Characterization of Piezo channels in T. cruzi

Two phylogenetically distinct genes are present in T. cruzi (Y C6 strain): TcPiezo1 (TcYC6_0088320, KAF8295942) in chromosome 7 and TcPiezo2 (TcYC6_0007880, KAF8281887) in chromosome 5, encoding proteins with 39 and 41 transmembrane domains (TMD), respectively, and containing the PFEW motif found in other Piezo channels [7]. The ORFs predict 2606- and 2514-amino acid-proteins with apparent molecular weights of 296 and 280 kDa, respectively, which share 20.4% identity and 35.3% similarity. Both TcPiezo1 and TcPiezo2 have typical Piezo features: a divergent N-terminal mechanotransduction module and a conserved C-terminal pore region. Protein sequence alignment of C-terminal pore regions of T. cruzi Piezo proteins with mammalian Piezo channels (S1 Fig) reveals that TcPiezo1 and 2 contain six conserved hydrophobic domains (TM33-TM38) and a glutamate residue (E2133) that are essential for ion (Ca²⁺) selectivity and pore blocking [33]. Recently, the CryoEM structures of mouse Piezo channels (mPiezo1 and 2) became available [34–36], but the low sequence identity between mPiezo and TcPiezo proteins (<19%) renders it unfeasible to perform conventional template-based homology modeling. We used AlphaFold II [37] to predict the structures of the Piezo conserved C-terminal sequences, as aligned in S1 Fig. As expected, the structures of mPiezo1 and mPiezo2 predicted by AlphaFold II (S2 Fig) resemble those obtained by CryoEM results [34–36]. Strikingly, the structures of TcPiezo1 and TcPiezo2 are very similar to those of mPiezo1 and mPiezo2 (S2 Fig), such as the critical E2133 residue located at the anchor and the inner helix-CTD region formed the transmembrane gate and cytosolic constriction neck of the ion (Ca²⁺) pathway [35].

Attempts to knockout (KO) TcPiezo1 and TcPiezo2 gene expression

We designed a CRISPR/Cas9 strategy (S3A Fig) to generate KO mutants of the pore region or whole TcPiezo1 and TcPiezo2 genes. The method involves the constitutive expression of Cas9 and specific single guide RNA (sgRNA) and the utilization of a DNA donor template to promote double-strand break repair by homologous-directed repair [38]. Three cassettes were utilized along with two unique TcPiezo1- or TcPiezo2- targeted sgRNA designs. Each of the two unique designs was used in technical duplicates and the complete co-transfection experiments were repeated a total of three independent times. Multiple CRISPR/Cas9 genome editing experiments to knock out the pore region or the whole TcPiezo1 and TcPiezo2 genes were attempted by using different sgRNAs with blasticidin (Bsd) or puromycin (Puro) resistance gene cassettes, but no epimastigotes survived the selection process with minimal concentration of these antibiotics for T. cruzi, suggesting the essentiality of these genes for cell growth.

Conditional knockout of TcPiezo1 and subcellular localization

We then used a newly developed conditional knockout method based on the tetracycline or theophylline-dependent activation of a hammerhead ribozyme (S3F Fig), recently described (Shark 1 and Shark3, respectively) [31].

In this system, we used the CRISPR/Cas9-mediated endogenous C-terminal tagging of T. cruzi epimastigotes [15] to introduce a Ty1 tag sequence into the TcPiezo1 gene, followed by the tetracycline/theophylline inducible ribozyme, a blasticidin deaminase (Bsd) gene and the TcPiezo1 3’UTR (Figs 1A and S3B–D). Fig 1B, top panels, is the immunofluorescence analysis (IFA) of TcPiezo1-Ty1-labeled epimastigotes showing punctate labeling that co-localizes with a plasma membrane marker (antibody TcHAf against a T. cruzi P-type H⁺-ATPase (α-HAf) [39]). To investigate the localization of TcPiezo1 in the infective stages we induced the differentiation of TcPiezo1-Ty1 epimastigotes into infective metacyclic trypomastigotes by incubating them in triatome artificial urine (TAU) medium as described previously [40]. We then infected Vero cells, collected the infective stages (trypomastigotes and amastigotes), and performed IFAs. Fig 1B, central panel, shows a surface punctate localization of TcPiezo1-Ty1 in trypomastigotes that does not perfectly coincide with the punctate surface localization of TcHAf. Fig 1B, lower panel, shows that TcPiezo1-Ty1 co-localizes with anti-TcHAf to the plasma membrane of amastigotes. Labelling of amastigotes was stronger and more continuous. When analyzed by western blot using antibodies against Ty1, a clear band of ~ 290 kDa (predicted size) was detected in the TcPiezo1-Ty1 cell lysates of two clones each of Tet-OFF and Theo-OFF epimastigotes (S4A Fig). The signal was weak because of the low expression levels of this protein. Knockdown of TcPiezo1, induced by addition of tetracycline, resulted in a marked growth defect in epimastigotes from day 4, with a correlative decrease in TcPiezo1 expression, as analyzed by western blot (Fig 1C). Similar results were observed when knockdown was induced by theophylline addition to epimastigotes (S4B Fig). Therefore, all further phenotypic analyses were conducted on day 4 of tetracycline or theophylline induction, unless indicated otherwise.

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Fig 1. Conditional knockout of TcPiezo1 and subcellular localization.

