FOP plays a negative role in ciliogenesis and promotes cell cycle re-entry by facilitating primary cilia disassembly

Primary cilia are microtubule-based, antenna-like organelles, which are formed in G0 phase and resorbed as cells re-enter the cell cycle. It has been reported that the length of primary cilia can influence the timing of cell cycle progression. However, the molecular links between ciliogenesis and cell cycle progression are not clear. FOP (Fibroblast growth factor receptor 1 Oncogene Partner, also known as FGFR1OP) has been implicated in ciliogenesis. Here, we show that the expression of FOP during cell cycle exit and re-entry is negatively correlated with ciliogenesis. Knockdown of FOP promotes cilia elongation and suppresses timely cilia disassembly. In contrast, ectopic expression of FOP inhibits cilia growth. Moreover, pharmacological inhibition of actin polymerization with Cytochalasin D abrogates FOP-induced cilia disassembly, suggesting that FOP facilitates cilia disassembly by promoting actin cytoskeleton formation. Lastly, knockdown of FOP delays cell cycle re-entry of quiescent cells following serum re-stimulation, and this can be reversed by silencing IFT20 (intraflagellar transport 20), an intraflagellar transport protein essential for ciliogenesis. Collectively, these results suggest that FOP plays a negative role in ciliogenesis and can promote cell cycle re-entry by facilitating cilia disassembly.


