هذا المشروع وأيده نبو الأول، LO1417 (وزارة التربية والتعليم والشباب والرياضة في الجمهورية التشيكية).

<ol>
	<li>Barbosa, I. C. R., Shikata, H., Zourelidou, M., Heilmann, M., Heilmann, I., Schwechheimer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+composition+and+a+polybasic+motif+determine+D6+PROTEIN+KINASE+polar+association+with+the+plasma+membrane+and+tropic+responses.">Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.</a> Development. 143 (24), 4687-4700 (2016).</li><li>Kato, M., Aoyama, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ca2+-binding+protein+PCaP2+located+on+the+plasma+membrane+is+involved+in+root+hair+development+as+a+possible+signal+transducer.">The Ca2+-binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer.</a> Plant J. 74 (4), 690-700 (2013).</li><li>Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence Microscopy: From Principles to Biological Applications. , (2013).</li><li>Janecki, A. J., Janecki, M., Akhter, S., Donowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+plasma+membrane+expression+of+a+fusion+protein+of+Na/H+exchanger+NHE3+and+green+fluorescence+protein+(GFP)+in+living+PS120+fibroblasts.">Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts.</a> J Histochem Cytochem. 48 (11), 1479-1492 (2000).</li><li>Peitzsch, R. M., McLaughlin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+acylated+peptides+and+fatty+acids+to+phospholipid+vesicles:+pertinence+to+myristoylated+proteins.">Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.</a> Biochemistry. 32 (39), 10436-10443 (1993).</li><li>Vosolsob&#283;, S., Petr&#225;&#353;ek, J., Schwarzerov&#225;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+plasticity+of+plasma+membrane+interaction+in+DREPP+family+proteins.">Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.</a> Biochim Biophys Acta - Biomembr. 1859 (5), 686-697 (2017).</li><li>Bolte, S., Talbot, C., Boutte, Y., Catrice, O., Read, N. D., Satiat-Jeunemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM-dyes+as+experimental+probes+for+dissecting+vesicle+trafficking+in+living+plant+cells.">FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells.</a> J Microsc. 214 (2), 159-173 (2004).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>R Development Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A language and environment for statistical computing. , (2008).</li><li>Logan, D. C., Domergue, O., Teyssendier de la Serve, B., Rossignol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+plasma+membrane+polypeptides+differentially+regulated+during+plant+development.">A new family of plasma membrane polypeptides differentially regulated during plant development.</a> Biochem Mol Biol Int. 43 (5), 1051-1062 (1997).</li><li>Ide, Y., Nagasaki, N., Tomioka, R., Suito, M., Kamiya, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+properties+of+a+novel,+hydrophilic+cation-binding+protein+associated+with+the+plasma+membrane.">Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane.</a> J Exp Bot. 58 (5), 1173-1183 (2007).</li><li>Nagasaki-Takeuchi, N., Miyano, M., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasma+membrane-associated+protein+of+Arabidopsis+thaliana+AtPCaP1+binds+copper+ions+and+changes+its+higher+order+structure.">A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure.</a> J Biochem. 144 (4), 487-497 (2008).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDP25,+A+Novel+Calcium+Regulatory+Protein,+Mediates+Hypocotyl+Cell+Elongation+by+Destabilizing+Cortical+Microtubules+in+Arabidopsis.">MDP25, A Novel Calcium Regulatory Protein, Mediates Hypocotyl Cell Elongation by Destabilizing Cortical Microtubules in Arabidopsis.</a> Plant Cell. 23 (12), 4411-4427 (2011).</li><li>Qin, T., Liu, X., Li, J., Sun, J., Song, L., Mao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+microtubule-destabilizing+protein+25+functions+in+pollen+tube+growth+by+severing+actin+filaments.">Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.</a> Plant Cell. 26 (1), 325-339 (2014).</li><li>Nagata, T., Nemoto, Y., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+BY-2+Cell+Line+as+the+&amp;#34;HeLa&amp;#34;+Cell+in+the+Cell+Biology+of+Higher+Plants.">Tobacco BY-2 Cell Line as the &amp;#34;HeLa&amp;#34; Cell in the Cell Biology of Higher Plants.</a> Int Rev Cytol. 132, 1-30 (1992).</li><li>Hellens, R. P., Anne Edwards, E., Leyland, N. R., Bean, S., Mullineaux, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pGreen:+A+versatile+and+flexible+binary+Ti+vector+for+Agrobacterium-mediated+plant+transformation.">pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.</a> Plant Mol Biol. 42 (6), 819-832 (2000).</li><li>Vermeer, J. E. M., Thole, J. M., Goedhart, J., Nielsen, E., Munnik, T., Gadella, T. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+phosphatidylinositol+4-phosphate+dynamics+in+living+plant+cells.">Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.</a> Plant J. 57 (2), 356-372 (2009).</li><li>La&#328;kov&#225;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Dynamics+of+Plant+Plasma+Membrane+Proteins+with+Fluorescence+Recovery+and+Raster+Image+Correlation+Spectroscopy.">Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.</a> Microsc Microanal. 22 (2), 290-299 (2016).</li></ol>