(A) Schematic representation of CRISPR/Cas9 mediated endogenous C-terminal tagging of TcPiezo1 with an aptazyme cassette Shark1 or Shark3 (composed of 3 × Ty tag, ribozyme, GAPDH 3’UTR, and Bsd gene) for conditional knockout (CKO) of TcPiezo1. (B) Ty1-tagged TcPiezo1 Tet-OFF colocalization with T. cruzi plasma membrane marker H⁺-ATPase (TcHAf or α-HAf) in the plasma membrane of epimastigote (Pearson’s correlation coefficient (PCC): 0.8750), trypomastigote (PCC: 0.6021) and amastigote (PCC: 0.9240), as detected by immunofluorescence analyses with antibodies against Ty1. Yellow in merged images indicates colocalization. DIC, differential interference contrast microscopy. DAPI staining in blue. Scale bars 5 and 10 µm, as indicated. (C) Growth of TcPiezo1 Tet-OFF epimastigotes in the absence (black line, − Tet) or presence (red line, + Tet) of 5 μg/ml tetracycline for the indicated number of days. Western blot analyses of TcPiezo1 Tet-OFF epimastigotes grown in the absence (0) or presence (2–6) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against Ty1 (top). One band of ~ 290 kDa was detected in epimastigote homogenates. Membranes were stripped and re-incubated with antibody against Alpha-tubulin as a loading control (bottom, Tub). (D) Percentage (%) of metacyclic trypomastigotes in TcPiezo1 Tet-OFF epimastigote cultures after incubation in TAU 3AAG medium in the absence (-Tet) or presence (+Tet) of 5 µ g/ml tetracycline after 96 h. Differentiation of epimastigotes to metacyclic trypomastigotes was quantified by staining with DAPI to distinguish the position of the kinetoplast by fluorescence microscopy. (E) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo1 Tet-OFF on trypomastigote infection of Vero cells after 4 h. (F) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo1 Tet-OFF on amastigote replication after 72 h. In panels C, D, E, F, values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

https://doi.org/10.1371/journal.ppat.1013105.g001

Knockdown of TcPiezo1 expression by addition of tetracycline (Figs 1D–F and S4F) or theophylline (S4C–E and S4G Fig) significantly inhibited metacyclogenesis, tissue culture-derived trypomastigote invasion of host cells and amastigote replication within Vero cells as compared with Tet-OFF or Theo-OFF cells. It has been reported before that there is no difference in the proliferation rate of wild type cells after treatment with Tet or Theo [31].

These results demonstrate that TcPiezo1 localizes to the plasma membrane of T. cruzi and is essential for its normal proliferation, differentiation and infectivity.

Expression of jGCaMP7s in TcPiezo1 transgenic cell lines

To investigate the effects of downregulation of TcPiezo1 and of Piezo activators and inhibitors on Ca²⁺ entry, we initially used Fura-2/AM loaded cells. However, although this method could be used with amastigotes to show inhibition of Ca²⁺ entry by downregulation of TcPiezo1 expression (S5A, B Fig), loading of epimastigotes resulted in inefficient esterase-catalyzed cleavage of the acetoxymethyl ester. We therefore expressed genetically encoded Ca²⁺ indicators (GECIs) in TcPiezo1 transgenic cell lines. GCaMP6 and jGCaMP7 are high performance sensitive GECIs with slow (s) and fast (f) Ca²⁺ binding kinetics in neurons [41,42]. To determine the sensitivity of those GCaMP variants to Ca²⁺ changes in T. cruzi, we transfected WT epimastigotes with GCaMP6s, GCaMP6f, jGCaMP7s and jGCaMP7f (S6A Fig), isolated cells with low fluorescence using cell sorting, and analyzed them for their response to ionomycin (S6B Fig). jGCaMP7s transfected cells gave maximal fluorescence response to the ionophore (S6B Fig). These cells were labeled by immunofluorescence analysis (IFA) with anti-GFP (S6C Fig) and showed bands of 50 kDa by western blot analyses (S6A Fig). Based on those results, we generated transgenic TcPiezo1 Tet-OFF/Theo-OFF cell lines expressing jGCaMP7s, which were verified by IFA and western blot analyses with anti-His antibody (S6D, E Fig). The TcPiezo1-CKO cell lines expressing jGCaMP7s released similar amounts of intracellular Ca²⁺ when either Tet-OFF or Theo-OFF cells were exposed to ionomycin (S6F Fig). We used them in our experiments.

Fig 2A shows that addition of 1.8 mM CaCl₂ to TcPiezo1-CKO transgenic epimastigotes expressing jGCaMP7s resulted in an increase in intracellular Ca²⁺, which was significantly reduced when tetracycline was added to downregulate TcPiezo1 expression. Similar results were obtained upon addition of theophylline to downregulate TcPiezo1 expression (S5C Fig).

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Fig 2. Downregulation of TcPiezo1 expression decreases Ca²⁺ entry in T. cruzi.

Intracellular Ca²⁺ concentrations of 5 × 10⁷ TcPiezo1 Tet-OFF epimastigotes (A-C) or 2 x 10⁷ trypomastigotes (D) expressing jGCaMP7s were measured by jGCaMP7s signal fluorescence in arbitrary units of fluorescence (AU). An increase in jGCaMP7s fluorescence indicates increasing cytosolic Ca²⁺. (A) Addition of 1.8 mM Ca²⁺ in Tet-induced TcPiezo1 cells (+Tet) elicited a lower increase in intracellular Ca²⁺ than in non-induced cells (-Tet). (B) Downregulation of TcPiezo1 expression showed a significant decrease of intracellular Ca²⁺ (+Tet). Addition of 10 µ M nifedipine (the TcCa_v inhibitor) slightly reduced intracellular Ca²⁺ in non-induced cells (-Tet + Nif) while it had an additive effect in Tet-induced cells (+Tet + Nif). (C) Addition of 10 µ M GsMTx4 showed a significant reduction of intracellular Ca²⁺ in non-induced cells (-Tet + GsM) comparable to downregulation of TcPiezo1 (+Tet) and had an additive effect in Tet-induced cells (+Tet + GsM). (D) Extracellular matrix (ECM) triggered TcPiezo1-mediated Ca²⁺ entry in tissue culture-derived trypomastigotes. Addition of 40 µg ECM to non-induced (-Tet) trypomastigotes significantly increased intracellular Ca²⁺, compared to Tet-induced cells (+Tet). In panels A-D, addition of buffer A with glucose (BAG), instead of 1.8 mM Ca²⁺, was used as a control baseline labeled with B. Values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

https://doi.org/10.1371/journal.ppat.1013105.g002

Addition of nifedipine, an inhibitor of the voltage-dependent Ca²⁺ channel (TcCa_V) on the surface of T. cruzi [43], enhances the inhibitory effect of TcPiezo1 downregulation (Fig 2B). This suggests that both TcPiezo 1 and TcCa_V channels are involved in Ca²⁺ entry. In addition, the Piezo inhibitor GsMTx4 has an inhibitory effect equivalent to the expression downregulation of TcPiezo1 (Fig 2C).

