tpHusion: An efficient tool for clonal pH determination in Drosophila.

Genetically encoded pH indicators (GEpHI) have emerged as important tools for investigating intracellular pH (pHi) dynamics in Drosophila. However, most of the indicators are based on the Gal4/UAS binary expression system. Here, we report the generation of a ubiquitously-expressed GEpHI. The fusion protein of super ecliptic pHluorin and FusionRed was cloned under the tubulin promoter (tpHusion) to drive it independently of the Gal4/UAS system. The function of tpHusion was validated in various tissues from different developmental stages of Drosophila. Differences in pHi were also indicated correctly in fixed tissues. Finally, we describe the use of tpHusion for comparative analysis of pHi in manipulated clones and the surrounding cells in epithelial tissues. Our findings establish tpHusion as a robust tool for studying pHi in Drosophila.

Introduction

Perturbations in intracellular pH (pHi) affect many cellular processes including cell growth, proliferation, differentiation, and metabolism [1]. Under normal conditions, pHi is maintained within a narrow physiological range, and deregulation of pHi is observed in various disease states. In contrast to regular physiology, a state of higher pHi than the extracellular pH (pHe) is conducive to the survival of tumor cells and metastatic progression [2]. Alterations in cytosolic pH have also been implicated to cause neurodegenerative diseases [3]. Exploration of pH dynamics can provide a better understanding of the fundamental cellular processes and help develop strategies for the prevention or treatment of human pathologies.

Over the last few decades, several techniques for pHi measurement have been established. These include the use of proton-permeable microelectrodes, NMR and fluorescence spectroscopy [4,5]. The pH-sensitive fluorescent dyes offer easy-to-use tools for pHi measurement. Although the generation of ratiometric pH probes could address certain shortcomings such as dye leakage, photobleaching and uneven loading of dyes, the application of these dyes is limited in specific cases including pHi analysis in whole-mount tissues because of heterogeneous dye uptake. Construction of the pH-sensitive GFP variant, pHluorin [6], has led to the development of genetically encoded pH indicators (GEpHIs) for pHi measurements in various cell types and organelles. In Drosophila, GEpHIs are typically based on the Gal4/UAS system-driven expression of the pH-sensitive superecliptic pHluorin (SEpHluorin) [7] and the pH-insensitive mCherry [8] in the region of interest [9-11]. Improved modifications of the sensors have also been described [12] but an organism-wide expression has not been exploited. This restricts the ability to examine pHi during developmental processes or in clonal situations within the same tissue, which is one of the major benefits of using Drosophila as a model system.

Overexpression of the Drosophila Na+-H+ exchanger, Nhe2, increases pHi in the retina [13]. However, the absence of an internal GEpHI expression and imaging control necessitates the generation of fluorescence ratio to pH calibration curves for every experiment. Ubiquitous expression of the GEpHI with a ubiquitously-expressed Gal4 in combination with other means to produce clonal manipulations (FLP/FRT, LexA or Q system) could provide ‘in-tissue’ control for a more accurate interpretation of results. However, this approach can be complicated by the prevalent use of the Gal4/UAS system and its possible toxicity [14]. To address these issues, we developed a Gal4/UAS-independent and ubiquitously-expressed GEpHI, called tpHusion. We describe its application for evaluation of pHi dynamics in living and fixed tissues, as well as for clonal analysis.

Materials and methods

Generation of tpHusion Act>CD2>Gal4 chromosome

The tubulin-3’-trailer was excised from the pKB342 plasmid (gift from Konrad Basler) using restriction sites XhoI and XbaI, and subcloned in-line with the pHusion-HRas sequence (gift from Gregory Macleod, [15]) in pJFRC14 vector [16] using BamHI and XbaI sites. The resulting pHusion-HRas-tubulin-3’-trailer sequence was excised by restriction digestion with NotI and XbaI and used to replace the Gal4 sequence under the tubulin promoter sequence in the pT2-attB-Gal4 vector (gift from Konrad Basler) using Acc65I and XbaI sites. The resulting plasmid tpHusion was injected into the fly line ΦX-86Fb [17]. The 3xP3-RFPattP landing site sequence was removed using Cre recombinase-mediated excision. The Act>CD2>Gal4 construct was recombined on the resulting 3R chromosome arm to generate the tpHusion Act>CD2>Gal4 chromosome.