الكتاب ليس لها علاقة بالكشف عن.

ينشئ الأسلوب الموصوفة هنا تقدير أكثر دقة لتقسيم غشاء البلازما للبروتينات المرتبطة محيطيا مقارنة بالنهج الأخرى استناداً إلى قياس كثافة الأسفار5. تحسن كبير لهذا الأسلوب أنه يأخذ بعين الاعتبار حيود الضوء والتراكب من الغشاء البلازما وإشارات هيولى. وعلى الرغم من هذه النتائج الأ?...

An erratum was issued for: <a href="https://www.jove.com/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.
One of the affiliations was updated from:
Department of Experimental Plant Biology, University of Science, Charles University
to:
Department of Experimental Plant Biology, Faculty of Science, Charles University

An erratum was issued for: <a href="https://www.jove.com/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.

Erratum: Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

غشاء البلازما المرتبطة محيطيا البروتينات هي المكونات الرئيسية للخلية إشارات المسارات. أحد أدوارها الأساسية هي رابطة عابر غشاء البلازما، والانفصال، وهامة لتوصيل الإشارة بين غشاء البلازما والسيتوبلازم. يمكن إرفاق البروتينات المرتبطة محيطيا غشاء البلازما في غشاء البلازما بالدهن المراسي (N-ميريستويليشن, S-أسيليشن، أو برينيليشن) أو بواسطة مجالات ملزمة الدهون (التفاعل مع الفوسفات فوسفاتيديلينوسيتول، وحامض فوسفاتيديك، إلخ).
درس ملزمة غشاء البلازما يمكن أن تكون خصائص هذه البروتينات في فيفو، مثلاً، عندما يتم تعديل بروتين فلوريسسينتلي معلم بالطفرات موقع الموجه من الأحماض الأمينية الأساسية، أو عند فإنه يتم التعامل مع الموانع المختلفة التي تؤثر على مما يشير إلى المادة الدهنية. توزيعات البروتينات الطرفية غشاء البلازما معظمها يجري تقييم نوعيا، ولا سيما في الحالات عند إعادة توزيع البروتين من الواضح. طريقة عرض الأمثل للحالات عند إعادة توزيع البروتين فقط جزئية والتقييم الكمي ضروري. اتباع نهج المستخدمة بشكل متكرر من عندما تقدر الرابطة غشاء البلازما من [كنفوكل] ليزر المسح المجهري الصور كنسبة شدة الأسفار في الغشاء البلازما وفي السيتوبلازم1،2، بسيط، ولكن ليس دقيقة. كثافات fluorescence في غشاء البلازما تعكس تراكب الإشارات غشاء البلازما والسيتوبلازم نظراً لأن الخاصية حيود الضوء لتقنية مجهرية خاصة الأسفار والعناصر البصرية تستخدم3. ونتيجة لذلك، يتم تضمين إشارة هيولى أيضا في منطقة الغشاء. ولهذا السبب، لا يمكن استخدام نمط المصبوغة FM 4-64 كقناع لغشاء إشارة تحديد4. وعلاوة على ذلك، القياسات