This work was supported by NIH DK092408 and DK100227 to MFR. AJR was supported by T32-DK007013. The authors wish to thank Dr. Julian A.T. Dow for the CapaR-GAL4 and c724-GAL4 Drosophila stocks. We also thank Jacob B. Anderson for assistance maintaining experimental fly crosses.

The authors have nothing to disclose.

The success of quantification of pHi in Drosophila MTs depends entirely on the health of extracted MTs and the quality of mounting and dissection (Figure A&#160;-&#160;C). Thus, the careful handling of tissue as described is imperative. Slides freshly coated in PLL substantially aid MT mounting as they tend to be much more adhesive than slides which have previously been exposed to solution. Careful mounting will also aid in identification of distinct MT segments (Figure D). Healthy MTs facilitate calibration of pHerry and functional assessment by reducing mCherry aggregation and yielding much more consistent quantification of acid extrusion, respectively. In some cases, avoiding mCherry aggregation is not possible as the experimental conditions may inherently damage MT epithelia or produce significant over-expression of the fluorescent reporter. In these cases, a pseudo-ratiometric calibration normalized such that fluorescence ratio 1.0 corresponds to pHi 7.0, and point calibrations will permit quantification (Figure E). Care should be taken when performing point calibrations to avoid exposing the permanent elements of the imaging and perfusion systems to nigericin as the ionophore will adhere to glass and plastic. Even pseudo-ratiometric calibration is not possible for circumstances in which an experimental manipulation induces cellular damage during an experiment, i.e.&#160;this damage will cause a progressive apparent increase in mCherry fluorescence throughout the experiment. In these later cases, the SEpH fluorescence signal can be used with a normalized calibration curve and point calibrations, with the caveat that the imaging will no longer correct for movement artifacts and focal shifts.
GEpHIs carry several general limitations when compared to quantification of pHi with fluorescent dyes. Dye retention can be used as an indicator of membrane integrity and cell health39, but no equivalent assay is available for GEpHIs use. As such, the preparation health must be monitored through independent means if cell damage is predicted to confound results. GEpHIs potentially permit imaging from minimally disturbed in vivo preparations but tissue integrity inherently limits experimental manipulations and can make point calibrations impossible. Another specific limitation inherent in using pHerry and other cytosolic dual fluorophore pH indicators (such as ClopHensor40) derives from the tendency of the two fluorophores to alter their fluorescence independently of both one another and pHi. RFP aggregation artifacts are the most significant manifestation of this limitation, but quantification can also be compromised by photobleaching of one or both fluorophores. Thus, imaging protocols much be adjusted to minimize photobleaching, which can lead to long exposure times and acquisition rates &#60;0.2 Hz. Long exposure times will fail to report rapid pHi shifts. SEpH fluorescence shows linear correlation to pHi from pH 6.8 - 7.8 in most preparations, but the accuracy of such measurements depends on the accuracy of the nigericin/high K+ technique. Nigericin acts as a K+/H+ ionophore and proper calibration relies on equilibrating extracellular [K+] with intracellular [K+]. Estimates of intracellular [K+] are not available or readily obtainable for all experimental systems. Accuracy of pHi quantification will only be as reliable as estimates of intracellular [K+], although rates of relative change in pHi will be consistent. Given this limitation and the inverse logarithmic relationship of pHi to intracellular [H+], it is always preferable to report data as rates of change in pHi, acid extrusion rate (JH+, Figure 6C), or acid flux (Figure 6D) rather than absolute changes in pHi. Analysis of data as acid flux has the added benefit of correcting for differences in surface are to volume ratio between cell types.
Several caveats must be appreciated when interpreting data from the adult fly MT preparation described in this protocol. The morphological distinction of the initial, transitional, and main segments is likely a simplification of the true diversity of functional and genetic domains present in the MTs2. Furthermore, while this protocol is designed to detect function of basolateral acid transporters it is possible that apical transport can influence pHi measurements as well. Sealing the ureters when mounting the MTs (step 5.9) ensures that solution exchange primarily occurs at the basolateral surface but para-cellular and apical movement of ions may still influence pHi as basolateral transport ultimately alters the cytoplasmic side of lumen/cytosolic ion gradients. Absolute separation of apical and basolateral function can be accomplished by independently perfusing the luminal and basolateral surfaces of the MT but such methods are substantially more technically demanding as they require micropipette cannulation41.
GEpHIs present many advantages over conventional methods of measuring pHi, and these strengths are amplified when combined with the genetic malleability and low cost of the Drosophila MT preparation. Quantification of pHi has historically relied upon fluorescent dyes such as (2&#39;,7&#39;-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein (BCECF)22 or complicated electrophysiological assessment via ion-selective electrodes20,21. As pHerry is genetically encoded, it can be expressed in specific cellular populations by specific promoters (as demonstrated here in principal cells and stellate cells of the MT, Figure 6) and is amenable to use in any tissue subject to transgenesis, transfection, or viral-mediated infection. Dyes are limited by cost of individual preparations and potentially complicated application protocols which convey no cell specificity in heterogeneous tissues. Ion-selective electrodes require specialized equipment for fabrication and measurement, while pHerry requires only widefield epifluorescent microscopy with conventional GFP and RFP filter sets. Use of dyes and electrodes necessitate physical as well as optical access to the tissue of interest while GEpHIs can be monitored in freshly extracted tissues and over time in vivo. The opportunity for live imaging in intact preparations is of particular interest when assessing the cellular physiology of pHi regulation as no other technology permits quantification of pHi in the presence of endogenous buffering mechanisms.
The Drosophila adult MT preparation presents many attractive features for those interested in cellular pH regulation and ion transport. Drosophila husbandry is inexpensive and tools such as genetically-encoded biosensor constructs and RNAi expression inserts are readily available from a variety of stock centers (Bloomington Drosophila Stock Center at Indiana University; Vienna Drosophila Research Center). Drosophila MTs are composed of a single layer of polarized epithelial cells, making them ideal for investigation of transepithelial ion transport. Basolateral transport can be easily assayed (as demonstrated here) but full assessment of apical and basolateral ion movement is possible with micropipette cannulation41. Additionally, organ function assays such as the Ramsay secretion assay17 and the luminal calcium-oxalate deposition18 are well-characterized and allow correlation of epithelial cellular physiology to models of fluid secretion and nephrolithiasis, respectively. While these features provide opportunities for robust analysis, the low-cost and wide availability of epifluorescent microscopy makes the Drosophila MT model ideal for demonstrations of cellular and whole-organ physiology in teaching laboratories.
Mastery of these methods permits quantification of pHi regulation by basolateral H+ flux in adult Drosophila MTs, an accessible yet robust model of transepithelial ion transport. Use of GEpHIs such as pHerry can be easily adapted to assess pHi regulation in other invertebrate cell types, cultured mammalian cells, and in vivo preparations. Development of new GEpHIs will likely follow that of genetically-encoded calcium indicators, with new generations spanning the visible spectrum and addressing current limitations such as aggregation artifacts30,42,43,44,45,46,47,48,49. GEpHIs have already been used extensively to report mitochondrial matrix pH50,51, and subcellular targeting strategies exist to localize biosensors to endoplasmic reticulum52, nucleus53, synaptic vesicles12,43, and the cytoplasmic54 and external surfaces of cellular plasma membrane55 (see Table 2 for a list of published reagents). As such tools become available they will permit vertical integration of sub-cellular pH regulation with other aspects of cellular physiology, such as Ca2+ handling and intracellular signaling, and whole organ function across a variety of vertebrate and invertebrate preparations.

