وكان باناب هدية نوع من الدكتور بيتر شولتز (معهد سكريبس للبحوث). وقد تم تمويل هذا العمل من قبل المعاهد الكندية لبحوث الصحة المنح موب-102689 و موب-136894 (إلى رب) والمؤسسة الكندية للابتكار منحة 950-225005.

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الكتاب ليس لديهم ما يكشف.

في أمينواسيلاتيون في الجسم الحي من ترناس التي يجري بشكل مستمر يجري نسخها جنبا إلى جنب مع الحمض الريبي النووي النقال-سينثيتاس، يجعل من الممكن الحصول على مستويات التعبير عالية لقياسات مضان. لدمج فوا كفاءة، فمن الأهمية بمكان أن يتم حقن باناب بشكل صحيح في النو?...

وقد تم دمج أكثر من 200 من الأحماض الأمينية غير الطبيعية من مختلف الخصائص الكيميائية والفيزيائية وراثيا في البروتينات في E. القولونية والخميرة وخلايا الثدييات 3 . يتم تضمين الأحماض الأمينية غير الطبيعية ردا على كودون وقف معين عن طريق زوج متعامد المهندسة الحمض الريبي النووي النقال / سينثيتاس. وقد وفرت النهج الوراثي لتعديل البروتينات رؤى قيمة في هيكل البروتين وظيفة. هنا، نقدم بروتوكول لاستخدام الجهد المشبك فلوروميتري (فف) في تركيبة مع وا الفلورسنت. في فف، ومراقبة في وقت واحد من البيانات الوظيفية وإعادة الهيكلة الهيكلي المترجمة حول مسبار الفلورسنت (~ 5 Å) يسمح لنا للحصول على معلومات ديناميكية مع ميلي ثانية واحدة القرار 1 . تحقيقات الفلورسنت تغير حالة التبريد الخاصة بهم على حركة محلية من البروتين. حركة فقط 1-2 Å كافية تؤدي إلى تغييرات كبيرة في مضانكثافة 4 . بعد تحديد موقع الفائدة في البروتين المستهدف، يتم تحور الموقع عن طريق طفرة نقطة. بشكل كلاسيكي، تم تحوير بقايا إلى السيستين في حين الآن، يتم إدخال كودون وقف العنبر (تاج) لدمج فوا الوراثية. ثم يتم في البروتين المختبر كتب في المختبر . في حين أن أنظمة التعبير الأخرى (على سبيل المثال، خلايا الثدييات) يمكن استخدامها 5 ، 6 ، 7 ، والبويضات القيطم هي الأفضل للدراسات هيكل وظيفة بسبب حجمها أكبر، مما يؤدي إلى التلاعب أسهل وكثافة أعلى مضان (أكثر فلوروفوريس)، وبالتالي، إلى نسبة الضوضاء. وعلاوة على ذلك، البويضات القيطم لها خلفية منخفضة من البروتينات الذاتية 2 ، 8 ، والتصبغ الظلام على الدروع القطب الحيواني ضد مضان الخلفية من رانه سايتوسول. يتم إزالة البويضات القيطم جراحيا ويتم حقن الحمض النووي ترميز الحمض الريبي النووي النقال متعامد / الحمض الريبي النووي النقال-سينثيتاس محددة ل فوا في نواة البويضات. بعد 6-24 ساعة ساعة الحضانة، يتم حقن الحمض النووي الريبي البروتين مع فوا في السيتوسول من البويضات، تليها فترة الحضانة 2-3 أيام. من أجل منع أي ضرر على فوا (فوتوبلاشينغ)، والإجراءات بما في ذلك أناب يجب أن تنفذ تحت الضوء الأحمر لتجنب فلورفور الإثارة. يتم دراسة البويضات على قطع مفتوحة البويضة الجهد المشبك الإعداد، والتي هي التي شنت على المجهر مضان تستقيم، وتسجيل التيار الكهربائي والتغيرات مضان في وقت واحد 9 ، 10 . بدلا من ذلك، اثنين من القطب الكهربائي المشبك الجهد 1 أو التصحيح المشبك تكوينات 11 يمكن استخدامها. مضان هو متحمس من قبل الأطوال الموجية المناسبة مع انخفاض الضوضاء رمز و<html> الانبعاثات المسجلة باستخدام الثنائي الضوئي مرتبطة