In agreement with previous reports on the effect of extracellular matrix (ECM) on Ca²⁺ increase in trypomastigotes [6], addition of ECM in the presence of extracellular Ca²⁺ resulted in Ca²⁺ increase that was greatly reduced in trypomastigotes in which TcPiezo1 expression was downregulated by addition of either tetracycline (Fig 2D) or theophylline (S5D Fig).

These results indicate that TcPiezo1 mediates Ca²⁺ entry in T. cruzi, which is inhibited by GsMTx4 and stimulated by the mechano-stimulant ECM.

Subcellular localization of TcPiezo2 to lysosomes

To investigate the localization of TcPiezo2 (Figs 3A and S7A),its C-terminus was tagged in epimastigotes with a high performance “Spaghetti monster” fluorescent protein (smFP, smV5) tag using homologous recombination with the endogenous gene locus and a pMOTag2mV vector, previously modified for endogenous tagging of weakly expressed membrane proteins in T. brucei [44]. Western blot analysis confirmed the expression of the protein of the expected size (S7B Fig) at higher levels of expression than those of TcPiezo1 (S4A Fig). TcPiezo2 localized to the reservosomes and lysosomes, as demonstrated by colocalization with antibodies against T. cruzi reservosomal/lysosomal markers cruzipain [45] (Fig 3A) and serine carboxypeptidase [46] (S7A Fig). Similar colocalizations were found in trypomastigotes and amastigotes (Figs 3A and S7A). These experiments also demonstrate the utility of pMOTag2mV for endogenous C-terminus tagging in T. cruzi without the use of CRISPR/Cas9 genome editing. Further confirmation of this localization was obtained by IFA of epimastigotes tagged with Ty1 used for conditional knockout of TcPiezo2 (S7I Fig), as described below.

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Fig 3. Subcellular localization and conditional knockout (CKO) of TcPiezo2.

(A) Endogenous C-terminally smV5-tagged TcPiezo2 colocalized with T. cruzi cruzipain (TcCZP) to the reservosomes/lysosomes of epimastigotes (PCC: 0.7294), trypomastigotes (PCC: 0.7443) and amastigotes (PCC: 0.8524). Yellow in merged images indicates colocalization. DIC, differential interference contrast microscopy. DAPI staining in blue. Scale bars 5 and 10 µm, as indicated. (B) Growth of TcPiezo2 Tet-OFF epimastigotes in the absence (black line, − Tet) or presence (red line, + Tet) of 5 μg/ml tetracycline for the indicated number of days. Western blot analyses of TcPiezo2 Tet-OFF epimastigotes grown in the absence (0) or presence (2–6) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against Ty1 (top). One band of ~ 280 kDa was detected in epimastigote homogenates. Membranes were stripped and re-incubated with antibody against Alpha-tubulin as a loading control (bottom, Tub). (C) Percentage of metacyclic trypomastigotes in TcPiezo2 Tet-OFF epimastigote cultures after incubation in TAU 3AAG medium in the absence (-Tet) and presence (+Tet) of 5 µ g/ml tetracycline after 96 h. (D) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo2 Tet-OFF on trypomastigote infection of Vero cells after 4 h. (E) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo2 Tet-OFF on amastigote replication after 72 h. In panels B, C, D, E, values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

https://doi.org/10.1371/journal.ppat.1013105.g003

Conditional knockout of TcPiezo2 and phenotypic changes

The same approach used with TcPiezo1 (Figs 1A and S3B–F) was applied to knockdown the expression of TcPiezo2. Knockdown of TcPiezo2 by addition of tetracycline resulted in a marked growth defect in epimastigotes with a correlative decrease in TcPiezo2 expression, as analyzed by western blot (Fig 3B). Similar results were observed when knockdown was induced by theophylline addition to epimastigotes (S7C Fig). All further phenotypic analyses of TcPiezo2 transfectants were also done on day 4 of tetracycline or theophylline induction.

As occurs with TcPiezo1, knockdown of TcPiezo2 expression by addition of tetracycline (Figs 3C–E and S7G) or theophylline (S7D–F and S7H Fig) significantly inhibited metacyclogenesis, trypomastigote invasion of host cells, and amastigote replication within Vero cells.

To investigate the role of TcPiezo2 in Ca²⁺ homeostasis in T. cruzi, we then expressed jGCaMP7s in TcPiezo2-CKO transgenic cell lines. The cells were labeled by immunofluorescence analysis (IFA) with anti-His antibody (S6G Fig), showed bands of 50 kDa by western blot analyses (S6H Fig), and released similar amounts of intracellular Ca²⁺ when either Tet-OFF or Theo-OFF cells were exposed to ionomycin (S6I Fig). We investigated the effects of downregulation of TcPiezo2 on intracellular Ca²⁺ stores in these epimastigotes (Fig 4). Experiments were done in the absence of extracellular Ca²⁺ (100 µ M EGTA added) to prevent Ca²⁺ influx. Fig 4A shows that addition of 70 µ M glycyl-L-phenylalanine 2-naphthylamide (GPN), a widely used lysosomotropic compound that releases Ca²⁺ from lysosomes [47], to TcPiezo2-CKO transgenic epimastigotes expressing jGCaMP7s, resulted in an increase in intracellular Ca²⁺. This increase was significantly reduced when tetracycline was added to downregulate TcPiezo2 expression (Fig 4A). Similar results were obtained upon addition of theophylline to downregulate TcPiezo2 expression (S8A Fig). In contrast, Ca²⁺ increase upon addition of cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca²⁺-ATPase [23], was not significantly reduced when downregulation of TcPiezo2 expression was obtained by addition of tetracycline (Fig 4B) or theophylline (S8B Fig). When 1.8 mM CaCl₂ was added to transgenic parasites, Ca²⁺ influx occurred, and was not affected when TcPiezo2 expression was downregulated in the presence of tetracycline (Fig 4C) or theophylline (S8C Fig). In contrast, when ionomycin was added in the absence of extracellular Ca²⁺ (100 µ M EGTA added), there was an increase in intracellular Ca²⁺ which was inhibited by downregulation of TcPiezo2 expression by either tetracycline (Fig 4D) or theophylline (S8D Fig). Ionomycin is not thought to mobilize Ca²⁺ from acidic organelles [48] and the increase in Ca²⁺ by ionomycin in the absence of Ca²⁺ entry in the control suggest Ca²⁺ release from a non-acidic or neutral compartment, like the ER. The lower release by TcPiezo2 knockdown cells suggest that less Ca²⁺ is in this compartment. It is possible that this could indicate that TcPiezo2 Ca²⁺ release from lysosomes could have a function in refilling Ca²⁺ neutral stores in the absence of Ca²⁺ entry. Taken together these results are consistent with the intracellular localization of TcPiezo2, its involvement in Ca²⁺ release from acidic compartments and its lack of involvement in Ca²⁺ entry or in Ca²⁺ release from the ER.

