Phagocytic and pinocytic uptake of cholesterol in Tetrahymena thermophila impact differently on gene regulation for sterol homeostasis

The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. This metabolic shift is mainly driven by transcriptional regulation of genes for tetrahymanol synthesis (TS) and sterol bioconversion (SB). The mechanistic details of sterol uptake, intracellular trafficking and the associated gene expression changes are unknown. By following cholesterol incorporation over time in a conditional phagocytosis-deficient mutant, we found that although phagocytosis is the main sterol intake route, a secondary endocytic pathway exists. Different expression patterns for TS and SB genes were associated with these entry mechanisms. Squalene synthase was down-regulated by a massive cholesterol intake only attainable by phagocytosis-proficient cells, whereas C22-sterol desaturase required ten times less cholesterol and was up-regulated in both wild-type and mutant cells. These patterns are suggestive of at least two different signaling pathways. Sterol trafficking beyond phagosomes and esterification was impaired by the NPC1 inhibitor U18666A. NPC1 is a protein that mediates cholesterol export from late endosomes/lysosomes in mammalian cells. U18666A also produced a delay in the transcriptional response to cholesterol, suggesting that the regulatory signals are triggered between lysosomes and the endoplasmic reticulum. These findings could hint at partial conservation of sterol homeostasis between eukaryote lineages.


Results
A secondary pathway for cholesterol transport in T. thermophila. Recent evidence suggests that cholesterol can primarily be internalized by the ciliate via phagocytosis 22 . In order to further characterize this process, we analyzed the kinetics of cholesterol intake. Radiolabeled cholesterol dissolved in ethanol was added to cultures at early exponential growth, and its incorporation into the cells was assessed at different times. Figure 1 shows that wild-type cells (CU428) avidly incorporated cholesterol with a hyperbolic behavior, reaching its maximum after approximately two hours. In contrast, the conditional mutant lacking phagosomes (II8G-IA) 23 exhibited a reduced uptake of cholesterol at the restrictive temperature (37 °C). This uptake was completely abol-  Fig. S1), which are known inhibitors of actin assembly and actin-dependent vesicle transport 29,30 . This suggests the presence of a secondary pinocytic entry of sterols aside from the main phagocytic mechanism.
Different signaling pathways triggered by cholesterol. The next question we addressed was whether the sterol intake route would influence the transcriptional response of genes known to be regulated by the presence of cholesterol in T. thermophila. In our previous transcriptomic analysis 19 , we found that cholesterol produced the up-regulation of 179 genes and the down-regulation of 177 genes. Among these, we selected several up-and down-regulated genes to quantify their transcripts by RT-qPCR in cells grown in the absence or presence of cholesterol. Figure 2 shows the variation produced by cholesterol in transcript levels of DES22B (TTHERM_00085010), MLup (TTHERM_00030420) and Δ6DES (TTHERM_00339850) as representative upregulated genes and SQS (TTHERM_00382150) and MLdw (TTHERM_00353379) as down-regulated genes 19 . RNA samples were obtained from CU428 and II8G-IA strains cultured at the restrictive (37 °C, Fig. 2a) or permissive (30 °C, Fig. 2b) temperatures. MLdw and SQS exhibited similar responses to cholesterol in both strains grown at 30 °C. Interestingly, these genes were only insensitive to cholesterol at the restrictive temperature in the II8G-IA strain, indicating that phagocytosis must be functional for a successful down-regulation of these genes. By contrast, up-regulated genes displayed similar responses to cholesterol in both strains and temperatures. The disparity in the transcriptional responses to cholesterol between these groups of genes further demonstrates that they are independently regulated by at least two signaling pathways. These results showed that up-regulated genes were induced in the II8G-IA strain despite the absence of phagosomal function. We considered the possibility that endocytosed cholesterol-loaded vesicles would arrive to an internal compartment where the signals would be triggered. Consequently, we examined the expression levels of up-regulated genes in both strains