Introduction
Primary cilia are microtubule-based organelles that protrude from the cell apical surface to sense environmental cues that regulate cell growth, development and homeostasis (Ishikawa and Marshall, 2011;Malicki and Johnson, 2017;Nigg and Raff, 2009;Satir and Christensen, 2007;Satir et al., 2010). As cell signaling centers, primary cilia coordinate with many cell signaling pathways, including those mediated by Hedgehog, Wnt and RTKs (Receptors of Tyrosine Kinases) (Bangs and Anderson, 2017;Christensen et al., 2017;Liu et al., 2018;Malicki and Johnson, 2017;Wallingford and Mitchell, 2011). Defects in cilia assembly and/or functions can lead to a wide range of human disorders, termed ciliopathies, characterized by mental retardation, polycystic kidney, retinal defects, obesity, diabetes and other development abnormalities (Hildebrandt et al., 2011;Reiter and Leroux, 2017).
The assembly and disassembly of primary cilia are tightly controlled in the cell cycle.
Primary cilia are formed in quiescent (G0 phase) cells and are resorbed as cells reenter the cell cycle (Sánchez and Dynlacht, 2016). Primary cilia emanate from the mother centrioles, and primary cilia and centrosomes share the same centrioles. When a quiescent cell enters the proliferative cycle, the centrioles need be released from primary cilia in order to act as spindle poles in mitosis. It is therefore assumed that primary cilia function as a structural checkpoint for cell cycle re-entry (Basten and Giles, 2013;Izawa et al., 2015;Ke and Yang, 2014). This hypothesis has been verified by several studies. For example, both Nde1 (Nuclear distribution gene homologue 1) and Tctex-1 (dynein light chain tctex-type 1, also known as DYNLT1) promote primary cilia disassembly and accelerate cell cycle re-entry in response to growth-factor stimulation, and they do not significantly influence cilia assembly (Kim et al., 2011;Li et al., 2011).
Moreover, knockdown of NEK24 or KIF24 induces primary cilia formation and inhibits cell proliferation in cycling RPE1 cells and breast cancer cells (Kim et al., 2015). In contrast, depletion of proteins required for ciliogenesis, such as IFT88 (intraflagellar transport 88, also known as Polaris and Tg737) and KIF3A (kinesin family member 3A), facilitates cell proliferation (Deng et al., 2018).
The dynamics of primary cilia assembly and disassembly is modulated by numerous regulators (Sánchez and Dynlacht, 2016). Recent studies have shown that the actin cytoskeleton is involved in this dynamic process. Pharmacological inhibition of actin polymerization with Cytochalasin D (CytoD) promotes primary cilia formation and elongation and inhibits cilia disassembly during cell cycle re-entry (Kim et al., 2011;Li et al., 2011;Sharma et al., 2011), suggesting that the actin cytoskeleton negatively regulates ciliogenesis. Moreover, actin related proteins such as cortactin, components of the ARP2/3 complex and Tctex-1 required for actin nucleation, branching, polymerization and stress fiber formation, also inhibit ciliogenesis and promote primary cilia disassembly (Bershteyn et al., 2010;Cao et al., 2012;Drummond et al., 2018;Kim et al., 2010;Ran et al., 2015;Saito et al., 2017).
While primary cilia assembly has been extensively studied, much less is known about the molecular mechanism underlying primary cilia disassembly (resorption). In addition to the proteins mentioned above, several other signaling axes, such as Aurora A-HDAC6 (Histone Deacetylase 6), responsible for cilia disassembly have been identified (Sánchez and Dynlacht, 2016). Aurora A can be activated by HEF1 (Human enhancer of filamentation 1, also known as NEDD9), Pitchfork (Pifo), trichoplein and calcium/calmodulin, and the activated Aurora A in turn phosphorylates HDAC6 and stimulates its tubulin deacetylation activity, resulting in the destabilization of the ciliary axoneme and thus cilia resorption (Inoko et al., 2012;Kinzel et al., 2010;Plotnikova et al., 2012;Pugacheva et al., 2007). FOP (Fibroblast growth factor receptor Oncogene Partner, also known as FGFR1OP) is a centrosomal and centriolar satellite protein (Lee and Stearns, 2013;Popovici et al., 1999;Yan et al., 2006). It associates with CEP350 and is required for microtubule anchoring (Yan et al., 2006). Knockout of FOP in chicken DT40 lymphocytes causes G1 arrest and eventual apoptosis (Acquaviva et al., 2009). Although several studies have suggested that FOP is involved on ciliogenesis, the role of FOP on ciliogenesis remains controversial. Some studies reported that knockout of FOP inhibited primary cilia formation (Kanie et al., 2017;Lee and Stearns, 2013;Mojarad et al., 2017); however, moderate knockdown of FOP had no effect on ciliogenesis (Graser et al., 2007;Lee and Stearns, 2013). Moreover, whether FOP regulates cilia length is unknown. In this study, in contrast to previous reports, we show that FOP plays a negative role in ciliogenesis. Knockdown of FOP increases cilia length, while ectopic over-expression of FOP suppresses cilia growth. Moreover, FOP induces actin polymerization-dependent cilia disassembly. We also demonstrate that knockdown of FOP delays cell cycle re-entry of quiescent cells following serum re-stimulation.
Disruption of ciliogenesis by IFT20 depletion abolishes the delay in cell cycle re-entry caused by FOP knockdown. Together, these data suggest a negative effect of FOP on ciliogenesis and reveal a novel role of FOP in cilia disassembly by promoting actin cytoskeleton formation and thereby linking the dynamics of ciliogenesis to cell cycle progression.

Cell Line and Culture
hTERT-RPE1 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM/F12 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 0.01 mg/mL hygromycin B in a humidified incubator at 37 °C with 5% CO2.

Flow Cytometry (FACS Analysis)
Cells were detached with trypsin, centrifuged at 3000 rpm for 10 min, washed twice with PBS and fixed overnight in 70% ethanol at -20°C. The cells were then resuspended and stained with PI staining buffer (5 μg/mL RNase A, 0.1% (v/v) Triton X-100, 10 mM EDTA (pH8.0) and 50 μg/mL propidium iodide [Sigma]) for 1 hr at room temperature. For each sample, at least 10,000 cells were analyzed by FACSort machine (Becton Dickinson). Data were analyzed using Flowing Software 2.