The goal of this protocol is to describe quantification of intracellular pH (pHi) using a Genetically-Encoded pH-Indicator (GEpHI) and demonstrate how this method can be used to assess basolateral H+ transport in a model insect (D. melanogaster) renal structure, the Malpighian tubule (MT). MTs serve as the excretory organs of the fruit fly and are functionally similar to the mammalian nephron in several key respects1. MTs are arranged as 2 pairs of tubules (anterior and posterior) in the thorax and abdomen of the fly. The single-cell epithelial tube of each MT is composed of metabolically active principal cells with distinct apical (luminal) and basolateral (hemocoel) polarity as well as intercalated stellate cells. Anterior MTs are composed of 3 morphologically, functionally, and developmentally distinct segments, notably the initial dilated segment, transitional segment, and secretory main segment, which joins to the ureter2. At the cellular scale trans-epithelial ion transport into the lumen is accomplished by an apical plasma membrane V-ATPase3 and an alkali-metal/H+ exchanger as well as a basolateral Na+-K+-ATPase4, inward-rectifier K+ channels5, Na+-driven Cl&#8722;/HCO3&#8722; exchanger (NDAE1)6, and Na+-K+-2Cl&#8722; cotransporter (NKCC; Ncc69)7, while stellate cells mediate Cl- and water transport8,9. This complex but accessible physiologic system provides excellent opportunities for investigation of endogenous ion transport mechanisms when combined with the diverse genetic and behavioral toolsets of Drosophila.
The rationale for this protocol was to describe a genetically malleable system for studying epithelial ion transport with potential for integration from cell to behavior and export of tools to other model systems. Expression of pHerry10, a GEpHI derived from a fusion of green pH-sensitive super-ecliptic pHluorin11,12 (SEpH) and red pH-insensitive mCherry13, in MTs permits quantification of H+ transport in single MT cells through the high K+/nigericin calibration technique14. As many ion transporters move H+ equivalents, quantification of intracellular pHi serves as a functional representation of ion movement via a variety of transporters. The Drosophila MT model system also offers powerful genetic tools in tissue-specific transgene15 and RNA interference (RNAi)16 expression, which can be combined with cellular imaging and whole-organ assays17,18,19 of tubule function to create a robust toolset with vertical integration from molecules to behavior. This stands in contrast to many other protocols for assessing epithelial biology, as historically such measurements have relied on intricate and daunting micro-dissection, sophisticated ion-selective electrodes20,21, and expensive pH-sensitive dyes22 with restrictive loading requirements and poor cellular specificity in heterogeneous tissues. GEpHIs have been used to extensively measure pHi in a variety of cell types23. Early work exploited the inherent pH-sensitivity of Green Fluorescent Protein (GFP) to monitor pHi in cultured epithelial cells24 but the past two decades have seen GEpHIs used in neurons25, glia26, fungi27, and plant cells28. The combination of the potential for cellular targeting of genetic constructs through the GAL4/UAS expression system15 and the physiologic accessibility of the Drosophila MT make this an ideal preparation for investigations of pHi regulation and epithelial ion transport.
pHi regulation has been studied for decades and is vital to life. The MT preparation offers a robust model to teach physiology of pHi regulation but also perform sophisticated investigations of pHi regulation ex vivo and in vivo. This protocol describes quantification of H+ movement across the basolateral membrane of the epithelial cells of the Drosophila MT using the NH4Cl pulse acid loading technique21, but as the pH-indicator is genetically encoded, these methods and their theoretical framework can be applied to any preparation amenable to transgenesis and live-imaging.

