Biz optimize etmek ve protokol doğrulamak katkıda laboratuarımızda tüm insanlara teşekkür ederim. Bu çalışma INSERM ve ulusal araştırma kurumu (ANR-09-JCJC-0116 VE ANR-11-BSV1-033-02) tarafından desteklenmiştir. ET yüksek öğrenim ve araştırma Fransız bakanlığından bir araştırma bursu ile onu doktora sırasında desteklenmiştir.

Çözümler 1. hazırlanması <ol><li> PBS 14.5 ml% 37 formaldehit 5.5 mi seyreltilmesiyle PBS (tuz) içinde% 10 formaldehit hazırlayın. PBS, 50 ml glisin 3.8 g çözülmesiyle, 1 M glisin, pH 8.0, hazırlanması; 1x PBS içinde 100 mM glisin elde etmek için bu seyreltilir. </li><li> 1x PBS içinde% 0.1 Triton-X100 hazırlayın. 3.0 M sodyum klorid ve 25 ° C de pH 7.0 ile, iyonu giderilmiş su içinde hazırlanan 0.30 M trisodyum sitrat ile 20x Tuzlu sodyum sitrat (SSC) hazırlayın. 1x bu tampon sulandırmak ve deiyonize su kullanılarak 0.01x. </li></ol> Hücreler 2. Sabitleme NOT: Bu çalışmada HuH7 hepatokarsinoma hücre çizgisi kullanılır, ancak bu yöntem diğer yapışık hücre kültürleri için de geçerlidir. <ol><li> kaplanmamış 35 mm&#39;lik cam alt tabak başına 150.000 hücre (% 10 fetal dana serumu ve% 0.01 penisilin-streptomisin stok ile desteklenmiş DMEM, 1 g / L glikoz kültürlenmiştir) Levha Huh7 hücreleri,. Birincil hücrenin üzerinde çalışırkenKültürler, kolajen kaplı yemekler kullanımı. </li><li> Sonraki gün, kültür ortamı çıkarın. PBS aspire 1 ml hücreleri yıkayın. Formaldehit% 10 1 ml ekleyerek hücreleri saptamak ve ajitasyon altında oda sıcaklığında (RT) 10 dakika süreyle inkübe edilir. </li><li> 1 M glisin, 1 ml reaksiyonu durdurun ve rotasyon karıştırılır. Dur reaksiyon çözüm çıkarın ve 1x PBS 1 ml ekleyerek hücreleri yıkayın; rotasyon ve aspirat ile çalkalayın. hücrelere 100 mM glisin 1 ml ilave edilir ve 15 çalkalanarak oda sıcaklığında dakika ve daha sonra aspire için inkübe. NOT: protokol burada durdurulabilir ve aşağıdaki adımlar başka bir güne ertelenebilir. Bu durumda, 100 mM glisin, 1 ml ilave edilir ve gerektiğinde, 4 ° C&#39;de tutun. </li></ol> 3. Hücrelerin Permeabilization <ol><li> 15 çalkalanarak oda sıcaklığında dakika ve daha sonra aspire inkübe edin, 1 x PBS içinde% 0.1 Triton-X100 içinde 1 ml ilave edilir. Primer hücre kültür üzerinde çalışırken 20 dakika - Bu inkübasyon süresi 15 kadar arttırılabilires (örneğin, birincil fare hepatositler). 1x PBS 1 ml ekleyerek hücreleri yıkayın; aspire. </li></ol> 4. Engelleme <ol><li> (Kit ile birlikte verilen) her bir örnek için bloke etme çözeltisi 40 ul ekle; Bu hacimli bir numunesi kapsayacak şekilde arttırılabilir. bir nemlilik bölmesi içerisinde 37 ° C&#39;de 30 dakika boyunca yemekler inkübe edin. </li><li> yemekler kapalı engelleme çözümü dokunun. Bu tekrarlanabilirlik etkileyecek gibi, her slayt için eşit kalan miktarlar elde etmek için çalışın. Numuneler kurumasına izin vermeyin! </li></ol> 5. Birincil antikorlar, <ol><li> 1x PBS birincil antikorlar seyreltin ve yemekler (VDAC1 fare