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Netrin-1 Peptide Is a Chemorepellent in Tetrahymena thermophila

Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation is not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism.

Much effort has gone into understanding the various signaling pathways through which netrin-1 works. Signaling mechanisms are complex, given the number of receptors netrin-1 binds to, as well as the multifaceted physiological roles attributed to netrin-1. Chemoattractant signaling is thought to involve a large number of growth and survival pathways, along with focal adhesion kinase, while chemorepellent signaling is thought to involve the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2 [1,2]. At present, chemorepellent signaling is less well understood than chemoattractant signaling.

International Journal of Peptides
Tetrahymena thermophila is free-living, eukaryotic ciliates which have been used as a model system for chemorepellent signaling for several decades [7][8][9][10][11]. When exposed to a chemorepellent, Tetrahymena reverse their cilia, causing them to exhibit avoidance behavior which is characterized by swimming in circles or jerky, back and forth swimming. This behavior is easily observed and characterized under a light microscope. Tetrahymena avoid a number of polycationic proteins and peptides that are derived from vertebrate systems, including lysozyme [9], PACAP-38 [12], and nociceptin [13]. This avoidance may sometimes be blocked using pharmacological inhibitors, allowing us to determine which signaling pathways are necessary for avoidance [12,13].
Since netrin-1 acts as a chemorepellent in some vertebrate cell types, and since netrin-1 peptide is polycationic under our assay conditions, we believe that netrin-1 peptide will act as a chemorepellent in Tetrahymena thermophila.  [15] without shaking or addition of antibiotics. One-day-old cultures were used for all behavioral and pharmacological assays.

Chemicals and Solutions.
Netrin-1 peptide and antinetrin-1 antibody were obtained from Abcore LLC, Ramona, CA. PACAP-38 was obtained from the American Peptide Company, Sunnyvale, CA. GTP--S and pharmacological inhibitors were obtained from Tocris Bioscience, Minneapolis, MN, USA. Anti--tubulin antibody was obtained from Abcam, Cambridge, MA, USA. QuantaRed6 enhanced chemifluorescent HRP substrate was obtained from Pierce Biotechnology, Rockford, IL, USA. All other chemicals were obtained from Sigma Chemical Co., St. Louis, MO, USA.
Phosphate buffered saline (PBS) was made by dilution of a 10x stock with distilled water. Behavioral buffer consisted of 10 mM Trizma Base, 0.5 mM MOPS, and 50 M CaCl 2 ; pH was adjusted to 7.0 with HCl. Blocking buffer consisted of 3% bovine serum albumin (BSA) and 0.3% Triton X-100, diluted in 1x PBS. Antibody dilution buffer consisted of 1% bovine serum albumin (BSA) and 0.3% Triton X-100, diluted in 1x PBS.

Behavioral Assays.
Behavioral assays were carried out as previously described [8,9,[11][12][13]. Tetrahymena thermophila was washed 3 times in behavioral buffer and then 300 L of cell suspension was transferred to the first well of a microtiter plate. Cells were then transferred individually using a micropipette into the second well of the microtiter plate, already containing 300 L of buffer as a control. Cells were then transferred to a third well containing 300 L of netrin-1 peptide. Behavior of the cells was observed (1-5 seconds), and the percentage of cells exhibiting avoidance behavior was noted. Varying concentrations of each peptide were used until we determined the minimum concentration at which 100% of the cells exhibited avoidance behavior (EC 100 ). In order to minimize variability due to cells conditioning the buffer, cells were used within 30 minutes after being washed in buffer.
Adaptation assays were carried out as previously described [9]. Cells were placed in 1 M netrin-1 peptide for a specified amount of time. Cells were then washed in buffer for 20 seconds and then transferred back into 1 M netrin-1 peptide to assay for repellent response. Cells were then individually scored for avoidance.
Cross-adaptation assays were carried out as previously described [16]. Cells were first placed in one of the test solutions for 10 minutes, until the cells were adapted to that solution. They were then washed briefly (20 seconds) in buffer and transferred to another test solution. Cells were then individually scored for avoidance.
Pharmacological inhibition assays were performed similar to the behavioral assays described above. After being washed in buffer, cells were exposed to pharmacological agents known to block specific signaling pathways and incubated for 15 minutes. Cells were then transferred to a solution containing netrin-1 peptide at EC 100 and then monitored for avoidance behavior.
2.4. Immunofluorescence. T. thermophila were washed 3 times in behavioral buffer and then fixed in 3.7% formaldehyde, diluted in behavioral buffer, for 15 minutes. Cells were then washed 3 times in PBS and incubated in blocking buffer at room temperature, overnight with shaking. Cells were then washed 3 times in PBS and incubated in a 1 : 100 dilution of either anti-netrin-1 or anti-tubulin antibody in the presence of antibody dilution buffer, for 2 hours at room temperature, with constant shaking. Cells were once again washed 3 times in PBS and then incubated with a 1 : 100 dilution of secondary antibody, in the presence of antibody dilution buffer for one hour at room temperature, with constant shaking. Cells were then washed three times in PBS, stained with DAPI, and viewed under a Nikon H550L Microscope using the Nikon Intensilight C-HGFI. Fluorescence images were obtained with a QI Click 74-0083-AO camera using NIS Elements BR 4.13.04 Software.

