Name: optical quantification of intracellular ph in drosophila melanogaster malpighian tubule epithelia with a fluorescent genetically-encoded ph indicator
Uploaded: 2017-08-11T12:30:00
Description: 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.

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.

All steps in this protocol comply with the Mayo Clinic (Rochester, MN) animal use guidelines.
1. Fly Husbandry
<ol>
	<li>Raise flies and set crosses according to standard husbandry29. 
	NOTE: Fluorescent reporter expression by the GAL4/UAS system is proportional to temperature and thus rearing temperature can be adjusted to alter expression level. While high expression levels often lead to a better signal-to-noise ratio this condition is also associated with incr...

     

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 ...

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 Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4832</td>
			<td>Store at 4 &#176;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 &#176;C.</td>
		</tr>
		<tr>
			<td>Adhesive Perfusion Chamber Covers, adhesive size 1 mm, chamber diameter &#215; thickness 9 mm &#215; 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&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>21720024</td>
			<td>Store at 4 &#176;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 mm 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 inch, OD 1/8 inch, thickness 1/32 inch</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 Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 10</td>
		</tr>
		<tr>
			<td>capaR-GAL4 driver line Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 32</td>
		</tr>
		<tr>
			<td>c724-GAL4 driver line Drosophila melagnogaster</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 uL 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 si...

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 ...