antikoru: 1/100, IP3R1 tavşan antikoru: 1/500) için çözüm ekleyin. Seçenek olarak ise, GRP75 ve CypD antikorlar (1/500 kullanılan her iki fare antikorları) kullanılarak VDAC1 arasında kullanılabilir. </li><li> 4 ° C&#39;de bir nem odasında gece boyunca inkübe edin. % 0.01 Tween (TBS-T) ile slaytlar Tris Tamponlu Salin kullanarak iki kez yıkayın. </li></ol><pclass = &quot;jove_title&quot;&gt; 6. Proximity ligasyon Testi Probları <ol><li> Yakınlık ligasyon tahlil sondaları kit tarafından sağlanmaktadır. Primer antikor türlere göre problar seçin. </li><li> Antikor seyreltici 5: İki yakınlık ligasyon tahlil Sensörler 1 hazırlayın. Karışım, oda sıcaklığında 20 dakika boyunca bekletin. seyreltilmiş yakınlık ligasyonu tahlil prob çözüm ekleyin. 37 ° C&#39;de 1 saat boyunca önceden ısıtılmış bir nem odasında yemekler inkübe edin. TBS-T ile yemeklere iki kez yıkayın. </li></ol> 7. ligasyonu <ol><li> In situ saptama reaktif maddesi Texas kırmızısı kiti kullanım. </li><li> Yüksek saflıkta su içinde 5 ve iyice karıştırın (kit ile birlikte verilen) 5x bağlama suyu 1 seyreltin. 1:40 ve vorteks (kit tarafından sağlanan) 1x ligasyon çözeltisi içinde ligaz sulandırmak. hemen örneklerin yanı sıra öncesine kadar ligaz eklemek için bekleyin. </li><li> önceden ısıtılmış bir nem Cham slaytlar her bir örnek için, bu solüsyonu (35 mm cam alt yemekler için 40 ul) ilave edilir ve kuluçkayaBER, 37 ° C&#39;de 30 dakika karıştırıldı. TBS-T ile slaytlar iki kez yıkayın. </li></ol> 8. Büyütme NOT: Dikkatli, ışığa duyarlı reaktifler olunuz. <ol><li> Yüksek saflıkta su içinde 5: (kit ile birlikte verilen) 5x büyütme stok 1 seyreltin. donma blok kullanarak dondurucu polimeraz çıkarın (-20 ° C). (Kit tarafından sağlanan) polimeraz 1x büyütme çözüm ve vorteks içinde 1:80 seyreltilir. karışımı kullanılarak hemen önce polimeraz ekleyin. </li><li> Her bir örnek için, bu solüsyonu (35 mm cam alt yemekler için 40 ul) eklenir. 37 ° C&#39;de 100 dakika boyunca önceden ısıtılmış bir nem odasında slaytlar inkübe edin. slaytlar kapalı Amplifikasyon-Polimeraz çözümü dokunun. </li></ol> 9. Final Yıkama <ol><li> 2 dakika için 1 x SSC yıkama tamponunda slaytlar yıkayın. 2 dakika boyunca 0.01x SSC yıkama tamponunda slaytlar yıkayın. Yemekler karanlıkta oda sıcaklığında kurumaya bırakın. </li></ol> Görüntüleme için 10. Hazırlık <ol&gt; <li> hava kabarcıkları kapak kayma altında yakalanmak sağlanması, (sulu ve katılaşmaya değildir) DAPI içeren montaj orta minimal hacmi kullanılarak slaytlar monte edin. Tırnak cilası kenarları mühür için kullanılabilir. (: 594 nm, emisyon: 624 nm, Büyütme: 63X uyarım) bir floresan veya konfokal mikroskop kullanılarak analiz etmeden önce yaklaşık olarak 15 dakika boyunca bekleyin. </li><li> görüntüleme sonra karanlıkta -20 ° C&#39;de slaytlar saklayın. </li></ol>

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The authors declare that they have no competing financial interests.