exposed to pharmacological treatment in order to disrupt any actindependent vesicular transport. DES22B and SQS were selected as representative reporters for up-and downregulated genes, respectively, for further RT-qPCR determinations. As can be seen in Fig. 3, cytochalasin D abolished the response to cholesterol of DES22B in II8G-IA cells. The persistent induction of DES22B observed in CU428 cells could be due to an incomplete inhibition of endocytosis. As shown in Fig. 1 and Supplementary  Fig. S1, the drugs completely prevented cholesterol internalization in mutant cells. These findings demonstrate that modulation of the transcriptional response to cholesterol requires its internalization by phagocytosis and pinocytosis, the latter being sufficient to induce DES22B. Furthermore, they exclude any involvement of signaling triggered from outside of the cell. Differential effect of cholesterol concentration in transcriptional regulation. The results of the previous section led to the hypothesis that only bulk incorporation of cholesterol can trigger the transcriptional repression of SQS. In contrast, DES22B up-regulation could be achieved with lower quantities of the sterol, which have been sufficiently provided by the pinocytic pathway. To support this idea we investigated the dependence of each signaling pathway on cholesterol concentration. Figure 4 shows that the cholesterol concentration required for a significant SQS down-regulation was approximately one order of magnitude higher than what was needed for DES22B up-regulation.   31 . In our radiolabeling experiments, we were able to estimate the time of trafficking to ER in approximately 30 min for wild-type cells and 120 min for mutant cells (Fig. 5a,b). The amino-steroid U18666A is a known inhibitor of intracellular cholesterol trafficking in mammalian cells 32 . It binds NPC1, thus blocking the trafficking between LE/LY and other organelles like the ER 33 . Treatment with U18666A inhibited the synthesis of cholesteryl-esters in both strains, indicating a blockage in the traffic between LE/LY (or similar organelles) and the ER. This effect persisted during the five hours of the assay (Fig. 5). Based on these results, it can be inferred that the two routes of cholesterol uptake share a common NPC1-containing compartment before reaching the ER.  www.nature.com/scientificreports/ To rule out a direct inhibition of ACAT by U18666A as a possible cause for these results, we assayed its activity in cell-free extracts prepared in two different ways: (i) cell-free preparations incubated with this inhibitor during the enzyme assay; (ii) preparations from cells treated with U18666A during 20 min before extract preparation. No significant inhibition was detected in any case (Fig. 5c).
In agreement with previous reports that U18666A induced a transient accumulation of free cholesterol in mammalian lysosomes 3,33 , we observed in the present study a similar effect on T. thermophila (Fig. 6). The amount of radioactivity in the cell pellets increased shortly after treatment with U18666A in CU428 cells but not in mutant II8G-IA cells (Fig. 6a). To better understand the nature of this transient blockage, cells were stained with Filipin, a fluorescent dye that binds free, non-esterified sterols, and were observed using fluorescence microscopy. Wild-type cells treated with cholesterol for 1 h before fixation exhibited few characteristically large, phagosome-like vesicles (Fig. 6b, upper central panel). Pretreatment with U18666A produced a marked increase in the number and fluorescence intensity of such large vesicles (Fig. 6b, upper right panel). This supports the hypothesis that U18666A inhibits cholesterol transport from phagosomes or LE/LY to other vesicles or organelles, such as the ER.
In contrast with what was observed in wild-type cells, we could not detect Filipin staining signals in II8G-IA cells, as cell fluorescence in either treatment was similar to autofluorescence levels (Fig. 6b, lower panels). This lack in detection likely reflects the limitation of the method to detect small amounts of free cholesterol putatively present inside pinocytes or inserted in the plasma membrane. Under normal growth conditions, it is likely that cholesterol is rapidly distributed to different organelles and membranes, including the ER where it is esterified (and stored in lipid droplets). The fluorescent signal of Filipin therefore becomes fainter and more difficult to detect.
