قسم الدفاع W81XWH - 05 - 1-0005 ، W81XWH - 06 - 1-0495 ، W81XWH - 08-1 0029 وW81XWH - 08-1 - 0356

1. إعداد GST الانصهار بروتين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مصنوعة من مخزونات الجلسرين JM109 البكتيريا المختصة التي تحتوي على أول محطة N - 90 الأحماض الأمينية في المجال rhotekin ملزمة رو (RBD) subcloned في ناقلات pGEX3x BamH1/EcoR1. لا بد منه لضمان كفاءة عالية يتم تنفيذ الخطوات التالية للحصول على كل تجربة. في تجربتنا ، التي أعدت حديثا GST - RBD هو المفتاح لمقايسة GTPase التنشيط قوية ودقيقة. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر (تقريبية ، لا الذوبان) من الأسهم الجلسرين إلى 50 مل أمبير LB وتنمو 37 درجة مئوية خلال الليل مع اهتزاز. </li><li style=";text-align:right;direction:rtl"> تمييع 01:10 LB في 500 مل أمبير وتنمو لمدة 1 ساعة. </li><li style=";text-align:right;direction:rtl"> حمل GST إنتاج البروتين بنسبة 0.1 ملي IPTG لمدة 2 ساعة. </li><li style=";text-align:right;direction:rtl"> توزع بالتساوي في زجاجات الطرد المركزي وأجهزة الطرد المركزي ، Sorval الدوار في 5000 دورة في الدقيقة GSA3 4 درجات مئوية لمدة 20 دقيقة. </li><li style=";text-align:right;direction:rtl"> Resuspend بيليه في زجاجات الطرد المركزي ، و 10 مل من تحلل العازلة *. * الاحتياطي تحلل الجرثومي : السكروز 20 ٪ ، 10 ٪ الجلسرين ، و 50 ملي تريس الرقم الهيدروجيني 8.0 ، 0.2 ملم نا 2 S 2 O 5 ، 2 مم MgCl 2 ، 2 DTT ملم ، إضافة PMSF الطازجة ، بينزاميد ، وأبروتينين leupeptin. </li><li style=";text-align:right;direction:rtl"> يصوتن 2-3 دقيقة (وهذا يعتمد على نوع sonicator. نستخدم سونيك فيشر Dismembranator موديل 500 علامة وضعت في 6 و 50 ٪ دورة). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي ، Sorvall SS34 ، 20 دقيقة 10000 دورة في الدقيقة في 4 درجات مئوية. عند هذه النقطة يمكن أن يتخذ من قسامة الصغيرة ويديرها SDS - PAGE. يمكن أن يكون coomassie الجل الملون والفرقة واضحة وينبغي على 46 كيلو دالتون. </li><li style=";text-align:right;direction:rtl"> طاف إزالة وإضافة 1 مل من 50 ٪ glutatione - 4B Sepharose الطين. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 30 دقيقة عند 4 درجات مئوية مع التناوب. تغسل 3 مرات مع تحلل العازلة. </li><li style=";text-align:right;direction:rtl"> Resuspend حبات الطين GST-RBD/glutatione-Sepharose إلى 50 ٪ (حوالي 1 مل) مع GST الأسماك العازلة **. ** GST السمك الاحتياطي : الجلسرين 10 ٪ و 50 ملي تريس الرقم الهيدروجيني 7،4 ، و 100 ملي مول كلوريد الصوديوم ، و 1 ٪ NP - 4 ، 2 مم MgCl 2 ، إضافة PMSF الطازجة ، بينزاميد ، وأبروتينين leupeptin </li></ol> 2. GST الانصهار هدم مقايسة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام 50-10 × 10 6 خلية / مقايسة </li><li style=";text-align:right;direction:rtl"> غسل خلايا مرة واحدة في برنامج تلفزيوني الجليد الباردة ، والحفاظ على لوحات على الجليد (نستخدم صحن الزجاج المسطح مملوءة الجليد) وليز مع 0.5 العازلة GST الأسماك مل. </li><li style=";text-align:right;direction:rtl"> احتضان 5 دقائق على الجليد ، وخلايا الحصاد باستخدام المزيل شرطي المطاط أو شقة ، ونقل إلى microfuge الأنابيب وأجهزة الطرد المركزي 20000 دورة في الدقيقة 5 دقائق على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> طاف لنقل أنبوب microfuge الطازجة. تأخذ 50 ميكرولتر لتحديد رو الكلي (أي الناتج المحلي الإجمالي GTP + ملزمة). </li><li style=";text-align:right;direction:rtl"> إضافة 0.5 مل GST-RBD/glutatione-Sepharose الخرز واحتضان عند 4 درجة مئوية ، بين عشية وضحاها مع التناوب. </li><li style=";text-align:right;direction:rtl"> يغسل مع 6X GST الأسماك العازلة. </li><li style=";text-align:right;direction:rtl"> Resuspend الخرز في 40 ميكرولتر العازلة عينة - 2X Laemelli عند هذه النقطة يجب أن يتم تحديد تركيز البروتين الكلي قسامة رو المتخذة في 2.4 و 30 ميكروغرام أعدت ليتم تشغيلها على هلام مع العينة التنشيط المقابلة. </li><li style=";text-align:right;direction:rtl"> عينات يغلي عند 90 درجة مئوية لمدة 5 دقائق ، طرد المركزي لفترة وجيزة ، واتخاذ طاف (أي تجنب الخرز) تحميل وتشغيل المعامل على 12 ٪ جيد 40-20 SDS - PAGE هلام. </li><li style=";text-align:right;direction:rtl"> تشغيل هلام في أي أعلى من 150 فولت ، ونقل إلى استخدام غشاء Towbin العازلة الحالية والمستمرة من 100 دبليو. </li><li style=";text-align:right;direction:rtl"> كتلة الغشاء بمحلول الحليب / TBST 5 ٪ ، والتحقيق مع RhoC - specfic الأضداد. </li></ol> 3. ممثل النتائج وينبغي أن تنتج نتائج جيدة في شريط واحد حوالي 22 كيلو دالتون. النتائج السيئة تنتج نطاقات متعددة أو خلفية عالية. هذا يدل على تدهور البروتين ، وغسل العينات غير مكتملة أو استخدام القديمة GST - RBD. <img src="/files/ftp_upload/2083/2083fig1.jpg" alt="الشكل 1" /> الشكل 1. تظهر ثلاثة خطوط منفصلة خلية التعبير عن مستويات مختلفة من GTPase RhoC لإظهار مستويات منخفضة من RhoC نشطة وإجمالا ، مستويات عالية من RhoC النشط والكلي أو RhoC لا. جردت ثم وصمة عار reprobed مع أجسام مضادة لهذا الجين أكتين منزل حفظ لتكون بمثابة سيطرة التحميل للبروتين RhoC الإجمالي. <img src="/files/ftp_upload/2083/2083fig2.jpg" alt="الشكل 2" /> الشكل 2. نطاقات متعددة أو خلفية نتائج عالية من البروتين التدهور ، وغسل العينات غير مكتملة أو استخدام القديمة GST - RBD.