Fly husbandry

All lines and crosses were maintained at 25°C on normal fly food unless otherwise stated. Normal fly food is composed of 100 g fresh yeast, 55 g cornmeal, 10 g wheat flour, 75 g sugar, 8 g bacto agar, and 1.5% antimicrobial agents (33 g/L nipagin and 66 g/L nipasol in ethanol) in 1 L water.

Fly lines used: ΦX-86Fb (gift from Johannes Bischof), Act>CD2>Gal4 (4780 Bloomington Drosophila Stock Center (BDSC)); UAS-ECFP-golgi (42710 BDSC), UAS-LacZ (control, gift from Johannes Bischof), UAS-Ras [18], Pten (101475 Vienna Drosophila Resource Center (VDRC)), foxo (107786 VDRC), and Tsc1 (31039 BDSC).

Cross setup and clone induction

Flies were crossed for two days before an overnight egg laying. For clone induction, a 15 min heat shock at 37°C was applied 36 h after egg laying (AEL) and the animals were allowed to develop at 25°C. Wing and eye imaginal discs, and brains were dissected from wandering L3 larvae 108 h AEL; ovarioles were dissected from fertilized females. For clonal analyses, only female larvae were used for dissection. Tissues were dissected in HCO3- buffer [19] for live imaging or in 4% paraformaldehyde (PFA) for fixed tissue imaging.

Microscopy and immunofluorescence staining

After dissection of live tissues in HCO3- buffer, the samples were mounted in the same buffer on 35 mm MatTek dishes coated with 0.1 mg/mL poly-L-Lysine. Fluorescence images were acquired on a Visitron Spinning Disk confocal microscope within one hour of dissection.

For fixed samples, tissues were dissected in 4% PFA, fixed for at least 30 min at room temperature (RT), and stored at 4°C until processing of all experimental conditions. The samples were washed in PBS for 10 min and mounted on glass slides in VECTASHIELD (Vector Laboratories H-1000) mounting medium. Confocal images were obtained within 24 h of dissection on a Leica SPE TCS confocal laser-scanning microscope.

For the immunofluorescence staining with Fasciclin III (FasIII), tissues were dissected in PBS. The samples were fixed in 4% PFA (30 min, RT), washed thrice in 0.3% Triton-X in PBS (PBT, 15 min, RT), blocked in 2% Normal Donkey Serum (NDS) in 0.3% PBT (2 h, 4°C), incubated with mouse anti-FasIII (1:15 in 2% NDS, 7G10 Developmental Studies Hybridoma Bank (DSHB), overnight, 4°C), washed thrice in 0.3% PBT (15 min each, RT), incubated with goat anti-mouse Alexa Fluor 647 (1:500, Thermo Fisher Scientific, 2 h, RT), washed thrice in 0.3% PBT (15 min each, RT), stained with DAPI in 0.3% PBT (1:2000, 10 min, RT), and washed once with PBS (10 min, RT). The samples were mounted on glass slides in VECTASHIELD and imaged using a Leica SPE TCS confocal laser-scanning microscope.

In vivo nigericin calibration

Nigericin calibration buffers and curves were generated as described previously [19]. Briefly, after acquiring images of live tissues, the HCO3- buffer was replaced with the first calibration buffer containing nigericin. Samples were imaged every 4 min after a minimum incubation of 10 min. After a total incubation time of 20 min, subsequent buffers were added for 6 min and images were acquired every 2 min.

Quantification and statistical analysis

Images were processed using ImageJ [20]. Background subtraction was performed for each channel. The images were converted to 32-bit and median filtering was applied with radius 2. A defined area encompassing cells of interest, clones or surrounding wild-type tissue was selected and mean gray values were measured for SEpHluorin and FusionRed. The pseudo color images were produced by auto-thresholding of individual channels, followed by division of SEpHluorin channel intensity with FusionRed. The calibration bar represents the relative ratio of SEpHluorin to FusionRed intensities within a tissue from low (blue) to high (red). Statistical analyses were performed using unpaired two-tailed Student’s t-test. p values are described in the Figure legends. All plots were generated in R Studio and Figures were assembled using Adobe Illustrator.