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Fig 4. Effects of TcPiezo2 expression downregulation on intracellular Ca²⁺ stores in T. cruzi.

Intracellular Ca²⁺ concentrations of 5 × 10⁷ TcPiezo2 Tet-OFF epimastigotes expressing jGCaMP7s were measured by jGCaMP7s signal fluorescence in AU. (A) TcPiezo2 mediated lysosomal Ca²⁺ release. Addition of 70 µ M Glycyl-L-phenylalanine 2-naphthylamide (GPN) for activation of lysosomal Ca²⁺ release in Tet-induced TcPiezo2 cells (+Tet) elicited less increase in intracellular Ca²⁺ than in non-induced cells (-Tet). (B) Addition of 30 µ M cyclopiazonic acid (CPA) for activation of ER Ca²⁺ release demonstrated no significant difference between Tet-induced (+Tet) and non-induced (-Tet) cells. (C) Addition of 1.8 mM Ca²⁺ showed no significant difference in intracellular Ca²⁺ between Tet-induced (+Tet) and non-induced (-Tet) cells. (D) Addition of 1 µ M ionomycin in Tet-induced cells (+Tet) elicited less increase of intracellular Ca²⁺ than in non-induced cells (-Tet). 100 µ M EGTA was added to remove extracellular Ca²⁺ in A-D. Addition of DMSO or BAG was used as control (baseline) labeled with B. Values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01).

https://doi.org/10.1371/journal.ppat.1013105.g004

Effects of Piezo activators and inhibitors on TcPiezo channels

Yoda1 was identified by a high-throughput screening as the first selective activator of mouse and human Piezo1 with no activity on Piezo2 [49]. A search for Yoda1 analogs led to the synthesis of Dooku1, which acted as antagonist of Yoda1-induced effects without the ability to activate Piezo channels [50]. However, further work revealed that Dooku1can also activate Piezo1 in red blood cells [51]. Two additional small molecules of different structure and known as Jedi1 and Jedi2 were also shown to activate mPiezo1 [52].

Addition of Yoda1 to transgenic epimastigotes expressing jGCaMP7s, in the presence of only contaminant Ca²⁺ (~10 µ M), resulted in a dose-dependent increase of two peaks of Ca²⁺ increase (Fig 5A). The first was tentatively attributed to Ca²⁺ entry through activation of TcPiezo1 and the second to Ca²⁺ release from an intracellular store by activation of TcPiezo2. Its analog Dooku1 has greater stimulatory activity (Fig 5B). To further demonstrate our peak assignments, we applied Dooku1 and 1.8 mM Ca²⁺ resulting in two peaks of Ca²⁺ increase (Fig 5C). Downregulation of TcPiezo1 expression with either tetracycline (Fig 5C) or theophylline induction (S9A Fig) decreased the first peak of Ca²⁺ due to TcPiezo1 activation but did not affect the Ca²⁺ increase due to TcPiezo2 activation (Figs 5C, D and S9A). In conclusion, both Yoda1 and Dooku1 can activate TcPiezo1 and TcPiezo2.

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Fig 5. Effects of activators and inhibitors on TcPiezo channels.

Intracellular Ca²⁺ changes of 5 × 10⁷ TcPiezo Tet-OFF epimastigotes expressing jGCaMP7s were measured by jGCaMP7s signal fluorescence in AU. (A) Yoda1 activated TcPiezo channels. Addition of 20 µ M or 40 µ M Yoda1 to TcPiezo1 Tet-OFF cells elicited two distinct peaks of Ca²⁺ increase, indicating that TcPiezo1 and TcPiezo2 were independently activated. (B) Addition of 20 or 40 µ M Dooku1 to the cells elicited two different peaks of Ca²⁺ rise, also suggesting that the two TcPiezo channels were successively activated. (C) Two Ca²⁺ spikes were generated in Tet-induced (+Tet) and non-induced (-Tet) TcPiezo1 Tet-OFF cells, respectively, by the additions of 1.8 mM Ca²⁺ and 40 µ M Dooku1. (D) Changes in jGCaMP7s fluorescence (first peak and second peak in Fig 5C) in Tet-induced (+Tet) and non-induced (-Tet) cells. Downregulation of TcPiezo1 expression significantly decreased Dooku1-evoked TcPiezo1-mediated Ca²⁺ entry (left bar graph) but had no effect on Dooku1-evoked TcPiezo2-mediated Ca²⁺ release (right bar graph). (E) 100 µ M EGTA was incubated with TcPiezo2 Tet-OFF cells to remove extracellular Ca²⁺, abolishing TcPiezo1-mediated Ca²⁺ entry. 70 µ M GPN was added to activate Ca²⁺ release from lysosomes. Downregulation of TcPiezo2 expression (+Tet) showed a significant decrease of lysosomal Ca²⁺ release as in Fig 4A. Addition of 10 µ M GsMTx4 showed a significant reduction of intracellular Ca²⁺ in non-induced cells (-Tet + GsM) like downregulation of TcPiezo2 (+Tet) and had an additive effect on TcPiezo2-mediated Ca²⁺ release in Tet-induced cells (+Tet + GsM). (F) Changes in jGCaMP7s fluorescence (left bar graph) and the rates of fluorescence increase (right bar graph) upon GsM inhibition in Tet-induced (+Tet ± GsM) and non-induced (-Tet ± GsM) cells in Fig 5E. (G) 100 µ M EGTA and 70 µ M GPN were added to TcPiezo2 Tet-OFF cells (±Tet), as indicated. Addition of 20 µ M Yoda1 (+Yoda1) dramatically stimulated TcPiezo2-mediated Ca²⁺ release from lysosomes. (H) Changes in fluorescence (left bar graph) and the rates of fluorescence increase (right bar graph) upon Yoda1 activation in Tet-induced (+Tet±Yoda1) and non-induced (-Te±Yoda1) cells in Fig 5G. Addition of DMSO, instead of Yoda1/Dooku1/GsM, was used as control (baseline) labeled with B. In panels D, F, H, values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (**P < 0.01, ***P < 0.001).