ELISA.
One-day-old cultures of T. thermophila were washed in behavioral buffer consisting of 1 mM Tris, 0.5 mM MOPS, and 50 mM CaCl 2 ; pH 7.0. 10 mL of culture was concentrated into 1 mL of buffer. For secreted proteins, cells were incubated at room temperature for 24 hours with constant shaking. The cells were then pelleted by centrifugation and the supernatant was saved as our secreted protein sample. For the whole cell extract, cells were incubated on ice in 1% Triton X-100 for one hour in the presence of protease inhibitor cocktail. Both extracts were used in ELISA, using 1 : 10,000 dilution of goat anti-netrin-1 IgG as the primary antibody and a 1 : 5,000 dilution of rabbit-anti-goat IgG, HRP conjugate, as the secondary antibody. QuantaRed6 enhanced chemifluorescent HRP substrate was used to develop the ELISA, and the plate was read using a Promega Glomax Multi Detection System.

Results and Discussion
Netrin-1 is a basic peptide, with a net charge of +6 at pH 7.0. The amino acid sequence of this peptide is shown below:

KFQQREKKGKCKKA.
Basic amino acids are shown in bold, while acidic amino acids are shown in italic. The net charge of the peptide is +6 at our assay pH of 7.0. In our behavioral assay, netrin-1 peptide was a chemorepellent (Figure 1), consistent with our hypothesis and with netrin-1 activity in other cell types [4,17]. Cells showed avoidance over a broad range of concentrations, with an EC 100 of 1 M, and an EC 50 of approximately 1 nM ( Figure 1). In contrast, similar concentrations of myelin basic protein (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), which has a charge of +4 under our assay conditions, did not elicit avoidance behavior (data not shown). The EC 100 of netrin-1 peptide is lower than what we have previously described for both lysozyme and nociceptin, which have an EC 100 of approximately 100 M (Table 3), but higher than what we have observed for PACAP-38 (0.1 M; Table 3). We were able to obtain a small quantity of recombinant human netrin-1 and found that Tetrahymena thermophila avoided this compound as well (data not shown). However, since netrin-1 peptide was more readily obtained and elicited robust avoidance, we chose to use netrin-1 peptide for the remainder of our experiments.
Tetrahymena adapt to netrin-1 peptide over the course of about 10 minutes (Figure 2), which is similar to the time course of adaptation to the chemorepellents lysozyme [9], GTP [9], and PACAP-38 [16]. Cross-adaptation assays ( Table 1) show that netrin-1 adapted cells still avoid PACAP-38, nociceptin, and GTP. Similarly, cells that adapted to any of these other compounds still show avoidance to Table 1: Cells that adapted to netrin-1 peptide are not cross-adapted to nociceptin, GTP, or PACAP-38. Percentage of avoidance, as listed below, represents the mean ± standard deviation of at least 6 trials. Each trial consisted of 10 cells which were individually observed and scored for avoidance within the first 5 seconds of exposure to the chemorepellent being tested. Adaptation to the same signal (e.g., GTP adapted to GTP, nociceptin adapted to nociceptin) was run as controls. Each of these controls showed adaptation, showing less than the baseline avoidance of 20% typically seen in behavioral assays. netrin-1 ( Table 1), suggesting that netrin-1 signaling is using a different receptor and/or second messenger pathways than those used by the other three repellents. Controls (netrin-1 adapted to netrin-1, PACAP adapted to PACAP, and GTP adapted to GTP) showed only baseline avoidance, indicating that adaptation to these compounds was occurring. Baseline avoidance in our behavioral studies indicates the amount of avoidance typically seen when moving cells from a well containing behavioral buffer to another well containing the same buffer and ranges from about 7 to 20% ( [8,11,12], current study). This is how baseline avoidance is defined in the pharmacological studies as well.