Results

Development and in vivo validation of tpHusion pH reporter

The use of the Gal4/UAS system is one of the most common ways to produce clonal manipulations in Drosophila [21]. To develop a ubiquitously-expressed GEpHI that would be useful for clonal analysis, the expression of the sensor was rendered independent of Gal4/UAS control. A translational fusion protein of SEpHluorin with FusionRed was cloned under the control of the tubulin promoter. FusionRed was used to replace mCherry due to its low cytotoxicity, better performance in fusions and increased stability in a monomeric state [22]. An HRas sequence was also included to tether the fusion protein to the cytosolic side of the plasma membrane. This reduces the pHi variability due to the presence of compartments of differing pH within the cytosol [23]. The construct is referred to as tpHusion. The expression and membrane localization of tpHusion was confirmed in various tissues (data shown for wing imaginal discs in Fig 1A).

Fig 1

Validation of tpHusion reporter.

(A) Fixed wing pouches of wandering L3 larvae depicting the cellular localization of tpHusion. FasIII labels the plasma membrane and DAPI stains the nuclei. Scale bar = 50 μm. (B) Changes in SEpHluorin (green) and FusionRed (red) fluorescence intensities, and the ratio of SEpHluorin to FusionRed intensities from live wing pouch cells upon incubation in nigericin buffers of varying pH. n > 7 larvae. Data are represented as mean ± standard deviation. (C) Calibration curve between intracellular pH (pHi) and the ratio of SEpHluorin to FusionRed intensities generated from experiments in B.

To verify that tpHusion is a suitable pH indicator, fluorescence intensity calibrations were performed on wing imaginal discs incubated in buffers containing nigericin, which is an ionophore used to clamp the pHi to the buffer pH (see Materials and Methods). The pH-sensitive SEpHluorin displayed the predicted changes in fluorescence based on the buffer pH, whereas the pH-insensitive FusionRed only showed minor alterations. The ratio of SEpHluorin to FusionRed reflected the buffer pH (Fig 1B), resulting in the generation of a calibration curve of pHi with the ratio of fluorescence intensities (Fig 1C). Thus, tpHusion can be used to indicate changes in pHi.

tpHusion reports pHi changes in living tissues

Apart from the clonal analyses, a major advantage of a ubiquitously-expressed GEpHI is the ability to compare pHi in different cells across various developmental processes and stages. Earlier studies have reported a higher pHi in the differentiated follicle cells of the Drosophila ovariole as compared to the follicle stem cells (FSCs) using the Gal4/UAS-dependent expression of SEpHluorin/mCherry probe [24]. This finding was confirmed using tpHusion, which showed a similar increase in pHi of follicle cells in contrast to the FSCs (Fig 2A and 2A’).

Fig 2

Comparison of pHi in different cell types of living tissues.

(A-D) SEpHluorin, FusionRed, and ratiometric images of live (A) ovarioles, (B) eye discs, (C) wing discs, and (D) brains. (A’-D’) Quantification of the ratio of fluorescence intensities in (A’) FSC (arrow, white solid square) and follicle (arrowhead, yellow dashed square) cells, (B’) cells anterior (amf, arrow, white solid square) and posterior (pmf, arrowhead, yellow dashed square) to the morphogenetic furrow (dashed line), (C’) pouch (arrow, white solid square) and notum (arrowhead, yellow dashed square) cells, and (D’) cells in optic lobe (OL, arrow, white solid square) and central brain (CB, arrowhead, yellow dashed square). n > 7 larvae. Data are represented as mean ± standard deviation. * p < 0.05, *** p < 0.001 and ns = not significant. Scale bar for A = 50 μm, scale bar for B-D = 100 μm. Calibration bar represents the ratio values of SEpHluorin to FusionRed intensities used to generate ratiometric images.