https://doi.org/10.1371/journal.ppat.1013105.g005

We then tested the effect of inhibitors (GsMTx4) and activators (Yoda1) on TcPiezo2 ability to alter Ca²⁺ release by GPN from acidic intracellular stores of T. cruzi epimastigotes. To prevent Ca²⁺ entry we incubated the cells in the presence of 100 µ M EGTA. GsMTx4 (GsM) was able to inhibit Ca²⁺ release from acidic compartments similar to observations resulting from downregulation of TcPiezo2 expression and both treatments together had an additive effect (+Tet, Figs 5E, 5F, + Theo, and S9B). On the other hand, Yoda1 stimulated Ca²⁺ release by GPN from acidic compartments either in control cells or in cells where TcPiezo2 expression was downregulated (+Tet, Figs 5G and 5H, + Theo, and S9C). Interestingly, Jedi1/Jedi2 had no stimulatory activity on TcPiezo channels (S9D Fig).

These results indicate that TcPiezo-mediated Ca²⁺ fluxes in T. cruzi, are also able to be elicited by Yoda1 and Dooku1 instead of a mechanosensitive stimulus and are blocked by GsMTx4.

Role of TcPiezo1 and TcPiezo2 in Ca²⁺ increase during osmotic stress

Previous studies reported an increase in intracellular Ca²⁺ in different T. cruzi stages during hypoosmotic stress caused by 50% reduction in osmolarity from 300 to 150 mOsm [28]. This Ca²⁺ increase was attributed to Ca²⁺ entry through the plasma membrane since it was prevented by incubation with 1 mM extracellular EGTA [28]. To investigate the role of TcPiezo1 in this Ca²⁺ increase we incubated TcPiezo1 Tet-OFF epimastigotes expressing jGCaMP7s with 100 µ M extracellular EGTA (Fig 6A) to abolish Ca²⁺ entry, but this treatment did not significantly affect the Ca²⁺ rise (Fig 6A). However, downregulation of TcPiezo1 expression resulted in a slight but significant decrease in intracellular Ca²⁺ rise (Fig 6B). Intracellular Ca²⁺ rise was more dramatically affected by treatment with the cell-permeable inositol 1,4,5-trisphosphate receptor (IP₃R) inhibitor xestospongin C or by the general Ca²⁺ channel inhibitor ruthenium red (Fig 6A). To investigate the role of TcPiezo2 we incubated TcPiezo2-OFF epimastigotes expressing jGCaMP7s with 100 µ M EGTA to suppress Ca²⁺ entry. Under these conditions we detected a significant decrease in Ca²⁺ rise by either xestospongin C or ruthenium red treatment (Fig 6C). Downregulation of TcPiezo2 expression by tetracycline addition (+Tet) had more dramatic effects (Fig 6D). To test TcPiezo-CKO responses to hyperosmotic stress, TcPiezo Tet-OFF epimastigotes expressing jGCaMP7s were submitted to hyperosmotic condition (800 mOsm) by adding D-mannitol (1,300 mOsm), but no intracellular Ca²⁺ increase was detected (S10 Fig). In summary, both TcPiezo1 and TcPiezo2, as well as the IP₃R, contribute to the increase in intracellular Ca²⁺ levels of epimastigotes when they are submitted to hypoosmotic stress.

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Fig 6. TcPiezo-CKO responses to hypoosmotic stress.

5 × 10⁷ TcPiezo-CKO epimastigotes expressing jGCaMP7s in 600 µl isosmotic buffer (300 mOsm) was submitted to hypoosmotic stress (150 mOsm) by adding 600 µl H₂O at 50 s. Upon hypoosmotic stress, the cells swelled and the intracellular Ca²⁺ rose (control, C or -Tet). No intracellular Ca²⁺ was increased by adding 600 µl isosmotic buffer (baselines, B). (A) Non-induced TcPiezo1 Tet-OFF intracellular Ca²⁺ responses to the addition of 100 µ M EGTA, 1 µ M Xestospongin C (XestC), or 4 µ M ruthenium red (RR). The addition of EGTA did not affect intracellular Ca²⁺ rise, but the addition of XestC or RR did. (B) Tet-induced TcPiezo1 Tet-OFF cells (+Tet) had slightly lower Ca²⁺ rise than non-induced cells (-Tet). (C) Non-induced TcPiezo2 Tet-OFF (-Tet) intracellular Ca²⁺ responses to the addition of 1 µ M XestC or 4 µ M RR. The addition of XestC or RR significantly affected intracellular Ca²⁺ rise. (D) Tet-induced TcPiezo2 Tet-OFF cells (+Tet) had a significant decrease of intracellular Ca²⁺ rise, compared to non-induced cells (-Tet). 100 µ M EGTA was added to remove extracellular Ca²⁺, abolishing Ca²⁺ entry, as indicated. Values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, *** P < 0.001).

https://doi.org/10.1371/journal.ppat.1013105.g006

Cell culture

T. cruzi epimastigotes (Y strain) were grown at 28°C in liver infusion tryptose (LIT) medium (0.5% [w/v] liver infusion, 0.5% [w/v] tryptose, 68 mM NaCl, 56 mM Na₂HPO₄, 5.4 mM KCl, 0.2% [w/v] glucose, 0.002% [w/v] hemin, pH 7.3 [73], supplemented with 10% (v/v) heat-inactivated newborn calf serum (NCS-HI) and 1% Gibco’s penicillin/streptomycin (10 kU/mL). Transgenic T. cruzi cell lines were maintained in LIT medium with appropriate antibiotics (250 μg/ml G418, 10 μg/ml blasticidin, 5μg/ml puromycin). Conditional knockout (CKO) T. cruzi was induced with 5 μg/mL fresh tetracycline or 250 μg/mL theophylline when the cells were at a density of 2 × 10⁶ epimastigotes/mL. The growth rate of epimastigotes was determined by counting cells in a Neubauer chamber.