مكبر للصوت مع التضخيم عالية. هناك العديد من المزايا من استخدام الفلورسنت الأحماض الأمينية غير طبيعية (فواس) في فلوريوميتري الجهد المشبك. واحد هو الوصول إلى الجانب عصاري خلوي من البروتينات الغشاء. وتقع العديد من العمليات التنظيمية هنا (على سبيل المثال، كا 2 + - أو المواقع النوكليوتيدات ملزمة، سريع وإغلاق الدولة تعطيل قنوات أيون الجهد بوابات، افتتاح المسام، اقتران وحدة). كل هذه العمليات يمكن الوصول إليها الآن لوضع العلامات الفلورسنت. ميزة أخرى هي صغر حجم التحقيق مما يؤدي إلى أقل اضطراب في البروتين. حتى الآن، تم تصميم اثنين من أزواج الحمض الريبي النووي النقال / ترنا سينثيتاس متعامد ل فواس 12 ، 13 ، حيث 3- (6-أسيتيلنافثالين 2-يلامينو) -2-أمينوبروبانويك حمض (أناب) هو فوا الوحيد الذي تم استخدامه في البويضات القيطم 2 ،&quot;كريف&quot;&gt; 8. أناب هو فلوروفور حساسة بيئيا مع الوزن الجزيئي من 272.3 ز / مول وهي أكبر قليلا فقط من التربتوفان 12 ( أرقام 1A، 1B ). نظرا لصغر حجمها، من المرجح أن يتم إدخال عدد أقل من التأثيرات المجسمة بواسطة الفلورفور مقارنة مع الفلوروفوريس التقليدي المرفق عبر رابط (عادة أكثر من 500 جم / مول). وعلاوة على ذلك، في حالة أناب، ويقع فلورفور أقرب إلى العمود الفقري البروتين من تلك المرتبطة سيستينس، وبالتالي، أناب هو البحث عن إعادة ترتيب أكثر محلية. وأخيرا، إزالة السيستين الذاتية في فف التقليدية من أجل ضمان وضع العلامات على الموقع لم يعد شرطا في وا-فف وبالتالي (1) يترك البروتينات في (تقريبا) حالتهم الأصلية و (إي) يسمح فف ليتم تطبيقها لدراسة مجموعة واسعة من البروتينات التي يمكن تغيير وظيفة من خلال استبدال السيستين. <img alt="شكل 1" src = &quot;/ فيليز / ftp_upload / 55598 / 55598fig1.jpg&quot; /&gt; الشكل 1 : أناب و مضان الأطياف. ( A ) التركيب الكيميائي لل أناب. ( B ) طيف الامتصاص تطبيع وأطياف الانبعاثات ل 1 نانومتر أناب، مما يدل على حساسية مضان أناب إلى مسعور المائي. تم الحصول على أطياف الانبعاث مثيرة في 350 نانومتر. <a href="https://www.jove.com/files/ftp_upload/55598/55598fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> العيب من استخدام وارس الفلورسنت هو أن السكان غير المتجانسة من البروتينات قد ينتج عن وقف كودون ريدثرو، إعادة التأهيل متعدية، C- محطة اقتطاع البروتينات أو الحديث المتبادل مع أمينواسيلاتيون الذاتية إذا كان كمية ترناس أمينواسيلاتد نادرة. وينبغي دائما التحقق من هذا التعبير تسرب لفي غياب فوا ورنا الحمض الريبي النووي النقال / الحمض الريبي النووي النقال سينثيتاس الزوج. لقد تناولنا مسألة الانتقالوكيفية تكرارها، وكيفية التحايل عليها من أجل مواقع الإدراج N-ترمينال سابقا 14 . ومع ذلك، عندما فوا، ترنا و ترنا سينثيتاس موجودة في كميات مشبعة، لا يزال هناك احتمال ضئيل للتعبير تسرب. الاختلاف الإجرائي الرئيسي بين فوا-فف و فف التقليدي هو الحقن والتعامل مع البويضات. حقن الحمض الريبي النووي النقال ترنا الحمض الريبي النووي النقال و ترنا سينثيتاس (باناب) يليه إدخال أناب، والتي هي إما شارك في حقن مع مرنا البروتين أو بدلا من ذلك إضافة إلى حل الحضانة كما استر أسيتوكسيميثيل (آم).