<ol>
	<li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase,+a+novel+transforming+oncogene+for+human+mammary+epithelial+cells+that+partially+recapitulates+the+inflammatory+breast+cancer+phenotype.">RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype.</a> Cancer Res. 60 (20), 5832-5838 (2000).</li><li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+overexpression+modulates+induction+of+angiogenic+factors+in+breast+cells.">RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells.</a> Neoplasia. 2 (5), 418-425 (2000).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+RhoC+GTPase-induced+inflammatory+breast+cancer+phenotype+by+treatment+with+a+farnesyl+transferase+inhibitor.">Reversion of RhoC GTPase-induced inflammatory breast cancer phenotype by treatment with a farnesyl transferase inhibitor.</a> Mol Cancer Ther. 1 (8), 575-583 (2002).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+activated+protein+kinase+pathway+is+involved+in+RhoC+GTPase+induced+motility,+invasion+and+angiogenesis+in+inflammatory+breast+cancer.">Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer.</a> Clin Exp Metastasis. 19 (4), 301-311 (2002).</li><li>Yao, H., Dashner, E. J., van Golen, C. M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+is+required+for+PC-3+prostate+cancer+cell+invasion+but+not+motility.">RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility.</a> Oncogene. 25 (16), 2285-2296 (2006).</li><li>Hall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+Collagen+receptor+(alpha2beta1)+signaling+promotes+prostate+cancer+cell+invasion+through+RhoC+GTPase.">Type I Collagen receptor (alpha2beta1) signaling promotes prostate cancer cell invasion through RhoC GTPase.</a> Neoplasia. 10 (8), 797-803 (2008).</li><li>Sequeira, L., Dubyk, C. W., Riesenberger, T. A., Cooper, C. R., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+PC-3+prostate+cancer+cell+morphology,+invasion+and+tumor+cell+diapadesis.">Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapadesis.</a> Clin Exp Metastasis. 25, 569-579 (2008).</li><li>van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL2+Induces+Prostate+Cancer+Transendothelial+Cell+Migration+via+Activation+of+the+Small+GTPase+Rac.">CCL2 Induces Prostate Cancer Transendothelial Cell Migration via Activation of the Small GTPase Rac.</a> J Cell Biochem. 104, 1587-1597 (2008).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int J Cancer. , (2010).</li><li>Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., Collard, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+downregulates+Rho+activity:+reciprocal+balance+between+both+GTPases+determines+cellular+morphology+and+migratory+behavior.">Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.</a> J Cell Biol. 147 (5), 1009-1022 (1999).</li></ol>