البسيطة من الغشاء إشارة في الموقف الذي حدده وزير الخارجية 4-64 تلطيخ الحد الأقصى دائماً بصورة منهجية المبالغة إشارة غشاء البلازما الحقيقية للبروتين غشاء البلازما المرتبطة محيطيا بسبب التراكب الغشاء ومجمع هيولى. الحد أقصى الإشارات الملحوظة لمعلم فلوريسسينتلي البروتينات المرتبطة محيطيا أيضا تعريب لا يشترك مع الحد أقصى علامة غشاء البلازما (أي، إف أم صبغ ستيريل 4-64)، ولكن هو تحول نحو السيتوبلازم. قيد آخر يستند إلى حقيقة أن وزير الخارجية ذروة الانبعاث 4-64 أوسع بالمقارنة مع قمم الانبعاثات للبروتينات الفلورية الخضراء مثل التجارة والنقل بسبب الطول الموجي-التبعية من حيود الضوء3.
في الأسلوب الموصوفة هنا، إشارة البروتين المعلمة مزودة باثنين من المهام التجريبية تصف توزيع افتراضية لغشاء البلازما وإشارة السيتوبلازم، على التوالي. يتم تطبيق هذا التحلل إشارة إلى الملامح الفلورية الخطية التي يتم تطبيقها على سطح الخلية عمودياً إلى غشاء البلازما في مصدر الصور، وأقسام [كنفوكل] العادية، والقناة الثانية للتعبير عن البروتين معلم فلوريسسينتلي الخلايا المسماة بصبغ FM 4-64.
وصف الدالة الأولى المستخدمة لتركيب حيود إشارة السيتوبلازم على حافة الخلية. يتم الحصول عليها من الملامح المكتسبة سابقا الأسفار التي قيست في الخلايا معربا عن علامة بروتين السيتوبلازم معلم قبل chromophore نفسه كالبروتين المرتبطة محيطيا غشاء البلازما للفائدة. الدالة الثانية تصف حيود إشارة غشاء البلازما مشتق من الأسفار FM 4-64. أولاً هو تقريب هذه الإشارات من دالة ضبابي الذي يتم استخدامه لنمذجة تقريبي من حيود الضوء من مصدر نقطة. وثانيا، هذا النموذج، صالحة للاحمر FM 4-64 الانبعاثات، رياضيا تتحول إلى النموذج المرتبط الطول موجي انبعاثات من chromophore المستخدمة لوضع علامات على البروتينات المرتبطة محيطيا للفائدة في غشاء البلازما. يتم تطبيع كلتا الوظيفتين بكثافة قصوى ويعني من 10 في المائة من أعلى القيم لإشارة FM 4-64 والبروتين هيولى إشارة، على التوالي. بهذا التحلل إشارة (الأسلوب المناسب مربعة أقل غير الخطية)، يمكن تقدير نسبة غشاء البلازما والكسر السيتوبلازم لفحص البروتين بسهولة ودقة. البعد المادي الحقيقي لمعامل التقسيم محسوب في نطاق ميكرومتر، نظراً لتركيز وحدة التخزين هيولى بالمقارنة مع تركيز السطحية في غشاء البلازما. وهو يحدد المسافة من غشاء البلازما إلى السيتوبلازم، الذي هو مترجم على نفس القدر من البروتينات كما هو الحال في المنطقة المتاخمة لغشاء البلازما. هذه القيمة مكافئة لتقسيم معامل ك2 قدم سابقا5. الطريقة سريعة