Collectively, our studies indicate that the in situ proximity ligation assay is truly a relevant strategy to follow and quantify endogenous ER-mitochondria interactions in fixed cells, without the need for using organelle-specific fluorophores or fluorescent proteins. The specific use of VDAC1/IP3R1 antibodies has been adapted to study ER-mitochondria interactions in HuH7 cells. However, alternative isoforms of VDAC and IP3R may be used, depending on the cell type. In this case, antibodies need to be validated b...

Mitokondri ve endoplazmik retikulum (ER) hücrede bağımsız organelleri olmayan, ancak mitokondri ilişkili endoplazmik retikulum zarlarının (MAM) olarak tanımlanan temas bölgelerinde yapısal ve fonksiyonel etkileşim. Aslında, MAM her iki taraftan da proteinler arasındaki etkileşimi sağlayan ER membranlar ve mitokondri yakın kapatılan bölgelere karşılık gelir. Bununla birlikte, bu organellerin membranlar bu bölgeler içinde kaynaştırmak yok, bu yüzden onların ayrı varlıklar korumak. MAM Enerji metabolizması ve hücre yaşamını 1-3 etkileyen, mitokondri ER bir kalsiyum açısından önemli bir rol (Ca 2+) ve fosfolipid transferi oynarlar. ER ve mitokondri arasındaki ilişki ilk elektron mikroskobu ile 1970&#39;lerde görüntülendi. O zamandan beri, transmisyon elektron mikroskobu 4,5, elektron tomografi 6,7 veya ER ve mitokondri özgü fluorofor immün lokalizasyonus / floresan proteinleri 8 klasik ER-mitokondri etkileşimleri çalışmak için kullanılmıştır. MAM analizi için yararlı bir araç içi fraksiyonasyon kullanımına dayanır. Bir Percoll degrade 9 bağlanmış diferansiyel Ultrasantrifügasyon tarafından MAM fraksiyonların izolasyonu sağlar. Bununla birlikte, nihai ürün zenginleştirilmiş MAM fraksiyonlar yerine, saf kesimlerin içerir. Toplamda, bu stratejiler özellikle hassas ve / veya kantitatif değildir ve onlar büyük tarama kolayca uygun değildir. Alternatif olarak ilaç indüklenebilir floresan arası organel bağlayıcılar kullanılarak genetik yaklaşımlar ortaya çıkmıştır, ancak protein 10 &#39;nın endojen ekspresyonunun seviyelerinde organel etkileşimlerin analizine izin vermez. MAM 11&#39;de IP3R / GRP75 / SSVD kompleksinin Szabadkai keşfinden dayanarak, ER-mitokondri etkileşimleri analiz etmek için kantitatif bir yöntem geliştirdi. In situ yakınlığı ligati kullanılantahlil üzerinde algılamak ve VDAC1 ve IP3R1 arasındaki etkileşimleri ölçmek için, sabit hücrelerde 12 MAM arayüzünde Ca 2 + -channeling kompleksi yer alan iki organel-yüzey proteinleri. Kısaca, bizler, ER membran dış mitokondriyal zarın (fare anti-VDAC1 birincil antikor) ve IP3R1 (tavşan anti-IP3R1 birincil antikor) (Şekil 1, bir panel) de VDAC1 incelenir. Daha sonra, deney göre, anti-fare ve tamamlayıcı oligonükleotid uzantıları konjuge edilmiş anti-tavşan IgG (fare ve tavşan proksimite ligasyonu deney Probes), her iki ilave edildi. İki hedef proteinler 40 nm&#39;nin altında bir mesafede ise, oligonükleotidler dairesel DNA şablonu (Şekil 1 Panel B) oluşmasına izin vermek için daha sonra ilave bağlayıcı oligo ile hibridize olabilir. Bu dairesel bir DNA molekülü kovalent yakınlığı probları birine bağlanmış bir tek-şeritli DNA ürününü oluşturmak, lige edilmiş ve amplifiye edilir (Şekil 1, Panel C) </stro&gt; Ng. MAM arayüzü ER ve mitokondri arasındaki mesafe mil 6, proksimite ligasyonu ve amplifikasyon bağlı Texas kırmızısı etiketli oligonükleotid sondalar (Şekil 1, Panel D hibridizasyonu sonraki tespiti yol yapılabilir 25-10 nm arasında değerler ). Her floresan nokta böylece tek tek hücrelerin in situ ER-mitokondri etkileşimleri sayılmasına olanak tanıyan, VDAC1 / IP3R1 arasındaki etkileşimleri temsil eder. <img alt="Şekil 1" src="/files/ftp_upload/54899/54899fig1.jpg" /> Şekil 1: Yerinde Proximity ligasyon Tahlili tarafından Endoplazmik Retikulum-mitokondri Etkileşimleri Algılama şematik İllüstrasyon. a) VDAC1 ve MAM arayüzü yakın kendi epitopuna bağlanabilir IP3R1 karşı bir tavşan primer antikora karşı yönlendirilmiş bir fare primer antikor, B) proksimite ligasyonu probları bir çift ilavesifare ve tavşan IgG karşı. Bu sondalar bağlayıcı oligo ligasyonu için şablonlar oluşturabilirler DNA ipliklerini eklenmiş. c) bağlanmasından sonra oluşturulan dairesel DNA zinciri Texas kırmızısı etiketli oligonükleotidler kullanılarak bir flüoresan nokta olarak mikroskopi ile görünür) yükseltilir ve D edilebilir. <a href="http://ecsource.jove.com/files/ftp_upload/54899/54899fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> In situ proksimite ligasyonu deneylerinde benzer antikorların GRP75 / IP3R1 çifti, hem de siklofilin D (CypD) ile yapılabilir / IP3R1 antikorlar CypD MAM arayüzü IP3R / GRP75 / SSVD kompleksi ile etkileştiği gösterilmiştir olduğu dikkate 12-14.

