Name: optical quantification of intracellular ph in drosophila melanogaster malpighian tubule epithelia with a fluorescent genetically-encoded ph indicator
Uploaded: 2017-08-11T12:30:00.000+00:00
Duration: 11 min 54 s
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.

这项工作得到了 NIH DK092408 和 DK100227 对生产商的支持. AJR 得到了 T32-DK007013 的支持。作者希望感谢 Dr. 科尔尼 CapaR-GAL4 和 c724-GAL4 的果蝇股票。我们还感谢雅各布 b. 安德森协助维持实验飞行十字架。

作者没有什么可透露的。

在果蝇mts 中, pH 值i的量化成功与否完全取决于提取的 mts 的运行状况以及安装和剥离的质量 (图 A - C)。因此, 仔细处理组织的描述是势在必行的。新涂在 PLL 上的幻灯片实质上有助于 MT 的安装, 因为它们比以前暴露在溶液中的幻灯片更容易粘合。小心安装也有助于识别不同的 MT 段 (图 D)。健康 MTs 分别通过减少 mCherry 聚合和产生更?...

本议定书的目的是用基因编码的 ph 指示剂 (GEpHI) 描述细胞内 ph 值 (ph 值i) 的量化, 并演示这种方法如何用于评估模型昆虫中的侧 H+传输 (D。腹氏管 (MT) 的肾脏结构。MTs 作为果蝇的排泄器官, 在几个关键方面与哺乳动物肾单位的功能相似,1。在苍蝇的胸腔和腹部, MTs 排列为2对小管 (前后)。每个 MT 的单细胞上皮管由新陈代谢活性主细胞组成, 具有明显的顶端 (腔) 和侧 (腔) 极性, 以及夹层星状细胞。前 MTs 由3形态、功能和发育的不同部分组成, 特别是最初的扩张段、移行段和分泌主段, 它们连接到输尿管的2。在细胞尺度的跨上皮离子传输到流明是由一个顶端的等离子膜 V atp 酶3和一个碱金属/H+换热器以及一个侧 Na+-K-atp 酶4,内整流 K+通道5, Na+驱动 Cl−/小贩3−交换器 (NDAE1)6和 Na+-K+2Cl− cotransporter (NKCC;Ncc69)7, 而星状细胞介导 Cl-和水传输8,9。这一复杂但容易接近的生理系统提供了极好的机会, 研究内源性离子转运机制时, 结合了不同的遗传和行为工具的果蝇。
这个协议的基本原理是描述一个遗传可塑性的系统研究上皮离子运输有潜在的集成从细胞到行为和出口的工具到其他模型系统。pHerry10的表达式, GEpHI 从绿色 ph 值敏感的 super-ecliptic pHluorin11、12 (SEpH) 和红色 ph 不敏感的 mCherry13的融合中派生而来, 在 MTs 中允许量化 H+传输单 MT 细胞通过高 K+/尼日利亚校准技术14。由于许多离子转运体移动了 H+当量, 细胞内 pH 值i的量化是通过各种转运体的离子运动的功能表征。果蝇MT 模型系统还提供了功能强大的基因工具在组织特异转基因15和 rna 干涉 (干扰)16表达式, 可以结合细胞成像和全身检测17,18,19的小管函数创建一个具有从分子到行为的垂直集成的健壮工具集。这与许多其他用于评估上皮生物学的协议相对照, 因为历史上这种测量依赖于复杂而艰巨的微解剖, 尖端的离子选择性电极20,21,和昂贵的 pH 敏感染料22 , 具有限制性的负载要求和不均匀组织中的细胞特异性差。GEpHIs 已被用来广泛测量 pHi在各种细胞类型23。早期工作利用绿色荧光蛋白 (GFP) 的固有 ph 敏感度来监测培养的上皮细胞中的 ph 值i 24但过去20年中, 在神经元中使用过 GEpHIs25、胶质26、真菌27和植物细胞28。通过 GAL4/UAS 表达系统15和果蝇MT 的生理可访问性, 将基因构造的细胞靶向性结合起来, 使之成为研究 pH 值的理想准备.调节和上皮离子转运。
pH 值i规则已进行了几十年的研究, 对生命至关重要。MT 制剂提供了一个健壮的模型来教授生理学的 ph 值i调节, 但也执行复杂的研究 ph 值i调节ex 体内和体内。本协议描述了使用 NH4Cl 脉冲酸加载技术21, 在果蝇MT 的上皮细胞的侧膜上进行 H+运动的量化, 但由于 pH 值是基因编码, 这些方法和他们的理论框架可以适用于任何准备转基因和活成像。