Tissue culture cell-derived trypomastigotes and amastigotes were obtained as previously described [3]. Vero cells were grown in RPMI 1640 medium supplemented with 10% fresh fetal bovine serum (fresh FBS) and 1% Gibco’s penicillin/streptomycin (10 kU/mL) and incubated at 37°C with 5% CO₂.

In vitro metacyclogenesis

Metacyclic trypomastigotes were obtained as described [74] with minor modifications. Mid-log epimastigotes were collected by centrifugation at 1,000 × g for 7 min and washed twice in triatomine artificial urine (TAU) medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 8 mM sodium phosphate buffer pH6.0), and incubated in TAU for 2 hours at 28^oC. The parasites were then diluted 100-fold in TAU 3AAG medium (TAU supplemented with 20 mM L-proline, 10 mM glucose, 50 mM sodium glutamate, and 2 mM sodium aspartate) in T25 flasks and incubated horizontally with or without 5 μg/mL fresh tetracycline or 250 μg/mL theophylline at 28^oC for 96 hours.

For quantification, assays samples were fixed for 1 hour at RT in 4% paraformaldehyde in PBS, attached to poly-L-lysine-coated coverslips, and washed three times with PBS. Then, parasites were incubated for 1 hour in 50 mM NH₄Cl in PBS, washed three times in PBS, and mounted onto glass slides with Fluoromount-G containing 15 μg/mL 4’,6-diamidino-2-phenylindole (DAPI), which stains the DNA present in the nucleus and the kinetoplast of parasites. Twenty fields/slides were analyzed on an Olympus IX-71 inverted fluorescence microscope with a 100 × objective in three independent experiments. Metacyclic trypomastigotes were distinguished from epimastigotes by the location of the kinetoplast in the cell body (posterior in metacyclic trypomastigotes; between the nucleus and the flagellum in epimastigotes). Metacyclic trypomastigotes without tetracycline or theophylline treatment were also collected to initially infect Vero cells to generate tissue culture-derived trypomastigotes.

In vitro infection assays

T. cruzi invasion and intracellular replication assays were performed as previously described [24], with some modifications. Gamma-irradiated (2,000 rads) Vero cells (4.5 x 10⁵ cells) were plated onto sterile coverslips in a 12-well plate and incubated overnight at 37°C in 5% CO₂ in RPMI medium plus 10% fresh FBS. Tissue culture-derived trypomastigote collections were incubated with or without fresh tetracycline (5 µg/ml) or theophylline (250 µg/ml) at 4°C overnight and then 1 hour at 37^oC to allow amastigotes to settle from swimming trypomastigotes. Trypomastigotes from the supernatants of these collections were counted and used to infect the mammalian cells at a 10:1 ratio of trypomastigotes to host cells in the absence and presence of tetracycline or theophylline. At 4 h post-infection, coverslips were washed extensively with Hanks’ solution, followed by PBS at pH 7.4 to remove any extracellular parasites. Coverslips were fixed immediately in 4% paraformaldehyde in PBS (pH 7.4) at 4°C for 30 min, washed once with PBS, and mounted onto glass slides in Fluoromount-G containing 15 µg/ml of DAPI, which stains host and parasite DNA (S4F, S4G, S7G, and S7H Figs). Coverslips were analyzed on an Olympus IX-71 inverted fluorescence microscope to quantify the number of host cells that contained intracellular parasites and the number of intracellular parasites per cell in 40 randomly selected fields. Three hundred host cells were counted per sample in three independent experiments.

To quantify amastigote replication, Vero cells were infected at a 10:1 ratio of tissue culture-derived trypomastigotes to host cells, and after they were washed at 4 h post-infection as described above, coverslips were allowed to incubate in the absence and presence of tetracycline (1 µg/ml) or theophylline (125 µg/ml) for 72 h post-infection at 37°C with 5% CO₂ prior to fixation and DAPI staining. Coverslips were mounted onto glass slides and analyzed on an Olympus IX-71 inverted fluorescence microscope. Amastigotes in infected host cells were counted using a 100 × objective.

To confirm that expression down-regulation of TcPiezos could be achieved in amastigotes and trypomastigotes we added 1 µg/ml tetracycline or 125 µg/ml theophylline to cultures releasing large amounts of these forms. After 2 days the cells were collected, trypomastigotes were separated from amastigotes by decantation (1 hour at 37^oC incubation as above) and analyzed by western blot (S11 Fig).

Cell transfections

T. cruzi transfections were performed as previously described [25]. Mid-log phase T. cruzi Y strain epimastigotes (5 x 10⁷ cells) were washed with sterile PBS, pH 7.4, at room temperature (RT) and transfected in ice-cold Cytomix buffer (120 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄, 25 mM Hepes, 2 mM EDTA, 5 mM MgCl₂, pH 7.6) containing 10–25 μg of each DNA construct in 4-mm electroporation cuvettes with three pulses (1500 volts, 25 μF each pulse) delivered by a Gene Pulser Xcell Electroporation System (Bio-Rad). Stable cell lines were obtained and maintained under drug selection with appropriate antibiotic(s) (250 μg/ml G418, 10 μg/ml blasticidin, 5 μg/ml puromycin). Transfected epimastigotes were cultured in LIT media supplemented with 20% NCS-HI until stable and clonal cell lines were established.

Knockout of TcPiezo1 and TcPiezo2

Single guide RNA (sgRNA) sequences to target the pore region or the whole TcPiezo1 and TcPiezo2 (TryTripDB gene ID numbers TcYC6_0088320, and TcYC6_0007880, respectively), as described in S3A Fig, were PCR amplified from plasmid pUC_sgRNA [38]. Selection of the protospacers was performed using EuPaGDT (Eukaryotic Pathogen CRISPR guide RNA Design Tool [http://grna.ctegd.uga.edu/]). The protospacer sequences were included into the forward primers (TcPZ1gRNA-koF1/-koF2, and TcPZ1gRNA-koF1/-koF2/-koF3), while a common reverse primer (Com-gRNA-R) (S1 Table) was used for sgRNA amplification. These primers also contained a BamHI restriction site for cloning into Cas9/pTREX-n [38] to generate the TcPiezo sgRNA/Cas9/pTREX-n constructs. The sgRNA orientation was verified by PCR using the TcPiezo-sgRNA forward primers and the HX1 reverse primer (HX1-R) (S1 Table). Positive clones that generate a 190-bp PCR fragment were also sequenced. DNA donor cassettes designed to promote homologous directed repair and replacement of the pore region or the whole TcPiezo genes were obtained by PCR using a set of long primers, containing 120 bp homology arms which correspond to the TcPiezo genes, as indicated in S3A Fig, with 20–22 nt annealing on blasticidin (Bsd) or puromycin (Puro) resistance gene (S1 Table). The TcPiezo-sgRNA/Cas9/pTREX-n constructs and their corresponding DNA donor cassettes were co-transfected into T. cruzi epimastigotes and then selected with 250 μg/ml G418 and 10 μg/ml blasticidin or 5 μg/ml puromycin for 5 weeks to obtain antibiotics resistance cell lines.