<table><tbody><tr><td>Formaldehyde</td><td>Sigma</td><td>F-8775</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G-8898</td><td></td></tr><tr><td>Triton</td><td>Sigma</td><td>T8532</td><td></td></tr><tr><td>35 mm Glass bottom culture dishes</td><td>MatTeK corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Blocking solution</td><td>Sigma</td><td>DUO-92004 or DUO-92002</td><td>provided in the Duolink PLA probes, Sigma</td></tr><tr><td>VDAC1 antibody</td><td>Abcam</td><td>ab14734</td><td></td></tr><tr><td>IP3R1-H80 antibody</td><td>Santa Cruz</td><td>sc28614</td><td></td></tr><tr><td>CypD antibody</td><td>Abcam</td><td>ab110324</td><td></td></tr><tr><td>Grp75 antibody</td><td>Santa Cruz</td><td>sc13967</td><td></td></tr><tr><td>TBS 10x</td><td>euromedex</td><td>ET220</td><td>Dilute to obtain 1x</td></tr><tr><td>Tween 100x</td><td>euromedex</td><td>2001-B</td><td>dilute in TBS to obtain 0,01%</td></tr><tr><td>PLA Probes Mouse MINUS</td><td>Sigma</td><td>DUO-92004</td><td>Duolink, Sigma</td></tr><tr><td>PLA Probes Rabbit PLUS</td><td>Sigma</td><td>DUO-92002</td><td>Duolink, Sigma</td></tr><tr><td>Duolink detection reagents red</td><td>Sigma</td><td>DUO-92008</td><td>Duolink, Sigma</td></tr><tr><td>Ligation solution</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Ligase</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Amplification solution</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Polymerase</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Duolink Mounting Medium</td><td>Sigma</td><td>DUO80102</td><td>Duolink, Sigma</td></tr><tr><td colspan="4">&lt;strong&gt;Softwares&lt;/strong&gt;</td></tr><tr><td>Blob-finder software</td><td></td><td></td><td>BlobFinder is a freely distributed software that can perform calculations on cells from fluorescence microscopy images. This software can be downloaded for free from The Centre for Image Analysis at Uppsala University who have developed the software and the work was supported by the EU FP6 Project ENLIGHT and Olink Bioscience. http://www.cb.uu.se/~amin/BlobFinder/index_files/Page430.htm</td></tr><tr><td>ImageJ software</td><td></td><td></td><td>Can be downloaded for free from: http://rsb.info.nih.gov/ij/download.html</td></tr></tbody></table>