<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

上皮离子转运对系统离子稳态和基本细胞电化学梯度的维持至关重要。细胞内 ph 值 (ph 值i) 受许多离子转运体的影响, 因此监测 ph 值i是评估转运体活动的有用工具。现代基因编码的 ph 指标 (GEpHIs) 提供了在细胞和亚细胞尺度上的完整单元的 ph 值i的光学定量。本议定书描述 real-time 定量的细胞 pHi调节在氏管 (MTs) 的果蝇通过ex 活体成像 pHerry, pseudo-ratiometric GEpHI 与 pK非常适合跟踪胞中的 pH 值变化。提取的成人蝇 MTs 是由形态和功能分明的单细胞层上皮的部分组成, 并可作为一个容易接近的和基因驯服的研究上皮运输模型。GEpHIs 提供了一些优于传统 pH 敏感荧光染料和离子选择电极的优势。GEpHIs 可以标记不同的细胞数量提供适当的启动子元素可用。此标记在ex 体内、体内和原位准备工作中特别有用, 它们本质上是异构的。GEpHIs 也允许定量的 pH 值i在完整的组织, 而无需重复染料治疗或组织外化。当前 GEpHIs 的主要缺点是在组织损伤和构造表达的反应中, 聚集在胞浆包裹体中的倾向。这些缺点, 它们的解决方案, 和 GEpHIs 的内在优势, 通过评估侧质子 (H) 在功能分明的主要和星状细胞中提取的飞行 MTs 的传输来证明.所描述的技术和分析可以很容易地适应各种各样的脊椎动物和无脊椎动物的准备工作, 而且化验的复杂程度可从教学实验室扩展到通过特定转运体对离子通量的复杂测定。

健康的组织和正确的识别前 MTs 是至关重要的成功的协议。在解剖过程中, 应注意不要直接接触 mts, 而只能由输尿管来处理, 因为握住 mts 直接会导致破损 (图 4A- B)。当 MTs 被平扫到滑梯上时, 小管必须尽可能少地接触, 避免多余的运动, 因为这会损害单细胞上皮层 (图 4C)。正确解剖前 MTs, 通过胞上皮细胞?...

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

细胞离子传输通常可以通过监测细胞内 ph 值(ph 值i)来评估。基因编码的 ph 值指标(GEpHIs)提供了细胞内 ph 值的光学定量。本议定书详细介绍了细胞内 ph 值的量化, 通过蜂窝状的前体活成像的氏管的果蝇与 pHerry, pseudo-ratiometric 基因编码的 pH 指标。

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

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

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

10.1K 次观看.  Mayo Clinic College of Medicine. 细胞离子传输通常可以通过监测细胞内 ph 值(ph 值i)来评估。基因编码的 ph 值指标(GEpHIs)提供了细胞内 ph 值的光学定量。本议定书详细介绍了细胞内 ph 值的量化, 通过蜂窝状的前体活成像的氏管的果蝇与 pHerry, pseudo-ratiometric 基因编码的 pH 指标。

视频: 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.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

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

科研

发育生物学

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.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

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

神经科学

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.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

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).

液泡和胞质pH值的测量<em&gt;在体内</em在酵母细胞悬浮液

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

生物学

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.

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

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

基于 pHluorin2 的布氏锥虫糖体 pH 传感器的开发

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.

作者聚焦：使用 pHluorin2 生物传感器监测 布氏锥 虫糖体 pH 值的进展

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.

测量布氏活锥虫的动态糖体 pH 值变化

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

免疫与感染

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.

使用拉姆齐试验来测量液体分泌和离子流率在<em&gt;果蝇</em&gt;马氏管

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.

监测成人 果蝇 肠道的肠道酸化

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

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

摘要

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此视频中的章节

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

液泡和胞质pH值的测量

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化

使用拉姆齐试验来测量液体分泌和离子流率在

监测成人果蝇肠道的肠道酸化

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

摘要

探索更多视频

此视频中的章节

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

液泡和胞质pH值的测量

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化

使用拉姆齐试验来测量液体分泌和离子流率在

监测成人 果蝇 肠道的肠道酸化

监测成人果蝇肠道的肠道酸化