<table><tbody><tr><td>Poly-L-Lysine (PLL) Solution</td><td>Sigma-Aldrich</td><td>P4832</td><td>Store at 4 &amp;deg;C, can be reused.</td></tr><tr><td>Nigericin Sodium Salt</td><td>Sigma-Aldrich</td><td>N7143</td><td>CAUTION: Handle with gloves. Store as aliquots of 20 mM stock solution in DMSO at 4 &amp;deg;C.</td></tr><tr><td>Adhesive Perfusion Chamber Covers, adhesive size 1 mm, chamber diameter &amp;times; thickness 9 mm &amp;times; 0.9 mm, ports diameter 1.5 mm</td><td>Sigma-Aldrich</td><td>GBL622105</td><td>Can be substituted as needed to match perfusion system.</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td>Available from multiple vendors.</td></tr><tr><td>Helping Hands Soldering Stands</td><td>Harbor Freight Tools</td><td>60501</td><td>Available from multiple vendors.</td></tr><tr><td>Open Gravity-fed Perfusion System with Valve Controller, 8 to 1 Manifold and Reserviors</td><td>Bioscience Tools</td><td>PS-8S</td><td>Any comparable perfusion system can be used.</td></tr><tr><td>Flow Regulator</td><td>Warner Instruments</td><td>64-0221</td><td>Can be substituted as needed to match perfusion system.</td></tr><tr><td>Schneider&#039;s Medium</td><td>Fisher Scientific</td><td>21720024</td><td>Store at 4 &amp;deg;C in sterile aliquots.</td></tr><tr><td>#5 Inox Steel Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Can be substituted based on experimenter comfort.</td></tr><tr><td>35 x 10 mm polystyrene Petri dish</td><td>Corning Life Sciences</td><td>Fisher Scientific 08-757-100A</td><td>Exact brand and size are unimportant.</td></tr><tr><td>75 x 25 mm Microscope Slides</td><td>Corning Life Sciences</td><td>2949-75X25</td><td>Exact brand and size can vary as long as perfusion wells are compatible.</td></tr><tr><td>Filimented Borosilicate Capillary Glass, ID 1.5 mm, OD 0.86 mm, thickness 0.32 mm</td><td>Warner Instruments</td><td>64-0796</td><td>Filiment not necessary, glass can be substituted to match perfusion tubing and perfusion wells.</td></tr><tr><td>Tygon Tubing, ID 1/16&quot;, OD 1/8&quot;, thickness 1/32&quot;</td><td>Fisher Scientific</td><td>14-171-129</td><td>Available from multiple vendors, can be substituted to match perfusion system.</td></tr><tr><td>Vacuum Silicone Grease</td><td>Sigma-Aldrich</td><td>Z273554</td><td>Available from multiple vendors.</td></tr><tr><td>Plastic Flow Control Clamp</td><td>Fisher Scientific</td><td>05-869</td><td>Available from multiple vendors, sterility not required</td></tr><tr><td>Glass rods, 5 mm diameter</td><td>delphiglass.com</td><td>9198</td><td>Exact size is personal preference, multiple vendors available</td></tr><tr><td>PAP Hydrophobic Pen</td><td>Sigma-Aldrich</td><td>Z377821</td><td>Available from multiple vendors.</td></tr><tr><td>Sealing Film</td><td>Sigma-Aldrich</td><td>P7668</td><td>Available from multiple vendors.</td></tr><tr><td>15 mL Falcon tube</td><td>BD Falcon</td><td>352096</td><td>Available from multiple vendors.</td></tr><tr><td>50 mL Falcon tube</td><td>BD Falcon</td><td>352070</td><td>Available from multiple vendors.</td></tr><tr><td>HEPES; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td><td>Sigma-Aldrich</td><td>H3375</td><td>Available from multiple vendors.</td></tr><tr><td>MES; 4-Morpholineethanesulfonic acid monohydrate</td><td>Sigma-Aldrich</td><td>69892</td><td>Available from multiple vendors.