The Drosophila imaginal discs have proven to be excellent systems for the identification of genes regulating cellular growth during normal development [25,26] or in perturbed states [27,28]. The pHi in these tissues has not been analyzed due to the unavailability of robust indicators. During the larval stages, the eye imaginal disc consists of proliferating cells anterior to the morphogenetic furrow (amf) and mostly differentiating photoreceptors posterior to the furrow (pmf) [29]. Investigation of SEpHluorin and FusionRed intensities using tpHusion demonstrated a higher ratio in the mitotically active amf region of the eye disc (Fig 2B and 2B’). The several compartments and cell lineages of the wing disc have also been described in great detail [26]. The larval wing disc is comprised of cells in the pouch region (which will form the wing blade) and the notum (which will form the body wall) [30,31]. pHi analysis using tpHusion displayed no difference in the pouch versus notum of the wing imaginal disc (Fig 2C and 2C’).

A major organ that is vital in neurobiological research is the Drosophila brain [32]. Tremendous advances have been made towards deciphering the circuitries of the adult and larval brains [33,34]. The information about pHi of different cell types could be fundamental to understand processes such as vesicular transport [35]. The larval brain can be subdivided into the central brain (CB), optic lobe (OL) and the ventral nerve cord (VNC) [36]. A brief evaluation in the larval brain revealed that cells in the OL have a higher pHi than cells in the CB (Fig 2D and 2D’). The above results establish the use of tpHusion for comparative analysis of in vivo pH differences in various Drosophila tissues during different developmental stages.

tpHusion reflects in vivo pH changes in fixed tissues

Culturing of Drosophila organs has been challenging, with specific culture condition requirements for different tissues [37-40]. For pHi measurements in live cells, tissues are dissected in a bicarbonate buffer (see Materials and Methods) to prevent changes in the physiological pHi. However, the tissues cannot be maintained in this buffer for an extended period, rendering the handling of many experimental conditions difficult. Fixing the conformational state of the GEpHI can help slow down changes in fluorescence until all samples are processed [41]. To examine if tpHusion can be used to monitor pH variations in fixed tissues, comparisons were performed in tissue regions depicted in Fig 2 after fixation. The tissues were dissected directly in 4% PFA to restrict changes in pHi and imaged within 24 h. Interestingly, differences in the ratio of SEpHluorin and FusionRed intensities in the various cell types of the tissues tested were the same as in the live tissues (Fig 3A–3D’). SEpHluorin retains sensitivity to acidic pH after fixation [42]. Since the samples were exclusively exposed to pH 7–7.4 in our experimental setup, the physiological pHi should be maintained. This does not exclude the possibility of slight alterations in pHi but the consistent changes in the ratio of intensities between different regions of the live and fixed tissues (compare Figs 2 and 3) suggest that tpHusion can be reliably used to study pHi variations in fixed tissues.

Fig 3

Comparison of pHi in different cell types of fixed tissues.

(A-D) SEpHluorin, FusionRed, and ratiometric images of fixed (A) ovarioles, (B) eye discs, (C) wing discs, and (D) brains. (A’-D’) Quantification of the ratio of fluorescence intensities in (A’) FSC (arrow, white solid square) and follicle (arrowhead, yellow dashed square) cells, (B’) cells anterior (amf, arrow, white solid square) and posterior (pmf, arrowhead, yellow dashed square) to the morphogenetic furrow (dashed line), (C’) pouch (arrow, white solid square) and notum (arrowhead, yellow dashed square) cells, and (D’) cells in optic lobe (OL, arrow, white solid square) and central brain (CB, arrowhead, yellow dashed square). n > 7 larvae. Data are represented as mean ± standard deviation. * p < 0.05, *** p < 0.001 and ns = not significant. Scale bar for A = 50 μm, scale bar for B-D = 100 μm. Calibration bar represents the ratio values of SEpHluorin to FusionRed intensities used to generate ratiometric images.

Using tpHusion to detect clonal pH changes

Deregulated pH is now considered a hallmark of cancer with a higher pHi and a lower pHe observed in cancer cells as compared to normal cells [43]. Many tumor models have been described in Drosophila [44]. Overexpression of activated Ras (Ras) causes hyperplastic overgrowth [45] and metastatic behavior in combination with loss of polarity genes [27]. It has also been shown to have an increased pHi compared to control cells in a 2D cell culture system of breast epithelial cells [13]. To analyze the pHi in Ras-overexpressing clones in Drosophila epithelial cells, tpHusion was recombined with an actin-FLP-out cassette [46]. Comparison of SEpHluorin and FusionRed intensities ratio in clones and the surrounding wild-type tissue revealed an elevated pHi in the clones (Fig 4A and 4A’).