جداً وتتطلب واحد فقط الأقسام [كنفوكل] اكتسب استخدام الليزر [كنفوكل] روتينية فحص المجهر، وأنها لا تطالب حسابياً. نفذت التحليل الأساسية في مجموعة R محمولة وتمت كتابة ماكرو إيماجيج إضافية لتوفير واجهة مستخدم رسومية لتشغيل التحليل من مربعات الحوار بديهية. البرمجيات ووصف أكثر تفصيلاً للطريقة (نشرت سابقا6) يمكن الاطلاع على http://kfrserver.natur.cuni.cz/lide/vosolsob/Peripheral/.
الأسلوب مناسبة لعزل الخلايا وبروتوبلاستس، والأنسجة، حيث يمكن تمييزها الواضح غشاء بلازما خلايا فردية، معربا عن بناء معلم فلوريسسينتلي لفحص البروتينات المرتبطة بالمحيط. Chromophore متوافقة مع وزير الخارجية يجب أن تستخدم تلطيخ 4-64. 4-64 FM تنبعث ومضان أحمر؛ ولذلك، يمكن الموسومة دراسة البروتين بروتين الأسفار مع الانبعاثات الأزرق أو الأخضر أو الأصفر (مثلاً، التجارة والنقل، والحركة، يفب). يوصي بتحويل مستقرة للمواد البيولوجية لأنها تمكن الملاحظات أقل مصطنعة واستنساخه بأكثر من توزيع البروتين. فمن الضروري أن فحص البروتين قد توزيع هيولى متجانسة نسبيا. إضفاء الطابع المحلي على البروتين في هيولى أو آخر داخل الخلايا غشاء المقصورة يمكن أن تنتج نتائج مصطنعة.
بالإضافة إلى ذلك، يجب أن تستخدم نفس المواد البيولوجية معربا عن علامة هيولى للمقارنة. يمكن أن تتحول خلايا تشوموفوري حرة (نفس المستخدمة للبروتين الطرفية العلامات، مثلالتجارة الحرة والنقل) أو البروتين المعلمة للفائدة مع القدرة الملزمة غشاء الملغاة. يمكن إلغاء القدرة الملزمة الغشاء، على سبيل المثال، بالتشذيب الملك ملزمة غشاء أو بواسطة الطفرات موقع الموجه للمتبقي من الأحماض الأمينية الأساسية (مثلاً، مواقع ميريستويليشن ن، ق-أسيلاتيون، أو برينيليشن، إلخ).
للفحص المجهري المسح [كنفوكل]، يجب تمييز الخلايا بعلامة غشاء مثل صبغ FM 4-64. إذا FM تلطيخ 4-64 ليست مناسبة للمواد المدروسة (بسبب أوتوفلوريسسينسي التدخل والاختراق صبغ الفقراء، إلخ)، غشاء البلازما يمكن أن يكون المسمى، على سبيل المثال، بالبروتين غشاء البلازما لا يتجزأ معلم تشوموفوري مناسبة ( مشري، طلب تقديم العروض، إلخ). فمن الضروري أن العلامة قد التعريب لا يعتد بها في المقصورات غشاء داخل الخلايا (اندوميمبرانيس).
إذا كان يعمل مع عينات ثابتة، والأجسام المضادة، ويمكن استخدام يمكن حلها التماثلية FM 4-64FX أو وسم غشاء البلازما من الأجسام المضادة ضد هدف مناسب. وفي هذه الحالة، من الضروري تقييم النتائج بعناية شديدة لأن إجراءات التثبيت يمكن أن يؤدي إلى فقدان الانتقائي للبروتينات من السيتوبلازم والغشاء البلازما.

