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 ...

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 ...

optical-quantification-intracellular-ph-drosophila-melanogaster

Research

JoVE Journal

Biology

10.1K Views.  Mayo Clinic College of Medicine. Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs) 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 mela...

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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

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 ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

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 ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...