Cilia Assembly and Disassembly Assays
To induce primary cilia assembly, cells (untreated or 24 hrs post transfection) were starved in serum-free medium for 48 hrs. In some experiments, cells were treated with DMSO or 1 µM Cytochalasin D (CytoD, Sigma) at the final 2 hrs of the serum-starvation.
To analyze cilia disassembly, cells were plated at less than 30% confluency before transfection. Cells were serum starved for 48 hrs immediately after transfection, and then 10% serum was added back to the medium to stimulate cilia resorption and cell cycle re-entry. Cells were fixed at different time points and immunostained with cilia makers. The length of the cilia was measured using Image J software (NIH).

EdU Incorporation Assay
EdU (10 μM) was added to the growth medium 2 hrs prior to fixation. The cells were subsequently stained according to the manufacturer's instructions (RiboBio). Mounted slides were observed and photographed using Elipse Ti-E microscopy using a 20X objective. Images were acquired with NIS-elements basis research (Nikon) and processed with Image J (NIH).

Statistical Analysis
Statistical analysis was performed with Prism version 5 (GraphPad). Statistical significance of the difference between two groups was determined as indicated.

The FOP Expression Level Is Negatively Correlated with Ciliogenesis
Although several studies have suggested that FOP may be involved in ciliogenesis

FOP Plays a Negative Role in Ciliogenesis
To confirm the negative role of FOP in ciliogenesis, we silenced FOP in RPE1 cells using siRNA ( Figure 2A) and examined the effects on ciliogenesis by Arl13b (ADPribosylation like protein) immunostaining ( Figure 2B-D). Surprisingly, the percentage of ciliated cells in the negative control cells and FOP knockdown cells were comparable ( Figure 2B and 2C). However, the FOP knockdown cells formed significantly longer cilia, compared to the negative control cells ( Figure 2B and 2D).
To collaborate these results, we examined the effects of FOP overexpression on  Figure S1E). Therefore, cilia elongation induced by FOP knockdown is not mediated through OFD1 degradation.

FOP-Promoted Cilia Disassembly Is Dependent on Actin Polymerization
The actin cytoskeleton has been established as a negative regulator of ciliogenesis We then asked whether disrupting the actin structure with CytoD could attenuate the negative effects of FOP in ciliogenesis. We found that after CytoD treatment, the FOPoverexpressed cells no longer showed a decrease of ciliation or cilia length compared to the vector control cells, (Figure 4A-C) indicating that the CytoD treatment negated the negative effects of FOP overexpression on ciliogenesis. Therefore, these data suggest that FOP promotes cilia shortening through enhancing actin polymerization.

FOP Is Required for Timely Cilia Disassembly during Cell Cycle Re-entry
Since FOP negatively regulates cilia length, we next determined whether FOP promotes cilia disassembly when quiescent cells re-enter the cell cycle upon serum restimulation. To this end, FOP-silenced RPE1 cells were serum starved to induce cilia formation and then re-stimulated with serum to induce cilia resorption and cell cycle re-entry. Consistent with the data shown in Figure , 2007). We therefore investigated the relationship between FOP and Aurora A during cilia disassembly and cell cycle re-entry. Interestingly, immunoblotting analysis showed that the expression levels of Aurora A and phosphorylated Aurora A at T288 (p-Aurora A) decreased in FOP-silenced cells, which was most obvious at 18 hrs and 24 hrs post serum re-stimulation ( Figure 5D), suggesting that FOP may modulate the expression and activation of Aurora A to promote cilia disassembly.