In order to determine the signaling mechanism of netrin-1 peptide in Tetrahymena thermophila, we used a broad spectrum of pharmacological inhibitors targeting a number of second messenger pathways that have previously been associated with netrin signaling in vertebrates. Since netrin-1 mediated repellent signaling in vertebrates involves src-1 and shp-2 [1, 2], we included tyrosine kinase inhibitors Table 2: Pharmacological studies suggest that a tyrosine kinase is involved in the avoidance of netrin-1 peptide in Tetrahymena. Inhibition of G-proteins, chelation of intracellular and extracellular calcium, and inhibition of a number of other kinases had no effect on avoidance behavior. The tyrosine kinase inhibitor, genistein, reduced avoidance to baseline levels. "Avoidance" represents the mean ± standard deviation of 6 trials. Each trial consisted of 10 cells which were individually observed and scored for avoidance within the first 5 seconds of exposure to netrin-1 peptide.  and tyrosine phosphatase inhibitors in our study (Table 2). We also included a number of inhibitors targeting G-protein linked receptors and associated pathways, as well as focal adhesion kinases, since these have been associated with netrin-1 signaling in some systems [1,2,18]. As seen in Table 2, none of these compounds effectively reduced netrin-1 avoidance, with the exception of the tyrosine kinase inhibitor, genistein. The phytoestrogen, genistein, inhibited avoidance to netrin-1 peptide at concentrations ranging from 20 to 100 g/ L (Table 2; Figure 3). Avoidance was reduced to baseline levels at 75 g/ L genistein; the IC 50 of genistein was approximately 50 g/ L (Figure 3). These IC 50 values are similar to those we have previously seen when inhibiting GTP avoidance [11]. Another phytoestrogen, daidzein, was used as a negative control for genistein. Daidzein did not affect netrin-1 avoidance (Figure 3), indicating that genistein's effects were specific to that compound. Immunolocalization of phosphotyrosine in control (a) and netrin-1 peptide (b) treated cells showed low levels of phosphotyrosine staining in both treatment groups ( Figure 4). The anti-tubulin control (c) showed much higher levels of staining than either of the phosphotyrosine treatment groups. Treatment with netrin-1 peptide did not increase phosphotyrosine levels in Tetrahymena, suggesting that tyrosine phosphorylation was not required for netrin-1 peptide signaling. This is consistent with the findings of Eisen et al. [19]. When Eisen and colleagues sequenced the Tetrahymena genome, they found no consensus sequences for tyrosine kinases [19]. However, other studies [11,20] have implicated tyrosine kinase signaling in Tetrahymena, both on the basis of pharmacological inhibition by genistein and on the basis of immunolocalization of phosphotyrosine. While our immunofluorescence data does not show an increased level of phosphorylation in the presence of netrin-1 peptide, genistein does inhibit netrin-1 avoidance. Together, these data suggest that genistein may have a different mechanism of action in Tetrahymena than it does in vertebrates; however, our experiments do not reveal what this mechanism might be. It is possible that genistein is simply blocking another kinase; however, the broad spectrum kinase inhibitors used International Journal of Peptides [Phytoestrogen] ( g/mL) Figure 3: Avoidance of netrin-1 peptide is inhibited by the tyrosine kinase inhibitor, genistein (closed squares), but not by daidzein (closed circles). The IC 50 of genistein is approximately 50 g/mL. Baseline avoidance was achieved at a genistein concentration of 75 g/mL. Daidzein had no effect on avoidance behavior. Percentages represent the mean ± standard deviation of at least 6 trials. Each trial consisted of 10 cells which were individually observed and scored for avoidance. in our assay had no effect on the avoidance of netrin-1 peptide ( Table 2). It is also possible that genistein is binding to another enzyme or second messenger involved in netrin-1 signaling. Additional experiments will be required in order to determine the mechanism of genistein action in this organism.