</td></tr><tr><td>TAPS; N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid</td><td>Sigma-Aldrich</td><td>T5130</td><td>Available from multiple vendors.</td></tr><tr><td>10X/0.45 Air Objective</td><td>Zeiss</td><td>000000-1063-139</td><td>Comparable objectives can be substituted. 40X objectives can be used for single cell imaging.</td></tr><tr><td>Dissecting Stereoscope</td><td>Zeiss</td><td>Discovery.V8</td><td>Any dissecting stereoscope can be used.</td></tr><tr><td>UAS-pHerry transgenic &lt;em&gt;Drosophila melagnogaster&lt;/em&gt;</td><td>Available from Romero Lab</td><td></td><td>First published: Citation 10</td></tr><tr><td>capaR-GAL4 driver line &lt;em&gt;Drosophila melagnogaster&lt;/em&gt;</td><td>Available from Romero Lab</td><td></td><td>First published: Citation 32</td></tr><tr><td>c724-GAL4 driver line &lt;em&gt;Drosophila melagnogaster&lt;/em&gt;</td><td>Available from Romero Lab</td><td></td><td>First published: Citation 2</td></tr><tr><td>Monochromatic High Sensitivity Digital Camera</td><td>Zeiss</td><td>Axiocam 506 mono</td><td>Exact brand and model can vary, can be replaced with any monochromatic high-sensitivity camera suited to live cellular imaging.</td></tr><tr><td>GFP/FITC filter set, 470/40 nm ex., 515 nm longpass em., 500 nm dichroic</td><td>Chroma</td><td>CZ909</td><td>Any GFP/FITC filer set can be substituted.</td></tr><tr><td>RFP/TRITC filter set, 546/10 nm ex., 590 nm longpass em., 565 nm dichroic</td><td>Chroma</td><td>CZ915</td><td>Any GFP/FITC filer set can be substituted.</td></tr><tr><td>Inverted Epifluoescent Microscope</td><td>Zeiss</td><td>Axio Observer Z.1</td><td>Any comparable microscope with motorized filter switching can be used. Upright microscopes can be used with open perfusion baths and water-immersion objectives.</td></tr><tr><td>Statistical Analysis Software</td><td>Microcal</td><td>Origin 6.0</td><td>Any software with comparable functionality can be substituted</td></tr><tr><td>Image Analysis Software</td><td>National Institutes of Health</td><td>ImageJ 1.50i</td><td>Any software with comparable functionality can be substituted</td></tr><tr><td>Image Acquisition Software</td><td>Zeiss</td><td>Zen 1.1.2.0</td><td>Any software with comparable functionality can be substituted</td></tr><tr><td>Single-edged Carbon Steel Razor Blade</td><td>Electron Microscopy Sciences</td><td>71960</td><td>Available from multiple vendors.</td></tr><tr><td>Microscopy Slide Folder</td><td>Fisher Scientific</td><td>16-04</td><td>Available from multiple vendors.</td></tr><tr><td>Bunsen Burner</td><td>Fisher Scientific</td><td>50-110-1231</td><td>Available from multiple vendors.</td></tr><tr><td>Polystrene Drosophila Rearing Vials with Flugs</td><td>Genesee Scientific</td><td>32-109BF</td><td>Comparable items can be substituted.</td></tr><tr><td>2.5 L Laboratory Ice Bucket</td><td>Fisher Scientific</td><td>07-210-129</td><td>Available from multiple vendors.</td></tr><tr><td>NMDG; N-Methyl-D-glucamine</td><td>Sigma-Aldrich</td><td>M2004</td><td>Available from multiple vendors.</td></tr><tr><td>200 uL barrier pipette tips</td><td>MidSci</td><td>AV200</td><td>Available from multiple vendors.</td></tr><tr><td>200 &amp;#956;L variable volume pipette</td><td>Gilson Incorporated</td><td>PIPETMAN P200</td><td>Available from multiple vendors.</td></tr></tbody></table>