Fig 4

tpHusion detects clonal pH changes in Drosophila imaginal discs.

(A) SEpHluorin, FusionRed and ratiometric images of wing pouches dissected from wandering female L3 larvae with Ras-overexpressing clones that are marked by ECFP. Calibration bar represents the ratio values of SEpHluorin to FusionRed intensities used to generate ratiometric images. Scale bar = 50 μm. (A’) Quantification of the ratio of fluorescence intensities in wild-type and Ras clones. (B) Quantification of the ratio of fluorescence intensities in wild-type and control, Pten, foxo, Tsc1 or Tsc1 foxo clones from wing pouches of wandering female L3 larvae. n > 10 larvae. Data are represented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 and ns = not significant.

Earlier studies have reported an enhanced cellular overgrowth upon clonal loss of tumor suppressors of the phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTORC1) signaling network. Loss-of-function mutations in the phosphatase and tensin homolog (Pten) or the tuberous sclerosis complex (TSC) subunit 1 (Tsc1) lead to an escalation in cell size and number [47,48]. We have also outlined the function of the transcription factor forkhead box O (FoxO) in limiting the proliferation of Tsc1 mutant cells under conditions of nutrient restriction [49]. The loss of foxo does not cause an overgrowth phenotype on its own [50]. Using the system mentioned above, the pHi of control, Pten, foxo, Tsc1, and Tsc1 foxo knockdown clones was compared to the surrounding wild-type tissue (Fig 4B). The ratio of SEpHluorin and FusionRed intensities in control and foxo knockdown clones was similar to the surrounding wild-type tissue, whereas the ratio was higher in Pten, Tsc1, and Tsc1 foxo knockdown clones. These data validate the use of tpHusion for clonal analysis of pHi in Drosophila epithelial tissues and suggest that loss of the tested tumor suppressors increases pHi.

Discussion

The ubiquitously-expressed GEpHI, tpHusion, facilitates in vivo pHi measurement and comparative analysis in diverse tissues and developmental stages in Drosophila. We have used this reporter to investigate pHi differences in developing organs and genetically manipulated clones compared to their surrounding cells in the same tissue.

Such an analysis was not feasible with the previously available tools for pHi determination. Our results demonstrate the applicability of tpHusion for the identification of new pHi modulators, and for studying pHi homeostasis during developmental processes or in disease models.

Minor imbalances in the intracellular and extracellular pH equilibrium can have major impacts on many cellular functions. The regulation of cell proliferation by pHi has been shown in a variety of species including sea urchin eggs, yeast, and mammalian cell culture systems [51]. Changing pHi can coordinate differentiation or lineage specification of certain stem cells [24,52,53]. pHi has also been found to change during cell cycle progression [54]. Given the crucial role of pH in physiology, it is not surprising that a loss of pH homeostasis is seen in many diseases including cancer [55]. One notable aspect of this observation that remains disputed in the field is if pH can signal to cellular processes leading to diseases or whether the observed pH imbalance is a result of the pathological state.

Studies addressing the role of pHi in the fundamental processes mentioned above have been limited in Drosophila due to the lack of adequate tools. The uniform expression of tpHusion with no apparent cytotoxicity throughout development can aid the analysis of pHi dynamics in various developmental contexts. The complete potential of tpHusion can be realized by combining it with the existing repertoire of genetic modification tools in Drosophila. The flexible use of tpHusion with the Gal/UAS and FLP-out systems to express UAS-based transgenes in clones led us to present, for the first time, an increase in pHi in cells with overexpression of an oncogene or knockdown of tumor suppressor genes as compared to the surrounding wild-type cells. One caveat is the inability to use the most commonly used fluorophores to label clones or cell lineages but this can be circumvented by the use of non-interfering fluorophores in the blue or the far-red channels. We conclude that tpHusion is a powerful tool for investigating cellular pH in Drosophila.