الإعلان عن أي تضارب في المصالح.

الخطوة الأكثر أهمية من هذا الإجراء هو الجيل الجديد من GST - RBD. عدم إيلاء اهتمام وثيق لهذه الخطوة هو السبب الرئيسي للفشل. جميع الخطوات ، من جيل لGST - RBD (1.2) إلى حضانة بين عشية وضحاها مع lysate الخلية (2.5) ، ينبغي أن يتم في يوم واحد. خطوة أخرى حاسمة هي التأكد من ان هناك ما يكفي من ا?...

<table><tbody><tr><td>Gluthione-sepharose 4B beads</td><td>GE Healthcare</td><td>17-0756-01</td><td></td></tr><tr><td>Criterion 4-20% Tris-HCl gels</td><td>Bio-Rad</td><td>345-0032</td><td></td></tr></tbody></table>

rhoc gtpase activation assay

RhoC GTPase وقد تماثل 91 ٪ إلى GTPase RhoA. بسبب انتشاره في الخلايا ، وقد وضعت العديد من الكواشف والتقنيات اللازمة لGTPase RhoA. ومع ذلك ، أعرب RhoC GTPase في خلايا السرطان النقيلي عند مستويات منخفضة نسبيا. لذا ، وضعت بعض الكواشف RhoC محددة. وقد تكيفت لدينا فحص للكشف عن التنشيط رو GTPase RhoC. هذا الأسلوب يستخدم لضريبة السلع والخدمات ، رو الانصهار المجال ملزمة بالانسحاب من البروتين النشط RhoC GTPase. بالإضافة إلى ذلك ، يمكننا أن محصول البروتين الكلي في بداية الفحص لتحديد مستويات من المجموع (GTP الناتج المحلي الإجمالي وملزمة) RhoC GTPase. وهذا يسمح لتحديد GTPase RhoC النشطة مقابل الإجمالي في الخلية. وقد وضعت العديد من الإصدارات التجارية من هذا الإجراء ومع ذلك ، هي الأمثل مجموعات تجارية لGTPase RhoA وعادة لا تعمل بشكل جيد لGTPase RhoC. تم تعديل أجزاء من الفحص وكذلك وضع الضد RhoC محددة.

Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

RhoC GTPase Activation Assay

هذا البروتوكول يستخدم لهدم مقايسة لتحديد مستويات GTPase RhoC نشطة داخل الخلايا.

RhoC الفحص GTPase التنشيط

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.4K Views.  University of Delaware. هذا البروتوكول يستخدم لهدم مقايسة لتحديد مستويات GTPase RhoC نشطة داخل الخلايا.

Video: RhoC GTPase Activation Assay

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

قياس إشارة مستقبلات G- البروتينات عبر الملزمة غتب المسمى راديو

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

Biochemistry

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

الكشف عن برينيليشن جتباسي صغيرة وأنشئ ملزمة باستخدام تجزئة غشاء ومقايسة الممتز GTPase مرتبطة

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

مقايسة مضان الكالسيوم "مزدوجة الإضافة" للفحص عالي الإنتاجية لمستقبلات البروتين G المؤتلف

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

<meta charset="utf8"></head><body>اختبار بسيط لقياس ملامسات ER-Mitochondria للشاشات عالية الإنتاجية

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

<meta charset="utf8"></head><body>أضواء الكاشف على المؤلف: تنظيم وخلل تنظيم جهات اتصال الميتوكوندريا ER - الآثار المترتبة على التسبب في الأمراض التنكسية العصبية

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

مقايسة إعادة تجميع لوسيفيراز سبليت لقياس ملامسات الشبكة الإندوبلازمية والميتوكوندريا في الخلايا الحية

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

التصوير G - مستقبلات البروتين يقترن (GPCR) بوساطة الأحداث يشير إلى أن التحكم في الانجذاب الكيميائي Dictyostelium Discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

تقارب الأمطار من GEFs-رو بالموقع باستخدام ضريبة السلع والخدمات الموسومة البروتين رو المسخ (GST-RhoA (G17A)) من Lysates الخلايا الظهارية

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

توصيف G مستقبلات البروتين يقترن بها الكالسيوم تعبئة الفحص القائم على الإسفار

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

مقارنة الانجذاب من البروتينات ملزم GTPase باستخدام فحوصات المنافسة

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

RhoC الفحص GTPase التنشيط

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قياس إشارة مستقبلات G- البروتينات عبر الملزمة غتب المسمى راديو

الكشف عن برينيليشن جتباسي صغيرة وأنشئ ملزمة باستخدام تجزئة غشاء ومقايسة الممتز GTPase مرتبطة

مقايسة مضان الكالسيوم "مزدوجة الإضافة" للفحص عالي الإنتاجية لمستقبلات البروتين G المؤتلف

مقايسة إعادة تجميع لوسيفيراز سبليت لقياس ملامسات الشبكة الإندوبلازمية والميتوكوندريا في الخلايا الحية

مقايسة إعادة تجميع لوسيفيراز سبليت لقياس ملامسات الشبكة الإندوبلازمية والميتوكوندريا في الخلايا الحية

مقايسة إعادة تجميع لوسيفيراز سبليت لقياس ملامسات الشبكة الإندوبلازمية والميتوكوندريا في الخلايا الحية

التصوير G - مستقبلات البروتين يقترن (GPCR) بوساطة الأحداث يشير إلى أن التحكم في الانجذاب الكيميائي Dictyostelium Discoideum

تقارب الأمطار من GEFs-رو بالموقع باستخدام ضريبة السلع والخدمات الموسومة البروتين رو المسخ (GST-RhoA (G17A)) من Lysates الخلايا الظهارية

توصيف G مستقبلات البروتين يقترن بها الكالسيوم تعبئة الفحص القائم على الإسفار

مقارنة الانجذاب من البروتينات ملزم GTPase باستخدام فحوصات المنافسة