Knockdown of FOP Delays Cell Cycle Re-entry
The disassembly of primary cilia is required for cell cycle re-entry (Kim et al., 2011;Li et al., 2011). As knockdown of FOP suppresses cilia disassembly, we therefore asked whether silencing of FOP inhibits cell cycle re-entry. After serum starvation, cell cycle re-entry was induced by re-addition of serum. Cells were labeled with ethynyldeoxyuridine (EdU) after serum re-stimulation, and the percentage of EdU positive cells was determined. The percentage of EdU positive cells in FOP-silenced cells was significantly lower than that of the negative control cells at 12 hrs, 18 hrs, and 24 hrs after serum re-stimulation ( Figure 6A-B). Immunoblotting analysis also showed that FOP-silenced cells had lower levels of the cell proliferation markers, including phosphorylated Rb at Ser807/811, phosphorylated cdc2 at Tyr15 and cyclin A during cell cycle re-entry ( Figure 6C). Correspondingly, flow cytometry analysis also revealed a delay in cell cycle progression ( Figure 6D). In addition, 24 hrs after serum restimulation, ~12.3% of control cells entered mitosis, as indicated by the nuclear morphology, whereas only ~3.6% of FOP knockdown cells were mitotic ( Figure 6E).
Collectively, these data indicate that the knockdown of FOP delayed cell cycle re-entry.

FOP Regulates Cell Cycle Re-entry through Modulating Primary Cilia
We then asked if the delay in cell cycle re-entry caused by FOP knockdown is mediated by primary cilia. We silenced IFT20, a component of intraflagellar transport, to disrupt primary cilia formation (Follit et al., 2006). Consistent with previous studies, the depletion of IFT20 strongly inhibited cilia assembly ( Figure S2A-C). In addition, the primary cilia were also shortened after IFT20 depletion in cells transfected with either negative control siRNA or FOP siRNA ( Fig. S2D and E).
We then investigated if the loss of cilia by IFT20 silencing can overcome the delay in cell cycle re-entry induced by FOP depletion. Knockdown of FOP without ITF20 silencing (siNC, which was the control siRNA for the IFT20 siRNA) led to a significant decrease in the percentage of EdU positive cells at 18 hrs after serum re-stimulation, confirming the delay in cell cycle re-entry as described above. In contrast, this delay was rescued by IFT20 depletion (Figure 7A and B). Correspondingly, the levels of phosphorylated Rb (p-Rb), phosphorylated cdc2 (p-cdc2) and Cyclin A in the FOPsilenced cells were also restored by IFT20 depletion ( Figure 7C). Flow cytometry analysis data also showed that the inhibition of ciliogenesis by IFT20 depletion rescued the delay of cell cycle progression induced by FOP knockdown ( Figure 7D). These results collectively suggest that the delay in cell cycle re-entry induced by FOP knockdown is dependent on primary cilia.

