of Lkb1 in MDCK cells did not affect markers of apico-basal cell polarity or cilia formation ( and Supplementary Information, Fig. S4
). As expected, phosphorylation of AMPK, the target of Lkb1, was significantly decreased (Supplementary Information, Fig. S5a, b
), thus validating the knockdown approach. Similarly to unciliated cells, Lkb1-depleted cells showed no difference in size when compared with non-induced cells, in the absence of flow (). However, when subjected to flow, Lkb1-i cells were significantly larger than the controls without induction (). Similar results were obtained in cells expressing dominant-negative mutant Lkb136
; and Supplementary Information, Fig. S5c
), confirming that Lkb1 has a role in cell-size regulation under flow. The phosphorylation of S6K was increased in Lkb1 knockdown cells under flow () demonstrating that the size increase of Lkb1-deficient cells is accompanied by mTOR activation. Notably, expression of Lkb1 (Supplementary Information, Fig. S5d
) in unciliated Kif3a-depleted cells did not rescue the absent-cilia phenotype ( and Supplementary Information, Fig. S5e
) and suggested that cilia are required for Lkb1 activation.
Figure 4 Lkb1 modulates flow-dependent mTOR and cell-size regulation and is localized in the basal body and the cilium. (a) Western blot of lysates from Lkb1-i MDCK cells. Cells were established so that expression of shRNA against Lkb1, and expression of GFP, (more ...)
We then investigated the link between Lkb1 and the cilium. Lkb1 was present in cilia and the basal body, as visualized by immunostaining () and by overexpression of an Lkb1–YFP (yellow fluorescent protein) fusion protein ( and Supplementary Information, Fig. S5f
). We hypothesized that the bending of cilia may activate the Lkb1 pathway, but levels of phosphorylated AMPK in whole cell lysates were similar in cells grown under flow and without (Supplementary Information, Fig. S5g, h
). Strikingly, we found an intense accumulation of phosphorylated AMPK at the basal body (Fig. 5a–c). Furthermore, quantitative image analysis revealed that phosphorylated AMPK was significantly increased at the basal body of cells under flow, compared with cells grown in stationary medium (Fig. 5d, e). In summary, these findings demonstrate that bending of the cilium by flow activates the Lkb1 pathway in the cilia-basal body compartment to inhibit mTORC1 activity and cell size (Fig. 5f for schematic representation of a model).
Our data establish that cilia function as regulators in the mTOR signalling cascade and provide important insight into the mechanism of how extracellular signals are transduced to regulate mTOR activity. It is currently unclear how input–output specificity is achieved for a large number of extracellular signals that are translated into different mTOR effectors4
. Our data suggest that the cilia-basal body compartment is where the Lkb1 pathway is activated on mechanical engagement of the cilium, and thus provide evidence of a spatially restricted regulatory site for mTOR signalling. Our data further demonstrate that, apart from calcium transients, mTOR regulation by Lkb1 in the cilium-basal body compartment represents an independent mechanosensory signal transduction mechanism that translates shear stress through bending of the cilium into cellular programmes. Our findings raise the intriguing question of whether cell size is important for tubular geometry of the kidney. Previous evidence links cell shape and surface tension with complex cellular programmes and morphogenesis37
. Perfusion studies have shown that fluid flow and shear forces depend on tubular diameter38
. It is conceivable that larger cells change the tubular diameter and thereby alter flow sensing. Whether cell-size-mediated changes in shear stress contribute to cyst formation remains to be shown in future work. Recent data indicate that polycystin 1 downregulates mTOR independently of flow20
, suggesting that other pathways exist to modulate mTOR in renal epithelial cells.
It is possible that the cilium might have a role in mTOR signalling in contexts other than mechanotransduction. The cilium is a signalling organelle equipped with an intraflagellar transport system providing fast access for signalling molecules to a restricted compartment ideally suited for environmental sensing11
, such as light in photoreceptors, smell in odorant receptors, and the regulation of Hedgehog and Wnt signalling11
. Interestingly, the glucose transporter, Glut2, is localized in ependymal cilia in the brain39
. Furthermore, cilia-deficient mice have impaired metabolic control after glucose challenge40
, thus providing the basis for a functional link between glucose metabolism and cilia. Insulin signalling activates mTORC1 and glucose control is, at least in part, regulated by Lkb1 and AMPK in a complex mechanism that is not fully understood34
. Together with these findings our results raise the possibility that, beyond mechanosensation, cilia may have a broader role in metabolic control through Lkb1, AMPK and mTOR signalling.