We then tested whether the effects of U18666A are associated with changes in the transcriptional response to cholesterol. In the absence of the inhibitor, both DES22B and SQS genes showed a maximal response in CU428 cells 30 min after the addition of cholesterol. Treatment with U18666A produced a 60-min delay in the response of DES22B and completely reverted SQS down-regulation (Fig. 7a), similar to what was found in the untreated mutant (Fig. 7b). DES22B induction was also delayed in the mutant strain (30 min) and the NPC1 inhibitor produced a further delay in its up-regulation (Fig. 7b). These results are compatible with the localization of the signaling origin between LE/LY and ER for both up-and down-regulation. Alternatively, such signaling could be mediated directly by NPC1 in LE/LY.

Effect of Brefeldin A on the transcriptional responses for both reporter genes.
In order to support the notion that the triggering of signals is confined to the ER or in NPC1-containing compartments, we examined the effect of Brefeldin A, which has been used in Tetrahymena as a Golgi apparatus-disrupting agent 34 . We speculated that this agent would block cholesterol trafficking beyond the ER, thus increasing the cholesterol content in this cellular compartment and exacerbating the transcriptional response of reporter genes. Unexpectedly, Brefeldin A did not produce a significant increase in SQS down-regulation, although DES22B up-regulation www.nature.com/scientificreports/ was slightly increased after 30 min of cholesterol addition (Fig. 8). This response was quickly dissipated, as transcript levels of the gene were similar either with or without Brefeldin A at later time points. We were also unable to detect a significant increase in cholesterol conversion to steryl-esters (not shown). This result demonstrates that the Golgi apparatus is not involved in these particular signaling events.

Discussion
T. thermophila sterol metabolism alternates between tetrahymanol synthesis and exogenous sterols uptake and bioconversion, likely as an adaptive mechanism to the changing content of oxygen in the aquatic medium and sterol abundance 9,10 . To produce this "switch", the cell must rely on an internal signaling system that quickly and adequately responds to sterol availability.
In the present work, we demonstrate that cholesterol enters the cells through two types of actin-dependent transport: phagocytosis and pinocytosis. Our results support that phagocytosis is the main mechanism for cholesterol uptake, a finding which had also been suggested in a recent report 22 . Similar observations were made in the related ciliate Paramecium primaurelia, in which the uptake of a fluorescent cholesteryl ester analogue occurred via phagocytosis and through the plasma membrane 35 . Although we could not detect cholesterol incorporation in phagocytosis-deficient cells with the fluorescence microscopy approach used in these reports, we did so by measuring the uptake of radiolabeled cholesterol, which was found to be a more sensitive method. Because of their complex cortical organization, pinocytosis in Tetrahymena and other ciliates takes place at particular cellular sites, such as the parasomal sacs located near the ciliary basal bodies 36 . The elucidation of the mechanisms involved in sterol uptake warrants further research.
Based on the evidence shown in cholesterol esterification and U18666A treatment experiments, we propose that most of the cholesterol internalized by both mechanisms converges on mature phagolysosomes before being www.nature.com/scientificreports/ delivered to the ER where it is bioconverted or esterified. Interestingly, the fusion between endocytic vesicles formed at the parasomal sacs and phagosomes has been described in Paramecium 37 . As suggested from the experiments with U18666A, a NPC1-like protein would be present in this compartment and involved in sterol transport. In mammalian cells, NPC1 is located in LE/LY, where it is thought to control cholesterol export to other organelles together with NPC2; inhibitors and mutations in these proteins lead to accumulation of sterols and other lipids in endosomes/lysosomes 3 . We observed a remarkably similar effect in Tetrahymena cells stained with Filipin after treatment with U18666A. In fact, a putative NPC1-like protein exists in the T. thermophila genome, and a proteomic study located it in phagososmes 38 . NPC1-like proteins were found in the apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum, where they play important roles in pathogenicity 39,40 . Interestingly, the Plasmodium ortholog is not located in endosomes/lysosomes but in the plasma membrane 40 . We are currently carrying out different experimental approaches in order to confirm its identity, localization and role in Tetrahymena sterol trafficking. In a previous study, we showed that cholesterol provokes a large-scale regulation of gene expression on T. thermophila 19 . Here, we demonstrated that this response depends mainly on intracellular sterol concentration, which correlates with the type of transport in the cell. In other words, phagocytic entry is the primary way through which large quantities of sterols can enter the cell and trigger the repression of tetrahymanol biosynthesis. This process was reflected in the transcriptional down-regulation of SQS-a key enzyme in the tetrahymanol biosynthetic pathway-, which only took place at higher cholesterol concentrations in the medium. Conversely, DES22 and other up-regulated genes required ten times less cholesterol, allowing a rapid enzymatic conversion of exogenous sterols to BDHC. This indicates that these processes involve at least two signaling pathways, each regulating a different set of genes. In principle, this double sterol signaling pathway could ensure that the sterol requirements of cell membranes would be met, and in so doing define when tetrahymanol could be completely replaced by BDHC.