Conditional knockout of TcPiezo1 and TcPiezo2

Downregulation of TcPiezo1 or TcPiezo2 was developed by a novel conditional expression system for T. cruzi using modified hammerhead ribozymes (HHR) (S3F Fig), named as Small Hammerhead Aptazyme-Regulated Knockdown-SHARK³¹. First, TcPiezo1 and TcPiezo2 aptazyme tagging cassettes (composed of 3 × Ty tag, Tet-OFF/Theo-OFF aptazyme sequence, GAPDH 3’UTR, and Bsd gene) were generated by PCR using the pMiniTrex-mCherry-aptazymes (Shark1 and Shark3) [31] as templates with gene-specific forward and reverse primers TcPZ1-kd-F and TcPZ1-kd-R, TcPZ2-kd-F and TcPZ2-kd-R (S1 Table), respectively. The forward and reverse primers contain 50 bp homology arms corresponding to regions close to the 3’ ends of ORFs (right upstream of the Cas9 sgRNA and downstream of the stop codon of TcPiezo1, and upstream of the stop codon and downstream of the Cas9 sgRNA of TcPiezo2) (S3B Fig), followed in frame by the 23–24 nucleotides (nt) of the backbone sequences of pMiniTrex-mCherry-aptazymes (Shark1 and Shark3). Second, chimeric sgRNA targeting 25 nt upstream of the stop codon of TcPiezo1 and 24 nt downstream of the stop codon of TcPiezo2 (S3B Fig) were PCR-amplified and cloned into Cas9/pTREX-n vector to generate the gene specific sgRNA/Cas9/pTREX-n vectors, as described above. Last, gel-purified TcPiezo1 and TcPiezo2 aptazyme cassettes (15 μg) and the corresponding sgRNA/Cas9/pTREX-n (15 μg) were co-transfected into T. cruzi epimastigotes, respectively, to generate TcPiezo Tet-OFF/Theo-OFF (also called as TcPiezo-CKO, or TcPiezo-Shark1/3) homozygous transgenic cell lines, which were confirmed by PCR (S3C–E Fig) using the pair primers TcPZ1-ORF-F and TcPZ1–3UTR-R, and TcPZ2-ORF-F and TcPZ2–3UTR-R (S1 Table), respectively.

PCR-mediated in situ epitope-tagging of TcPiezo2

The one-step epitope-tagging protocol reported by Oberholzer et al. [75] was used to produce C-terminal smV5 tagging cassette of TcPiezo2 for transfection of T. cruzi epimastigotes. In brief, the PCR forward and reverse primers (S1 Table) included terminal 142 nt of ORF before its stop codon and the reverse complement of the 112 nt of the 3’UTR respectively, followed in frame by the 21–26 nucleotides of the backbone sequences of pMOTag2mV vector [44]. The smV5 tagging cassette containing an antibiotic selection marker (puromycin resistance gene) was generated for cell transfection by PCR using the pMOTag2mV as template with the primers TcPiezo2-TF and TcPiezo2-TR (S1 Table).

Expression of GCaMP Ca²⁺ sensors in T. cruzi

The coding sequences of genetically encoded Ca²⁺ indicators (GECIs) GCaMP6f, GCaMP6s, jGCaMP7f and jGCaMP6s (composed of 6 × His-T7 epitope-Xpress tag, circularly permuted GFP, the Ca²⁺-binding protein calmodulin (CaM), and CaM-interacting M13 peptide) were PCR-amplified by using pGP-CMV-GCaMP6f/s [41] or pGP-CMV-jGCaMP7f/s [42] as template, respectively, with primers GCaMP-F and GCaMP-R (S1 Table). The PCR products were gel-purified, XbaI-HindIII-digested, and then clone into XbaI-HindIII-cut pTREX-n or pTREX-p [25], which contains a neomycin (G418) or puromycin resistance gene, respectively. After confirmed by sequencing, the correct GCaMP variant constructs were transfected into T. cruzi WT or TcPiezo-CKO cell lines. The transgenic cell lines constitutively expressing cytosolic GCaMP Ca²⁺ sensors were confirmed by immunoblotting and IFA assays and used for Ca²⁺ entry and release assays, as described below.

Ca²⁺ entry and release assays

1. (i). Fura-2/AM

Tissue culture cell-derived TcPiezo1-CKO amastigotes in the absence or presence of tetracycline or theophylline for 2 days were collected in 50 ml sterile centrifuge tube and settled by incubation at 4^oC overnight to separate from swimming trypomastigotes. Amastigotes were washed twice with buffer A with glucose (BAG), which contained 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 5.5 mM D-glucose and 50 mM Hepes at pH7.3, at 1000 × g for 7 min at room temperature and loaded in BAG with 5 μM Fura-2/AM (Molecular Probes) plus 1.5% sucrose for 30 min at a 30°C water bath with mild agitation and rinsed twice by centrifugation to remove extracellular dye. The cells were resuspended to a final density of 1 x 10⁹ cells per ml in BAG. For fluorescence measurements, a 50 μl-aliquot (5 × 10⁷ cells) of the cell suspension was added to 2.45-ml BAG containing 100 μM EGTA (<10 μM extracellular Ca²⁺) in a cuvette. Ca²⁺ entry was monitored by adding 1.8 mM Ca²⁺ to suspension of amastigotes in the cuvette on a Hitachi F7000 spectrofluorometer with excitation wavelength 340/380 nm and emission wavelength 510 nm on the Hitachi F7000 spectrofluorometer, as previously described [76].