<table><tbody><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Barth&#039;s solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S7653</td><td>90 mM</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>BP366-500</td><td>3 mM</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M-9397</td><td>0.82 mM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C-7902</td><td>0.41 mM</td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C-1396</td><td>0.33 mM</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td>5 mM</td></tr><tr><td>NaOH hydrate</td><td>BDH</td><td>BDH7225-4</td><td>pH 7.6</td></tr><tr><td>Penicilin</td><td>Invitrogen</td><td>15140122</td><td>100 U/mL</td></tr><tr><td>Streptomycin</td><td>Invitrogen</td><td>15140122</td><td>100 &amp;micro;g/mL</td></tr><tr><td>Kanamycin</td><td>Invitrogen</td><td>15160054</td><td>10 mg/100mL</td></tr><tr><td>Horse Serum (HS)</td><td>Invitrogen</td><td>16050122</td><td>5%</td></tr><tr><td>&lt;strong&gt;SOS Standard Oocyte Solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>746398</td><td>102 mM</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>746436</td><td>3 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M9272</td><td>1 mM</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td>5 mM</td></tr><tr><td>&lt;strong&gt;External recording solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Alfa Aesar</td><td>L14282</td><td>115 mM</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td>10 mM</td></tr><tr><td>Calcium hydroxide</td><td>Sigma-Aldrich</td><td>239232</td><td>2 mM</td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>258105</td><td>pH 7.2</td></tr><tr><td>&lt;strong&gt;Internal recording solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Alfa Aesar</td><td>L14282</td><td>115 mM</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td>10 mM</td></tr><tr><td>Ethylenediamine Tetraacetic Acid (EDTA)</td><td>Fisher Scientific</td><td>E478-500</td><td>2 mM</td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>258105</td><td>pH 7.2</td></tr><tr><td>&lt;strong&gt;Labeling solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KOH</td><td>Fisher Scientific</td><td>P250-1</td><td>115 mM</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td>10 mM</td></tr><tr><td>Calcium hydroxide</td><td>Sigma-Aldrich</td><td>239232</td><td>2 mM</td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>258105</td><td>pH 7.2</td></tr><tr><td>&lt;strong&gt;TMR stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tetramethylrhodamine-5-maleimide (TMR)</td><td>Molcular Probes by Life Technologies</td><td>T6027</td><td>5 mM in DMSO</td></tr><tr><td>&lt;strong&gt;Anap stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anap</td><td>ABZENA (TCRS)</td><td>Custom synthesis TCRS-170</td><td>1 mM in nuclease-free water and 1% NaOH 1 N</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material/Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pAnap</td><td>Addgene</td><td>48696</td><td></td></tr><tr><td>High Performance Oocyte Clamp</td><td>Dagan Corporation</td><td>CA-1B</td><td></td></tr><tr><td>Gpatch Acquisition software</td><td>Department of Anesthesiology, University of California, Los Angeles</td><td></td><td></td></tr><tr><td>Analysis software</td><td>Department of Anesthesiology, University of California, Los Angeles</td><td></td><td></td></tr><tr><td>Recording Chamber</td><td>Custom machined</td><td></td><td></td></tr><tr><td>Photo diode detection system</td><td>Dagan Corporation</td><td>PhotoMax-200/PIN</td><td></td></tr><tr><td>Electrical shutter driver</td><td>UNIBLITZ</td><td>VCM-D1</td><td></td></tr></tbody></table>

voltage-clamp fluorometry in xenopus oocytes using fluorescent unnatural amino acids