optical quantification of intracellular ph in drosophila melanogaster malpighian tubule epithelia with a fluorescent genetically-encoded ph indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Healthy tissues and proper identification of anterior MTs are vital to the success of this protocol. During dissection, care should be taken to not directly touch the MTs and to only handle them by the ureter as gripping the MTs directly will lead to breakage (Figure 4A&#160;-&#160;B). When MTs are swept flat onto the slide, the tubules must be touched as little as possible and excess motion avoided as this will damage the single-cell epithelial layer (Figure 4C). Properly dissected anterior MTs will show even distribution of both red and green fluorescence through the cytosol of epithelial cells and morphologically distinct tubule segments. Tubules damaged by improper perfusion or mishandling will display aggregation of red fluorescence with no paired green aggregates, and misidentified posterior MTs will show uniform morphology from the proximal blind end to the distal ureter (Figure 4D).
Proper function of pHerry must be confirmed though physiologic assessment as well as morphology. The most expedient method of confirming proper pH-sensing is to apply an NH4Cl pulse. Under these conditions the green SEpH signal should report the expected pH changes (a rise in pHi during the pulse as NH3 enters the cell, a gradual decline during the pulse as NH4+ enters through K+ transporters and channels, and a rapid acidification and gradual recovery upon NH4Cl withdrawal21, while the red mCherry signal should remain constant (Figure 5A&#160;-&#160;B). The magnitude of changes in the SEpH signal will vary with protocol and cell type, but the mCherry signal should be stable in all cases. Changes in the mCherry signal during individual experiments indicate movement artifacts or progressive generation of sensor aggregates due to cell damage. The latter will prevent quantification of pHi and must be avoided. Upon completing the NH4Cl pulse it is important to perform a 2-point calibration (Calibration iPBS, pH 7.4 and 9.0, 10 &#181;M nigericin) to confirm a resting pHi near 7.4 and ensure that the current imaging parameters do not lead to saturation of fluorescence detection when fluorescence is maximized at pH 9.0 (Figure 5C). If resting pH is significantly lower than 7.4, the protocol should be repeated with healthy MTs; and if saturation occurs at pH 9.0, the protocol should be repeated with lower light intensity or exposure time. Once adequate imaging parameters are determined, they should not be changed between experiments or calibrations if the absolute fluorescence ratios are to be used. While the pseudo-ratiometric nature of pHerry can provide a method of movement correction in preparations prone to movement or changes in cell diameter, the absolute quantification of pHi requires full systematic correlation of the pHerry SEpH/mCherry ratio to pHi through the nigericin/high K+ technique. The calibration of pHerry in healthy preparations should produce consistent calibration curves with an apparent pKa of 7.1-7.4 depending on the cell type and calibration conditions (Figure 5D). For preparations in which mCherry aggregation is unavoidable, normalization of ratio values such that a fluorescence ratio of 1.0 corresponds to pHi 7.0 should yield similar results (Figure 5E). If point calibrations and normalized curves are used, imaging parameters can be optimized for each preparation.
Calibrated pHi traces can be used to compare pH regulatory mechanism between cell types. The GAL4/UAS expression system in Drosophila can be used to express pHerry in principal cells and stellate cells of the anterior MT (Figure 6A). pHi regulation can be assessed by acid-loading cells with NH4Cl pulses and quantifying the rate of pHi recovery. This can be accomplished by fitting exponential functions to the recovery phase in different experimental conditions to extract the decay constant (&#964;) as cells with more rapid H+ efflux will display a more rapid recovery (and thus lower values of &#964;). Based on this analysis, stellate cells of the MT appear to have more robust acid extrusion than principal cells (Figure 6B). This analysis will hold as long as the resting pHi, extent of acid loading, and buffering capacity are similar between experimental groups. However, when these conditions are not met, it is necessary to account for the observation that intrinsic buffering capacity of the cytosol (&#946;i) of many cells is itself pH-dependent35,37,38 and thus the rate of pHi change at different pHi may not be directly comparable. In such cases, exponential curves fit to the acid extrusion phase following an NH4Cl pulse and previously determined estimates of intrinsic buffering capacity (&#946;i) can be used to plot the acid extrusion rate (JH+) as a function of pHi and determine compensated rate of acid extrusion (Equations 3 &#38; 4). Once differences in acid loading and resting pHi are accounted for, it is evident acid extrusion in principal cells of the transitional segment exceed that of stellate cells (Figure 6C). While compelling, this analysis does not address that measured pHi is a function of volume while acid extrusion across the plasma membrane is a function of membrane surface area. Dividing JH+ by the surface area to volume ratio of the cell of interest will yield values in moles of acid equivalents per unit surface area per unit time (Equation 5), thus allowing correction for differences in cell size and morphology. Approximately two principal cells comprise the circumference of the MT in the transitional segments and thus single cells can be modelled as half of a tube (inner diameter 24 &#181;m; outer diameter 48 &#181;m; height 50 &#181;m). Stellate cells are smaller and tend to be bar-shaped in the transitional segment of the MT2. Exact quantification of surface area and volume is difficult but even conservative approximations of transitional stellate cell shape (cylinder with a height of 50 &#181;m and a diameter of 10 &#181;m) indicate a surface area to volume ratio at least 2x that of principal cells. Taking this into account reveals that the stellate cell acid flux is significantly below that of transitional principal cells, and in fact approaches that of initial segment principal cells (Figure 6D).