Discussion
Although recent studies reported that near complete silencing of FOP inhibited primary cilia formation (Cabaud et al., 2018;Kanie et al., 2017;Mojarad et al., 2017), others found that moderate knockdown of FOP had no effect on ciliogenesis (Graser et al., 2007;Lee and Stearns, 2013 Expression of the proteins involved in ciliogenesis must be precisely regulated to exert different roles during cilia assembly and disassembly. For example, CP110 displays complex roles in ciliogenesis through the ubiquitin-proteasome system as well as transcriptional programs. High levels of CP110 suppress primary cilia formation, while optimal levels of CP110 promote ciliogenesis (Cao et al., 2012;Kobayashi et al., 2011;Song et al., 2014;Spektor et al., 2007;Walentek et al., 2016;Yadav et al., 2016).
Notice that FOP does not completely degrade during ciliogenesis. Therefore, it is possible that a small fraction of FOP is necessary and sufficient for the recruitment of the CEP19-RABL2 complex to the ciliary base, allowing IFT entry and initiation of ciliogenesis. As such, complete absence of FOP would compromise the essential role of FOP in the early steps of primary cilia assembly. In the present study, we identified a novel function of FOP in ciliogenesis. We found that FOP suppresses ciliary axoneme elongation and promotes timely cilia disassembly during cell cycle re-entry, while knockdown of FOP by 80% does not impair primary cilia assembly. Together with the data from others' previous studies, our results suggest that FOP may play multiple and dose-dependent roles in ciliogenesis, being required for primary cilia formation and also playing a negative role in the regulation of cilia length. It will be interesting to elucidate how FOP's levels are precisely controlled to produce optimal levels for ciliogenesis and disassembly.
Recently, many studies have suggested that actin dynamics regulate cilia assembly and disassembly (Bershteyn et al., 2010;Cao et al., 2012;Drummond et al., 2018). Together, these data suggest that FOP promotes cilia disassembly by enhancing actin cytoskeleton formation.
The Aurora A-HDAC6 signaling pathway is essential for cilia disassembly when cells re-enter the cell cycle (Pugacheva et al., 2007). Aurora A has several activators including HEF1, Pifo and trichoplein (Inoko et al., 2012;Kinzel et al., 2010;Pugacheva et al., 2007). Our data suggest that FOP may serve as an upstream regulator of Aurora A and modulate the expression and activation of Aurora A, thereby promoting cilia disassembly. Although It has previously been suggested that Aurora A-HDAC6 and actin dynamics are two independent pathways that regulate cilia disassembly (Bershteyn et al., 2010), a recent study demonstrated that HDAC6 also deacetylates another substrate, cortactin, which further enhances actin polymerization, thus inducing cilia disassembly (Ran et al., 2015;Zhang et al., 2007). Together with these previous findings, our data on the regulatory role of FOP on Aurora A in the current study further strengthen the connection between the Aurora A-HDAC6 pathway and actin dynamics in the regulation of cilia disassembly.
The link between ciliogenesis and the cell cycle has been well established (Kim et al., 2011;Li et al., 2011;Pugacheva et al., 2007). Primary cilia have to be completely disassembled prior to mitosis, releasing the centrioles to form the mitotic spindle poles.
Therefore, the length of primary cilia regulates the duration of the cell cycle. For example, depletion of Nde1 or Tctex-1 accelerates cell cycle progression by triggering timely cilia disassembly (Kim et al., 2011;Li et al., 2011). The role of FOP in cell cycle progression has previously been implicated (Acquaviva et al., 2009). The data we present here suggest that FOP, like Nde1 and Tctex-1, also facilitates cell cycle reentry by promoting cilia disassembly. Given that FOP is a centrosomal protein, and loss of centrosome integrity causes G1-S arrest (Mikule et al., 2007;Yan et al., 2006), it could be argued that the cell cycle delay in FOP knockdown cells originated from a loss of centrosome integrity. However, as the cell cycle re-entry delay induced by FOP knockdown can be rescued by the depletion of IFT20 (which is essential for cilia assembly but not centrosome integrity; Follit et al., 2006), a possible loss of centrosome integrity cannot be the reason for cell cycle re-entry delay induced by FOP knockdown. Furthermore, in chicken DT40 lymphocytes, FOP knockout did not impair centrosome integrity (Acquaviva et al., 2009). By immunostaining g-tubulin, we also did not detect any apparent centrosome defects. Therefore, the delay in cell cycle re-entry induced by FOP knockdown is mediated by primary cilia.
Recent studies have suggested that primary cilia are involved in tumorigenesis and tumor progression, as the loss of cilia is frequently observed in various types of cancer such as breast cancer, prostate cancer and pancreatic ductal adenocarcinoma (Hassounah et al., 2013;Menzl et al., 2014;Seeley et al., 2009;Yuan et al., 2010).
Although the mechanism is still unclear, the loss of cilia probably provides a growth advantage and promotes malignant transformation and metastasis during the early stages of tumor development (Basten and Giles, 2013;Deng et al., 2018;Zingg et al., 2018). Upregulation of FOP has been observed in lung cancer tissues and cell lines (Mano et al., 2007). Here, we demonstrate that FOP promotes cilia disassembly and accelerates cell cycle progression. It is likely that the upregulation of FOP induces cilia loss and provides growth advantages for cancer cells.