وكان الجهد المشبك فلوروميتري (فف) تقنية الاختيار للتحقيق في هيكل ووظيفة البروتينات الغشاء الإلكتروجيني حيث القياسات في الوقت الحقيقي من مضان والتيارات تقرير في وقت واحد على إعادة ترتيب المحلية والوظيفة العالمية، على التوالي 1 . في حين أن تقنيات عالية الدقة الهيكلية مثل المجهر الإلكتروني البرد أو الأشعة السينية البلورات توفر صور ثابتة للبروتينات من الفائدة، فف يوفر البيانات الهيكلية الديناميكية التي تسمح لنا لربط إعادة الهيكلة الهيكلية (مضان) لبيانات وظيفية ديناميكية (الكهربية). حتى وقت قريب، والكيمياء ثيول رد الفعل المستخدمة في وضع العلامات الموجهة فلورزنت الموقع من البروتينات يقيد نطاق النهج لأنه سيتم وصفها جميع سيستينس الوصول إليها، بما في ذلك الذاتية. وبالتالي كان مطلوبا لبناء البروتينات خالية من السيستين الذاتية. كما تم وضع العلامات على المواقع التي يمكن الوصول إليها من خارج الخليةجانب. هذا تغير مع استخدام الفلورسنت الأحماض الأمينية غير طبيعية (فوا) لدمج على وجه التحديد مسبار الفلورسنت الصغيرة ردا على وقف قمع الكودون باستخدام الحمض الريبي النووي النقال متعامد ورنا الحمض الريبي النووي النقال سينثيتاس 2 . تحسين فف يتطلب فقط إجراء حقن من خطوتين من حقن الحمض النووي (الحمض الريبي النووي النقال / زوج سينثيتاس) تليها الحمض النووي الريبي / فوا شارك في الحقن. الآن، وضع العلامات على كل من الخلايا والمواقع المدفونة ممكن، واستخدام فف توسعت بشكل كبير. وبالتالي فإن تقنية فف تصبح جذابة لدراسة مجموعة واسعة من البروتينات، والأهم من ذلك - يسمح التحقيق في العديد من الآليات التنظيمية عصاري خلوي.

ويبين الشكل 4 مثالا على تسجيلات فف التي تم الحصول عليها من البويضة معربا عن قنوات شاكر مع تعطيل سريع إزالتها (إر)، L382stop-W434F في وجود باناب و أناب. يحول الطفرة W434F تيارات البوتاسيوم الأيونية، مما يجعل من الممكن قياس تشريد تهمة النابضة العابرة (ت...

Watch this Scientific Journal Video about Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids at JoVE.com

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

توضح هذه المقالة تعزيز التقليدية الجهد المشبك فلوروميتري (فف) حيث يتم استخدام الفلورسنت الأحماض الأمينية غير طبيعية (فوا) بدلا من الأصباغ ماليميد، لتحقيق إعادة ترتيب الهيكلي في القنوات الأيونية. ويشمل الإجراء زينوبوس البويضات حقن الحمض النووي، رنا / فوا سوينجكتيون، والقياسات الحالية والضوضاء في وقت واحد.

الجهد المشبك فلوروميتري في<em&gt; القيطم</em&gt; البويضات باستخدام الفلورسنت الأحماض الأمينية غير طبيعية

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

voltage-clamp-fluorometry-xenopus-oocytes-using-fluorescent-unnatural

Research

JoVE Journal

Biochemistry

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

10.4K Views.  University of Montréal.  توضح هذه المقالة تعزيز التقليدية الجهد المشبك فلوروميتري (فف) حيث يتم استخدام الفلورسنت الأحماض الأمينية غير طبيعية (فوا) بدلا من الأصباغ ماليميد، لتحقيق إعادة ترتيب الهيكلي في القنوات الأيونية. ويشمل الإجراء زينوبوس البويضات حقن الحمض ا?...

Video: Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

التصحيح تسجيل المشبك من القنوات ايون معبرا عنه البويضات القيطم

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Biology

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

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

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

استخدام pHluorin لتقييم ديناميات مستقبلات الإرشاد أكسون في زراعة الخلايا وفي الجنين الفرخ

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

Neuroscience

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

تصوير الكالسيوم الفلوري والتهجين في الموقع اللاحق لتوصيف السلائف العصبية في زينوبوس laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Developmental Biology

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

التصوير غشاء المحتملة مع اثنين من أنواع مشفرة وراثيا نيون الجهد مجسات

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

دراسة ديناميكية بتكوين جزئي للبروتينات الغشاء في الموقع مع الموقع الموجه وصفها الإسفار

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

قياس إمكانات العمل البصري أحادي الخلية في خلايا القلب المشتقة من الخلايا الجذعية المتعددة القدرات

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Bioengineering

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

تقييم وظيفي لمسارات حاسة الشم في المعيشة النمو الضفادع الصغيرة

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

قياس الكاتيونات النقل من نا، K-H و، K-أتباز في<em&gt; القيطم</em&gt; البويضات بواسطة الامتصاص الذري الطيفي: بديل لفحوصات النظائر المشعة

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

ال<em&gt; القيطم</em&gt; سيت قص مفتوحة الفازلين جاب الجهد المشبك تقنية مع التألق

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

قياس الفلورسنت في الوقت الحقيقي للوظائف المشبكية في نماذج التصلب الجانبي الضموري

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

الجهد المشبك فلوروميتري في

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