Since calcium-based depolarizations have been implicated in Tetrahymena avoidance [13,14,21], and since calcium has been implicated in netrin signaling [22], we used chelators of intracellular and extracellular calcium to try to inhibit avoidance. EGTA was used to chelate extracellular calcium, and thapsigargin was used to deplete calcium from intracellular stores. Neither chelator affected avoidance to netrin-1 peptide ( Table 2). Table 3 compares three polycationic compounds that are chemorepellents in Tetrahymena. Calcium is required for the avoidance of all of the compounds listed except for netrin-1 peptide. Calcium-based depolarizations have previously been shown in the presence of lysozyme [14] and nociceptin [13] as well as the repellents GTP [10] and ATP [21]. The absence of calcium dependence in the avoidance of netrin-1 peptide suggests a calcium-independent signaling mechanism for ciliary reversal that has not previously been described in this organism, which is unlikely to involve phosphorylation ( Table 2).
Netrin-1 peptide has one of the lowest EC 100 values of the polycationic compounds studied in Tetrahymena thermophila to date (Table 3), suggesting a relatively high affinity for its putative receptor when compared with other chemorepellents in this organism. However, pharmacological studies lead us to believe that the Tetrahymena receptor is unlikely to show conservation with the previously described vertebrate receptors, since blocking signaling pathways used in netrin-1 signaling in vertebrates did not impact the avoidance of netrin-1 peptide in Tetrahymena (Table 2). The physiological role of netrin-1 peptide in Tetrahymena thermophila is also an open question. Tetrahymena thermophila is thought to respond to chemorepellents in an attempt to escape predation and possibly to warn other cells of danger [7,13]. We hypothesized that Tetrahymena might secrete a netrin-1 like protein in order to communicate with other cells. Our ELISA data (Table 4) indicate that a netrin-like protein is present in both whole cell extracts and secreted protein obtained from Tetrahymena thermophila. The secreted protein also caused avoidance behavior in Tetrahymena thermophila (data not shown). Since netrin-like protein appears to be biologically active in terms of causing avoidance, it is likely that this protein is used in intercellular communication. We are currently doing experiments to further characterize this netrin-1-like protein. Though netrin appears to be conserved throughout the animal kingdom [2], this is the first time such a protein has been described in kingdom Protista.
Further studies are needed in order to elucidate the signaling mechanism of netrin-1 peptide in Tetrahymena thermophila, as well as to characterize the netrin-like protein that is secreted by Tetrahymena. Additional data will help us understand whether Tetrahymena could be a useful model system for netrin-1 signaling in vertebrates. In addition, since most chemorepellents in Tetrahymena require calcium in order for avoidance to occur, understanding netrin-1 signaling may uncover a previously unknown mechanism for ciliary reversal in Tetrahymena thermophila.  Figure 4: Tyrosine phosphorylation levels are not affected by netrin-1 peptide. Indirect immunofluorescence using PT-66 antiphosphotyrosine antibody shows no difference in fluorescence intensity between control (a) and netrin-1 exposed cells (b). This indicates that tyrosine phosphorylation is not required for netrin-1 signaling. In contrast, cells stained with an anti-tubulin antibody (c) show a high level of fluorescence intensity.