The overall goal of this imaging protocol is to describe the quantification of intracellular pH, using a genetically-encoded pH indicator and demonstrate how this method can be used to assess basolateral proton transport in an insect renal structure model, the malpighian tubule. This method can help answer key questions in the cellular transport field such as, how specific transporter proteins contribute to epithelial proton movement in intracellular pH regulation. The main advantage of this technique is that cellular transport can be quickly and easily assessed across multiple functionally distinct zones of intact epithelial.Though this method can provide insight into pH regulation and insect epithelial, it can also be applied to other systems such as, the mammalian nephron and epithelial cells in culture. To begin this procedure, position two sets of soldering helping hands clamps on the imaging microscope stage, with one clamp on each side. Next, insert the respective bent glass capillaries and the inflow line, and the vacuum connected, outflow line.Then, mount then in the helping hands and align them with the imaging stage of the microscope. Spread vacuum grease onto the ceiling tape and press the adhesive profusion well divider onto the tape to coat the bottom with grease. Peel off the adhesive perfusion well divider and place it grease side down, on top of a poly l lysine coated slide.Following that, remove the perfusion well divider to leave individual specific wells traced in hydrophobic grease. After that, place 200 microliters of room temperature IPBS or insect phosphate buffer saline in the greased and circle well on the poly l lysine coated slide. Then, place the slide under the steroscope, pour ice cold Schneider's medium into the dissecting dish and transfer a single anesthetized female fly to it.Hold the fly by the thorax with a set of forceps and use the other to gently grip the posterior abdomen. Pull open the posterior end of the fly in short deliberate motions. Once the hind guard is visible, grip the distal end and free the gut and antes from the underlying tracheales, by pulling the hind guard away from the body through repetitive brief tugs.Then, pinch off the anterior antes of the ureter with fine forceps, once the second set of antes is free from the abdomen. Pick up the free anterior antes with the pole glass rod, by sliding the rod under the ureter, such that the tubules fall to either side. Following that, lift the antes straight up, out of the solution, after that, turn the glass rod such that the antes and ureter are adhered to the underside of the rod.Lower the ureter straight down onto the slide, then, affix the ureter and seal the distal ends of the antes by further pressing the ureter onto the glass slide. Use the fine end of the glass rod to gently sweep each tubule across the slide surface. Graze the rod against the slide to avoid crushing the tubule and slide the rod over the top of the tubule, moving from distal to proximal, to attach the full length of each tubule to the surface of the poly l lysine coated slide.Success of this technique depends entirely on accurate identification and careful handling of the anterior malpighian tubules. Damaged tubules will produce inconsistent results. Next, place the adhesive profusion well divider back on the slide to form a small fluid filled well over the mounted tubule.After that, place the specimen on the microscope stage. Position the inflow and outflow capillaries over the inlet and outlet opening of the profusion well, respectively. In this procedure, turn on the microscope, light source and imaging system, then, open the imaging software.Look through the eyepieces and manually adjust the focus until the lumen of the antes is clearly visible under the transmitted light. Then, click the acquisition tab and select, 2x2 in the dimming pull down menu, in the acquisition load section. Insert a 5%neutral density filter into the light path, to reduce illumination light and minimize photo-bleaching.Click the GFP channel in the channels menu, then click live to observe the fluorescent signal via the camera. After that, adjust the time slider to set the exposure time, such that the brightest pixel values in the intensity histogram are approximately 40%of the maximum value. Then click stop, to stop the illumination.Repeat the procedures in the RFP channel and confirm the presence of the dilated initial segment of the anterior ante and the absence of cytosolid m cherry aggregates, which indicates tissue damage or over-expression. Afterwards, enable a timelapse imaging protocol by clicking the time series checkbox. Adjust the duration in the pull down menu, in the time series section to 10 minutes, and the interval slider to zero, to set the total capture time with the maximum image acquisition.Then, check both the GFP and RFP boxes in the channel section. Open the IPBS line of the profusion system by activating the appropriate valve controller and start the imaging protocol by clicking, start experiment. After one minute, switch to ammonium chloride post solution for 20 seconds, by opening the appropriate valve and closing the IPBS line.Then, return to IPBS by closing the ammonium chloride line and reopening the IPBS valve. To perform a two point calibration, remove the well divider by peeling it away from the underlying slide and remove the profusion capillaries in clamps from the imaging well. Following that, apply 200 microliters of calibration IPBS buffer to pH 7.4.Then, remove the solution from the imaging well and replace it with another 200 microliters of calibration solution. Repeat this process four times to ensure complete solution exchange. Next, incubate the preparation and calibration solution for 30 minutes before imaging.Repeat the imaging protocol using the same parameters determined previously, with the modification of only one minute of image capture. Then add 200 microliters of calibration IPBS buffer to pH nine. Remove the solution from the imaging well and replace it with another 200 microliters of calibration solution.Subsequently, incubate the preparation in the second calibration solution for 10 minutes before imaging and repeat the imaging protocol. After that, review the captured image stacked in the image analysis software to confirm that no pixels in either channel are saturated, by clicking MeanROI and that no values reported in the intensity histogram, reached the maximum detectable value, while scrolling through the image stack with the frame slider. Next, analyze the image stack by plotting fluorescent intensity and fluorescence ratio is a function of time, here, we see the expected fluorescent response to the ammonium chloride pulse in the pH sensitive and pH insensitive channels of the indicator.The ratio of these signals reveal the intracellular pH transience. To perform this analysis, first click meanROI and select the free form tool. Hold the left click on the mouse, to trace a 50 micrometer MT and right click to finish drawing the ROI.Then, repeat that in an area adjacent to the MT, to define background ROI. Next, click mean intensity under measurements, create a table of intensity values by clicking export, data table, create. Click the configuration clock wheel icon and deselect all parameters, except time and mean intensity.Right click the tab for the newly created data table, select save as, and export the data as a csv file. Shown here, is a wide field image, a super-elliptic fluorin fluorescence in principle cells of the anterior ante and stellate cells of the anterior ante. 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.This graph shows the calibrated intercellular pH changes in response to 20 seconds, 40 millimolar ammonium chloride pulse in regions of interest. Dashed curves denote single exponential fits applied to the acid recovery phase, following ammonium chloride withdrawal. From which, the decayed constant values are derived.This graph shows the acid extrusion rate plotted as a function of intracellular pH and this graph shows the acid flux plotted as a function of intracellular pH, derived from the exponential fits from the previous figure. Once mastered, the malpighian tubule extraction can be done within 10 minutes, if it is performed properly. While attempting this procedure, it's important to remember the success depends entirely on the quality of dissection and accurate calibration of the pH indicator.This preparation can be combined with other genetically encoded biosensors such as, fluorescent calcium and halide indicators to study solute movement and intracellular signaling in epithelial cells. After watching this video, you should have a good understanding of how to prepare healthy drosophila malpighian tubules, image a genetically encoded pH indicator and quantify proton movement in epithelial cells. Don't forget that working with nitroizene can be hazardous and damage preparations and thus, precautions should be taken while performing this procedure, to ensure that it does not contaminate any equipment that is to be reused.

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