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55698/55698fig4.jpg" /> 
Figure 4: Dissection of Adult Drosophila Anterior Malpighian Tubules. 
A. Schematic representation of anterior MT removal with 2 pairs of fine forceps in chilled Schneider&#39;s medium. &#34;A. Tubule&#34; = Anterior MT. &#34;P. Tubule&#34; = posterior MT. B. Process of retrieving and mounting extracted MTs using thin glass rods. C. Process of adhering the full length of extracted MTs to a slide for imaging and physiologic assessment. D. Representative widefield images of the SEpH (470/510 nm ex/em) and mCherry (556/630 nm ex/em) components of UAS-pHerry driven by capaR-GAL4 depicting healthy anterior MTs, anterior MTs damaged by insufficient perfusion, and misidentified posterior MTs. Note that healthy anterior MTs display a clear dilated blind initial segment, a constricted transitional segment with relatively increased expression of UAS-pHerry when driven by capaR-GAL4, and a distal main segment. Damaged MTs display noticeable aggregates of mCherry fluorescence with no corresponding SEpH fluorescence. Posterior MTs are uniform in diameter with no morphologically distinct segments. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55698/55698fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55698/55698fig5.jpg" /> 
Figure 5: Validation and Calibration of pHerry in Malpighian Tubules. 
A. Representative widefield images of the SEpH (470/510 nm ex/em) and mCherry (556/630 nm ex/em) components of UAS-pHerry driven by capaR-GAL4 depicting healthy anterior MTs. &#34;ROI&#34; marks signal region of interest. &#34;BG&#34; marks background region of interest which was subsequently subtracted from the signal ROI in the same channel. Scale bar = 50 &#181;m. B. Relative fluorescence changes in SEpH and mCherry signals of pHerry in response to a 20 s 40 mM NH4Cl pulse. Note that the mCherry signal is stable while the SEpH signal displays a characteristic increase during the pulse (indicative of alkalization, i.e.&#160;increased pHi) and a sharp decrease upon washout (indicative of acidification, i.e.&#160;decreased pHi). C. Fluorescence ratio of pHerry (SEpH/mCherry) calculated from data in B with additional data after 30 min incubation in Calibration iPBS (10 &#181;M nigericin, 130 mM K+, pH 7.4 and 9.0). D. Calibration curve constructed from absolute pHerry ratio (SEpH / mCherry) as a function of imposed pHi during exposure to Calibration iPBS buffered to one of eight pH values. Gray circles are individual values form 8 preparations. Black squares and bars are mean &#177;SD. Curve is Boltzmann fit. E. Same data as in D normalized such that fluorescence ratio at pH 7.0 is 1.0. Curve is modified sigmoidal curve fit (see step 7.4, Equation 1). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55698/55698fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55698/55698fig6.jpg" /> 
Figure 6: Quantification of Acid Extrusion in Malpighian Tubule Epithelia. 
A. Widefield image of SEpH fluorescence (from pHerry) in principal cells of the anterior MT (left, driven by capaR-GAL4 driver) and stellate cells of the anterior MT (right, driven by c724-GAL4). Note that stellate cells are bar-shaped in the initial segment, variable in the transitional segment, and display distinct cellular projections in the main segment. Scale bar = 100 &#181;m. B. Calibrated pHi changes in response to a 20 s 40 mM NH4Cl pulse in the regions of interest denoted in A (principal cells of the transitional segment, principal cells of the initial segment, and stellate cells of the transitional segment). Dashed curves denote single exponential fits applied to the acid recovery phase following NH4Cl withdrawal from which stated decay constant (&#964;) values are derived. C. Acid extrusion rate (JH+) plotted as a function pHi derived from the exponential fits seen in B. See step 8.3.1 for JH+ calculation (Equation 3). Dashed curves are exponential fits applied to each data plot within the area of overlapping pHi denoted by the gray box. D. Acid flux plotted as a function pHi derived from the exponential fits seen in B and Equation 5. Dashed curves are exponential fits applied to each data plot within the area of overlapping pHi denoted by the gray box. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55698/55698fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>iPBS</td>