<table><tbody><tr><td>FM 4-64</td><td>ThermoFisher Scientific</td><td>T13320</td><td>Plasma membrane dye</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D4540 Sigma</td><td>Dye solvent</td></tr><tr><td>Ordinary equipment (microscopic slides, pipettes, tips, tubes)</td><td></td><td></td><td>Equipment for cell labelling and microscopy</td></tr><tr><td>Confocal laser scanning microscope</td><td></td><td></td><td></td></tr><tr><td>Ordinary computer</td><td></td><td></td><td></td></tr></tbody></table>

determination of plasma membrane partitioning for peripherally-associated proteins

يوفر هذا الأسلوب نهجاً سريعة للبت في غشاء البلازما تقسيم أي معلم فلوريسسينتلي المرتبطة محيطيا البروتين باستخدام التشكيلات الجانبية لشدة الأسفار عبر غشاء البلازما. التشكيلات الجانبية للأسفار المقاسة مزودة بنموذج لتوزيع الأسفار الغشاء والسيتوبلازم على طول خط تطبيق هامش الخلية عمودياً. يتكون هذا النموذج من قيم الكثافة fluorescence في خلايا مرجع معربا عن علامة معلم فلوريسسينتلي السيتوبلازم، ومع وزير الخارجية 4-64-المسمى غشاء البلازما. الطريقة التي يمكن تطبيقها على مختلف أنواع الخلايا والكائنات الحية؛ ومع ذلك، يمكن تقييم فقط البلازما في أغشية الخلايا غير المجاورة. هذا الأسلوب السريع القائم على الفحص المجهري ومناسبة للتجارب، حيث يتوقع تغييرات خفية ودينامية العلامات المرتبطة بغشاء البلازما والحاجة إلى القياس الكمي، مثلاً، في تحليل الإصدارات متحولة من البروتينات، والعلاجات المانع، وتدل الملاحظات توصيل. يتم تطبيق الأسلوب في صفقة R منصة متعددة ويقترن مع ماكرو إيماجيج الذي يعمل كواجهة سهلة الاستخدام.

درب10 هو بروتين الخاصة بالنباتات المرتبطة محيطيا بلازما-غشاء الذي يرتبط بغشاء البلازما عبر N-ميريستويليشن وتفاعل الالكتروستاتيكي مع فوسفاتيديلينوسيتول الفوسفات11، 12-درب وصف بأنه مكون آلات مما يشير إلى الكالسيوم في الخلية ال?...

Watch this Scientific Journal Video about Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins at JoVE.com

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

نقدم هنا، بروتوكولا لإجراء تحليل الكمي لمستوى رابطة غشاء البلازما لمعلم فلوريسسينتلي البروتينات المرتبطة بالمحيط. الأسلوب يستند إلى عنصر هيولى من الإشارات التي لوحظت في الخلايا المسماة بعلامة نيون غشاء البلازما وتحلل الغشاء الحسابية.

تحديد التقسيم غشاء البلازما للبروتينات المرتبطة محيطيا

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

determination-plasma-membrane-partitioning-for-peripherally

Research

JoVE Journal

Biology

8.3K Views.  Faculty of Science, Charles University. نقدم هنا، بروتوكولا لإجراء تحليل الكمي لمستوى رابطة غشاء البلازما لمعلم فلوريسسينتلي البروتينات المرتبطة بالمحيط. الأسلوب يستند إلى عنصر هيولى من الإشارات التي لوحظت في الخلايا المسماة بعلامة نيون غشاء البلازما وتحلل الغشاء الحسابية.

Video: Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

طريقة لتصور وتحليل غشاء متبادلة البروتينات التي نقل المجهر

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biochemistry

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

غشاء الدماغ تجزئة: أ<em&gt; إكس فيفو</em&gt; نهج لتقييم توطين البروتين سوبسينابتيك

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

التجويف النيتروجين والطرد المركزي التفاضلي يسمح بمراقبة توزيع البروتينات الغشاء المحيطي في الخلايا المستزرعة

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

غشاء الخلية الأصلية نظام الجسيمات النانوية لتحليل التفاعل البروتينية الغشاء

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

التحليل الطيفي للارتباط الفلوري للتباين الموضعي لتحليل الانتشار الجزيئي في الغشاء البلازمي للخلايا الحية

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

إعادة تشكيل نشاط استخراج Msp1 مع مكونات منقى بالكامل

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

بيب على رقاقة: دراسة خالية من التسمية من التفاعلات البروتين فسفوانوسيتيد

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Bioengineering

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

تحديد غشاء طوبولوجيا البروتين عن طريق الإسفار البروتياز الحماية (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Membrane Proteins