6 Dec 2019

PONE-D-19-31154

tpHusion: An efficient tool for clonal pH determination in Drosophila

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Reviewer #1: This manuscript by Gupta and Stocker examines the usefulness and efficiency of a new genetic tool that assesses the intracellular pH of cells. The study describes a practical follow-up to pH detectors that are already in use. The major difference of the tool generated in this work is that it functions independent of the Gal4/UAS binary gene expression system. The authors demonstrate the use of tpHusion in live, fixed, and clonal tissues.

Overall, this study is relevant for three reasons. (1) The tpHusion tool can detect intracellular pH in live and fixed tissues independent of Gal4/UAS. (2) tpHusion can be used along with binary expression systems to evaluate intracellular pH in mosaic tissue. (3) tpHusion can be used to assess intracellular pH across different tissues, developmental stages, and biological processes. However, there are a few issues that should be addressed:

(1) The figures and figure legends can be better annotated to highlight different regions of the tissue. Dashed lines, arrowheads, or brackets can be used to indicate various regions within the tissue. For example, on the eye discs, a dashed line along the morphogenetic furrow can more clearly show the amf versus the pmf. Similarly, arrowheads pointing out the FSCs versus the follicle cells allows for better interpretation of the distribution of the tpHusion signal in the ovarioles. The wing disc and the larval brain images can also benefit from outlining and labeling the different compartments.

(2) The dashed squares in the figures are difficult to see. Replace them with solid squares of a different color or represent another way. It would be helpful to have the squares on the images in the single channels too.

(3) Explain the calibration bar a bit more – specifically the colors and the associated numbers.

(4) Increase the magnification of the ovarioles in figure 2. The magnification of the ovarioles differs between figure 2 and figure 3. At the higher magnification used in figure 3, it is easier to see the cells and the tpHusion signal.

(5) Include low magnification images of the single channels of figure 4 for an unbiased view of the distribution of the tpHusion signal across clones throughout the entire wing disc.

(6) The authors state that the detection of intracellular pH in fixed tissue is similar to that observed in live tissue. However, there appears to be a significant reduction of tpHusion signal in the images of the fixed eye discs. Based on the images presented, the tpHusion signal in the fixed larval brain appears expanded and more intense. Finally, the tpHusion signal in fixed ovarioles seems shifted with more intense FusionRed signal compared to SEpHluorin signal in fixed compared to live tissues. Discuss these differences.

Additional minor points

(1) Change HCO3 to HCO3-.

(2) Page 6, Line 122: Insert (NDS) after Normal Donkey Serum.

Reviewer #2: Gupta et al. describe the development of tpHusion, a membrane associated cytosolic pH sensor. The authors describe their strategy, which relies on expressing the sensor under the control of the tubulin promoter in order to obviate the necessity of the GAL4/UAS system. The utility of the strategy was validated by measurements of pH in different tissues during development, and in both fixed as well as live tissues.

The manuscript is technically sound, and the authors’ conclusions are backed by their data. This tool will certainly be useful to researchers interested in studying cytosolic changes during development. There remains, however, one minor concern that could be addressed by additional experiments as detailed below. tpHusion is tagged to the membrane using an HRAS-sequence. Though this is a reasonable strategy to attach the sensor to the PM, there is a concern regarding localization of the sensor to specific domains at the PM. Attachment of HRAS to the membrane is dependent upon the levels of membrane cholesterol (i.e. lipid rafts). Under conditions of alterations in PM cholesterol levels, the sensor could fall off the membrane. How would this change the pH measurements? It is recommended that the authors describe the effects of removing PM cholesterol with beta-cyclodextrin on the recordings made with tpHusion. The goal of this endeavor would be to provide a framework that future researchers could use should they worry about expressing the sensor in backgrounds that exhibit alterations in cholesterol trafficking.

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24 Jan 2020

Please see our Response to Reviewers where we address all concerns in detail (including two figures).

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29 Jan 2020

tpHusion: an efficient tool for clonal pH determination in Drosophila

PONE-D-19-31154R1

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4 Feb 2020

PONE-D-19-31154R1

tpHusion: an efficient tool for clonal pH determination in Drosophila

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