To date, it is unknown how Tetrahymena detects the presence of sterols and which proteins and mechanisms participate in the modulation of the transcriptional response triggered by these compounds. Genome analysis of T. thermophila shows no evidence of proteins homologous to those that constitute the SREBP/SCAP pathway (sterol regulatory element-binding protein/SREBP cleavage-activating protein), which in mammalian cells acts as a sterol sensor and regulates the expression of key enzymes required for cholesterol homeostasis 3,41 . In contrast, several putative members of other sterol-binding protein families have been identified in the Tetrahymena genome, such as START domain-containing proteins and ORPs. These proteins have been implicated in nonvesicular transport of sterols and in modulation of signaling processes 24,25 , but their role in ciliate cellular physiology remains to be elucidated. Despite these uncertainties, our results dismiss the participation of extracellular signaling, as inhibition of both phagocytosis and pinocytosis impaired the transcriptional response. The experiments with U18666A as well as the lack of effect of Brefeldin A further suggest that the internal signaling pathways originate between LE/LY and the ER. Alternatively, signal(s) could be triggered at the plasma membrane: (i) after the arrival of cholesterol delivered directly from LE/LY or (ii) after the arrival of modified cholesterol (BDHC) from the ER, without intervention of the Golgi apparatus.
Overall, the present work highlights some similarities in cholesterol transport and distribution in distantrelated organisms such as ciliates and metazoans. Further study on the way in which transport events take place in T. thermophila, a simple but powerful organism in terms of experimental malleability, would aid in the www.nature.com/scientificreports/ understanding of the mechanisms involved in cholesterol transport and homeostasis in higher eukaryotes. In addition, this ciliate lacks any endogenous synthesis of sterols, which in other models can mask the interpretation of sterol traffic/signaling experiments.

Materials and methods
Cell culture and drug treatment. T. thermophila strains CU428 (mpr1-1/mpr1-1, VII) and II8G-IA (presumed phg − /phg −) 23  TLC and count of lipid fractions. Neutral lipids were extracted according to the Bligh and Dyer method 43 and resuspended in chloroform. Each extract was seeded in 1 cm lanes on a 13 × 15 cm silica-aluminum plate (Merck). The mobile phase consisted of a mixture of acetic acid:diethyl ether:hexane (1:20:80 v/v/v). The plate was left to air dry and was exposed on a photosensitive screen overnight (Imaging Plate BAS-MS 2025, 20 × 25 cm, Fujifilm). Autoradiography scanning was performed on Typhoon FLA 7000 (GE Life Biosciences). For better visualization, minor adjustments in brightness and contrast were performed on the entire image using the Fiji software (ImageJ) 44 . For radioactivity measurement, each spot was first visualized with a sublimated iodine spray, then scraped and directly resuspended in scintillation liquid. Full-length versions of the autoradiograms are included in Supplementary Fig. S2 and S3.
ACAT Activity in cell-free extracts. Measurement of ACAT activity in cell-free extracts was performed using a modification of the method of Billheimer et al. 31