Plasma membranes have integral transmembrane proteins involved in facilitated transport. These proteins are collectively referred to as transport proteins, and they function as either channels for the material or as carriers themselves. Channel proteins have hydrophilic domains exposed to the intracellular and extracellular fluids and a hydrophilic channel through their core that provides a hydrated opening for solutes to pass through the membrane layers. Passage through the channel allows polar compounds to avoid the nonpolar central layer of the plasma membrane that would otherwise slow or prevent their entry into the cell.
Aquaporins are channel proteins that allow water to pass through the membrane at a high rate. Channel proteins are either open at all times or &#34;gated,&#34; with the opening of the channel being regulated. The attachment of a particular ion to the channel protein may control the opening, or other mechanisms or substances may be involved. In some tissues, sodium and chloride ions pass freely through open channels, whereas in other tissues, a gate must be opened to allow the passage. An example of this occurs in the kidney, where both forms of channels are found in different parts of the renal tubules. Cells involved in the transmission of electrical impulses, such as nerve and muscle cells, have gated channels for sodium, potassium, and calcium in their membranes.
Another type of protein embedded in the plasma membrane is a carrier protein. This protein binds a substance and, in doing so, triggers a change of its own shape, moving the bound molecule from the outside of the cell to its interior; depending on the gradient, the material may move in the opposite direction. Each carrier protein is specific to one substance, and there are a finite number of these proteins in any membrane. For example, glucose, water, salts, ions, and amino acids needed by the body are filtered in one part of the kidney. This filtrate, which includes glucose, is then reabsorbed in another part of the kidney. Because there are only a finite number of carrier proteins for glucose, if more glucose is present than the proteins carriers can handle, the excess is excreted from the body in the urine. In a diabetic individual, this is described as &#34;spilling glucose into the urine.&#34; A different group of carrier proteins called glucose transport proteins, or GLUTs, are involved in transporting glucose and other hexose sugars through plasma membranes within the body. Channel proteins transport much more quickly than carrier proteins.
This text is adapted from <a href="https://openstax.org/books/biology-2e/pages/5-2-passive-transport">Openstax Biology 2e, Section 5.2</a>

بروتينات الغشاء

Plasma membranes have integral transmembrane proteins involved in facilitated transport. These proteins are collectively referred to as transport ...

Education

JoVE Core

Cell Biology

Unit 1

Membrane Structure and Components

KEY TERMS AND CONCEPTS

Introduction to Membrane Proteins

The cell membrane, or plasma membrane, is an ever-changing landscape. It is described as a fluid mosaic where various macromolecules are embedded in the phospholipid bilayer. Among the macromolecules are proteins. The protein content varies across cell types. For example, mitochondrial inner membranes contain ~76% protein content, while myelin contains ~18% protein content. Individual cells contain many types of membrane proteins&#8212;red blood cells contain over 50&#8212;and different cell types have distinct membrane protein sets.Membrane proteins have wide-ranging functions. For example, they can be channels or carriers that transport substances, enzymes with metabolic roles, or receptors that bind to chemical messengers. Like membrane lipids, most membrane proteins contain hydrophilic (water-attractive) and hydrophobic (water-repelling) regions. The hydrophilic areas are exposed to water-containing regions inside the cell, outside the cell, or both. The hydrophobic regions face the hydrophobic tails of phospholipids within the membrane bilayer.Membrane proteins can be classified by whether they are embedded (integral) or associated with the cell membrane (peripheral). Most integral proteins are transmembrane proteins, which traverse both phospholipid layers and span the entire membrane. Their hydrophilic regions extend from both sides of the membrane, facing the cytosol on one side and the extracellular fluid on the other. In contrast, the hydrophobic regions consist of coiled amino acid groups (&#945;-helices or &#946;-barrels). Integral monotopic proteins are attached to only one side of the membrane.Peripheral proteins are not embedded in the phospholipid bilayer and do not extend into its hydrophobic core. Instead, they temporarily adhere to the outer or inner surfaces of the membrane, attached to integral proteins or phospholipids. Membrane proteins that extend from a cell&#39;s external surface often carry carbohydrate chains, forming glycoproteins. Some glycoproteins facilitate cell-to-cell recognition by functioning as &#34;ID tags&#34; that can be recognized by membrane proteins of other cells.

Cell Membrane: Integral and Peripheral Proteins

ارتباطات البروتينات

The cell membrane, or plasma membrane, is an ever-changing landscape. It is described as a fluid mosaic where various macromolecules are embedded in the ...