			<td>NH4Cl Pulse iPBS</td>
			<td>Calibration iPBS</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>121.5</td>
			<td>81.5</td>
			<td>0</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>0</td>
			<td>40</td>
			<td>0</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>20</td>
			<td>20</td>
			<td>130</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td>HEPES; 8.6</td>
			<td>HEPES; 8.6</td>
			<td>MES, HEPES, or TAPS; 8.6</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>10.24</td>
			<td>10.24</td>
			<td>0</td>
		</tr>
		<tr>
			<td>NaH2PO4 (1H2O)</td>
			<td>4.5</td>
			<td>4.5</td>
			<td>0</td>
		</tr>
		<tr>
			<td>NMDG</td>
			<td>0</td>
			<td>0</td>
			<td>30.5</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>6.8</td>
			<td>6.8</td>
			<td>Varies</td>
		</tr>
		<tr>
			<td>Osmolarity</td>
			<td>350 &#177;5</td>
			<td>350 &#177;5</td>
			<td>350 &#177;5</td>
		</tr>
	</tbody>
</table>
Table 1: Experimental Fly Solutions. 
iPBS solutions are prepared at room temperature and pH is set by titration with HCl and NaOH. Calibration solution is titrated with HCl and NMDG. Buffer of calibration solution is varied based on desired pH [2-(N-morpholino)ethanesulfonic acid (MES) for pH = 4.0 - 6.0; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for pH = 6.5 - 7.5; N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS) for pH = 8.0 - 9.0]. All values in mM, except pH (unitless) and osmolality (mmol/kg). Stock nigericin in DMSO is added to calibration solutions to a final concentration of 10 &#181;M just before use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GEpHI</td>
			<td>Excitation (nm)</td>
			<td>Emission (nm)</td>
			<td>pKa</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>Superecliptic pHluorin (SEpH)11</td>
			<td>395, 488</td>
			<td>530</td>
			<td>7.2</td>
			<td>Large linear range, large fold (50x) increase in pH-sensitive fluorescence across linear range</td>
		</tr>
		<tr>
			<td>Pt GFP42</td>
			<td>390, 475</td>
			<td>540</td>
			<td>7.3</td>
			<td>Validated for use in plant cells</td>
		</tr>
		<tr>
			<td>Superecliptic pHluorin - mCherry fusion31</td>
			<td>488, 556</td>
			<td>530, 620</td>
			<td>7.2</td>
			<td>Produces unpaired mCherry aggregates in some cells</td>
		</tr>
		<tr>
			<td>ClopHensor40</td>
			<td>488, 545</td>
			<td>525, 590</td>
			<td>6.8</td>
			<td>pH and Cl- sensor. Updated ClopHensorN30 varient shows less aggregation in neurons</td>
		</tr>
		<tr>
			<td>pHerry10</td>
			<td>488, 556</td>
			<td>530, 620</td>
			<td>7.2</td>
			<td>Updated SEpH-mCherry fusion with linker from ClopHensor</td>
		</tr>
		<tr>
			<td>mNectarine44</td>
			<td>558</td>
			<td>578</td>
			<td>6.9</td>
			<td>Correction for photobleaching is often necessary</td>
		</tr>
		<tr>
			<td>pHluorin245</td>
			<td>395, 475</td>
			<td>509</td>
			<td>6.9</td>
			<td>Variant of Ratiometric pHluorin12</td>
		</tr>
		<tr>
			<td>pHred47</td>
			<td>440, 585</td>
			<td>610</td>
			<td>7.8</td>
			<td>Updated varient of long-Stokes shift mKeima49, compatible with FLIM NIR 2-photon imaging</td>
		</tr>
		<tr>
			<td>pHuji43</td>
			<td>566</td>
			<td>598</td>
			<td>7.7</td>
			<td>Varient of mApple; lower than expected pH sensitivity in some cells</td>
		</tr>
		<tr>
			<td>pHtomato46</td>
			<td>550</td>
			<td>580</td>
			<td>7.8</td>
			<td>Validated to track vesicular endocytosis, poor cytocolic pH sensitivity</td>
		</tr>
		<tr>
			<td>pHoran443</td>
			<td>547</td>
			<td>561</td>
			<td>7.5</td>
			<td>Enhanced pH-sensitive orange fluorescent protein</td>
		</tr>
		<tr>
			<td>SypHer-248</td>
			<td>427, 504</td>
			<td>525</td>
			<td>8.1</td>
			<td>Brighter variant of ratiometric SypHer51, originally for mitochondrial measurements</td>
		</tr>
	</tbody>
</table>
Table 2: List of Published Cytosolic GEpHIs 
Excitation maxima, emission maxima, and apparent pKa values are approximate and can vary depending on expression system, imaging technique, and calibration method. FLIM = fluorescence lifetime imaging microscopy. NIR = near infrared.