1. (ii). GCaMPs

TcPiezo-CKO epimastigotes or trypomastigotes expressing GCaMP-type Ca²⁺ indicators were incubated in the absence or presence of tetracycline (or theophylline) for 2 days. The epimastigotes were collected by centrifugation at 1000 × g for 7 min at RT. Tissue culture cell-derived trypomastigote collections were incubated at 4°C overnight to allow amastigotes to settle from swimming trypomastigotes. Trypomastigotes from the supernatants were collected by centrifugation at 1,600 × g for 7 min. The epimastigotes and trypomastigotes were rinsed twice, resuspended to a final density of 1 x 10⁹ cells per ml in BAG, and kept on ice. For intracellular Ca²⁺ measurements, a 50 μl-aliquot (5 x 10⁷ epimastigotes or 2 × 10⁷ trypomastigotes) of the cell suspensions was added to 1.95-ml BAG in a cuvette on a Hitachi F7000 spectrofluorometer with excitation wavelength 488 nm and emission wavelength 510 nm on the Hitachi F7000 spectrofluorometer. Ca²⁺ entry was monitored in the TcPiezo1-CKO cells by adding 1.8 mM Ca²⁺, whereas Ca²⁺ release was detected in the TcPiezo2-CKO cells by adding 70 μM glycyl-l-phenylalanine 2-naphthylamide (GPN). Ca²⁺ ionophore (ionomycin), Piezo activators (Yoda1, Dooku1, Jedi1, Jedi2, Extracellular Matrix-ECM), Piezo inhibitor (Grammostola spatulata mechanotoxin 4 (GsMTx4), or SERCA inhibitor cyclopiazonic acid (CPA) were also added as described in Figures. Ca²⁺ fluxes were measured using jGCaMP7s signal fluorescence changes (∆) in arbitrary units of fluorescence (AU), which were obtained by subtracting the values at the turning point of the baseline from those at the peaks (S1 Data).

Osmotic stress assays

TcPiezo Tet-OFF epimastigotes expressing jGCaMP7s were grown in the absence and presence of 5 μg/mL tetracycline for 2 days. Exponentially growing parasites were collected by centrifugation at 1,000 g for 7 min and washed twice with BAG. Cells were resuspended to a final density of 1 × 10⁹ cells per ml in the isosmotic BAG buffer (300 mOsm) and kept on ice. A 50-μl aliquot (5x10⁷ cells) of cell suspension was added to the 550-μl isosmotic buffer and incubated without or with 100 μM EGTA. Osmolarity of the reaction buffer was changed by adding 600 μl of Millipore H₂O to achieve the hypoosmotic (150 mOsm) condition or by adding 600 μl of 1,300 mOsm D-mannitol (Sigma Aldrich) to make the hyperosmotic (800 mOsm) condition. The cells were incubated with 100 μM EGTA to remove extracellular Ca²⁺. 1 μM Xestospongin C (Xest-C) was added to monitor Ca²⁺ release from acidocalcisomes by blocking inositol 1,4,5-trisphosphate (IP₃) receptor. 4 μM ruthenium red (RR) was added. The cytosolic Ca²⁺ concentration of cells was monitored under hypoosmotic or hyperosmotic stress in a fluorometer with excitation at 488 nm and emission at 510 nm, as described above.

Immunofluorescence microscopy

T. cruzi cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS pH 7.4 for 1 hour at RT. The fixed cells were washed twice with PBS, allowed to adhere to poly-L-lysine-coated coverslips and permeabilized with 0.3% Triton X-100/PBS for 3 min for the parasites. After blocking with PBS containing 3% BSA, 1% fish gelatin, 50 mM NH₄Cl and 5% goat serum for 1 h, cells were stained in 1% BSA/PBS with the purified clone BB2 monoclonal antibody against Ty1 (1:100), monoclonal antibody against V5 (1:100), monoclonal antibody against His (1:100), rabbit polyclonal antibody against T. cruzi H⁺ -ATPase (α-TcHAf, 1:200), rabbit polyclonal antibody against T. cruzi cruzipain (α-TcCZP, 1:2,500), rabbit polyclonal antibody against T. cruzi serine carboxypeptidase (α-TcSCAR, 1:1,000), or rabbit polyclonal antibody against GFP (1:100) for 1 h. After thoroughly washing with PBS containing 3% BSA, cells were incubated with Alexa 488-conjugated goat anti-mouse or anti-rabbit, and Alexa 546-conjugated goat anti-rabbit antibody at 1:1,000 for 1 h. The cells were counterstained with DAPI before mounting with Gold ProLong Gold antifade reagent (Molecular Probes). Differential interference contrast and fluorescent optical images were captured using an Olympus IX-71 inverted fluorescence microscope with a Photometrix CoolSnap^HQ charge-coupled device camera driven by DeltaVision software (Applied Precision, Seattle, WA). Images were deconvolved for 15 cycles using Softwarx deconvolution software. Pearson’s correlation coefficients were calculated using the Softwarx software by measuring the whole-cell images.

Western blot analyses

T. cruzi cells were harvested and washed twice in BAG. The washed cells were lysed with RIPA buffer (150 mM NaCl, 20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1% Triton X-100) containing protease inhibitor tablet in ice for 1 h. The protein concentration was determined by using Pierce BCA protein assay kit with the microplate reader. Total cell lysates were mixed with 2 × Laemmli sample buffer (BioRad) at 1:1 ratio (volume/volume) and directly loaded. The separated proteins were transferred onto nitrocellulose membranes using a Bio-Rad transblot apparatus. The membranes were blocked with 10% non-fat milk in PBS-T at 4^oC overnight. The blots were incubated with mouse antibodies against Ty1 (1:2,500), rabbit antibodies against GFP (1:2,500), mouse antibodies against V5 (1:2,500), monoclonal antibody against His (1:1,000), and mouse antibodies against tubulin (1:10,000) for 1 h. After five washings with PBS-T, the blots were incubated with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG (H + L) antibody at a dilution of 1:15,000 for 1 h. After washing five times with PBS-T, the immunoblots were visualized using Pierce ECL Western blotting substrate according to the manufacturer’s instructions. Full-size images of immunoblots are shown in S12 Fig.