Watch this Scientific Journal Video about Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator at JoVE.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

optical-quantification-intracellular-ph-drosophila-melanogaster

Nanyang Technological University

Research

JoVE Journal

Biology

10.1K Views.  Mayo Clinic College of Medicine. Cellulaire ion vervoer kan vaak worden geëvalueerd door het toezicht van de intracellulaire pH (pHik). Genetisch een Encoded pH-indicatoren (GEpHIs) optische kwantificering van intracellulaire pH in onbeschadigde cellen. Dit protocol gegevens de kwantificering van de intracellulaire pH via cellulaire ex vivo wonen-imaging van Malpighian tu...

Video: Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Developmental Biology

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Neuroscience

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Development of pHluorin2-Based Sensor for Glycosomal pH in Trypanosoma brucei

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

Author Spotlight: Advancements in Glycosomal pH Monitoring in Trypanosoma brucei Using pHluorin2 Biosensor

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

Immunology and Infection

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ

Source: Rossano, A. J. and Romero, M. F. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55698/" target="_blank">Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator</a>. J. Vis. Exp. (2017).
This article describes the dissection of the anterior Malpighian tubules (MT) from a female Drosophila fly and an example protocol from Rossano et al. (2017) in which the MTs are prepared for live imaging in a perfusion chamber.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video