study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Bu protokolü kullanarak bizim deneyime dayalı, biz güvenle sabit hücrelerde ER-mitokondri etkileşimleri görselleştirme ve miktar tayini için bu yöntemi tavsiye ederim. Antikorların çeşitli çiftleri kullanılarak HuH7 hepatokarsinoma hücre çizgisinde in situ proksimite ligasyonu deneyi-görüntülenmiştir ER-mitokondri etkileşimlerine temsili görüntüleri gösterilmektedir. Flüoresans mikroskopisi ile, Şekil 2&#39;de gösterildiği g...

Watch this Scientific Journal Video about Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells at JoVE.com

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Burada, sabit hücrelerde endoplazmik retikulum ve mitokondri arasındaki endojen etkileşimleri görselleştirmek ve yüksek hassasiyet ile ölçmek için bir prosedür açıklanmaktadır. Protokol, bir mitokondri ilişkili zar arayüzünde inositol 1,4,5-trifosfat reseptörü / glukoz-regüle proteini 75 / voltaj bağımlı anyon kanalı / siklofilin D kompleksi hedefleyen in situ yakınlık ligasyonu deneyinde optimize özellikleri.

Endoplazmik retikulum ve mitokondri Etkileşimleri Çalışma ile<em&gt; In Situ</em&gt; Proximity ligasyon Testi Sabit Hücrelerde

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

study-endoplasmic-reticulum-mitochondria-interactions-situ-proximity

Research

JoVE Journal

Biology

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

24.5K Görüntüleme.  Lund University.  Burada, sabit hücrelerde endoplazmik retikulum ve mitokondri arasındaki endojen etkileşimleri görselleştirmek ve yüksek hassasiyet ile ölçmek için bir prosedür açıklanmaktadır. Protokol, bir mitokondri ilişkili zar arayüzünde inositol 1,4,5-trifosfat reseptörü / glukoz-regüle proteini 75 / voltaj bağımlı anyon kanalı / siklofilin D kompleksi hedefleyen in situ yakınlık ligasyonu deneyinde optimize özellikleri. 

Video: Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Co-immunoprecipitation tarafından Nükleer ve Sitoplazmik Kesirler Protein-protein Etkileşim görselleştirme ve<em&gt; In Situ</em&gt; Proximity ligasyon Testi

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Biyokimya

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Heterodimerization in Situ yakınlık ligasyonu tahlil kullanarak Protein izoformlarının tespiti

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Göreli ışık ve hacim elektron mikroskobu ile mitokondri ve endoplazmik reticulum görüntüleme

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Yakınlık Ligasyon Tayini kullanılarak C. elegans Germline Ribonükleoprotein Kompleksi Montaj Yerinde Algılama

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Gelişim Biyolojisi

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Algılama<em&gt; In Situ</emEn&gt; Protein-protein kompleksleri<em&gt; Drosophila</emProximity ligasyon Testi Kullanımı&gt; Larva Nöromüsküler Junction

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Nörobilim

<meta charset="utf8"></head><body>Yüksek Verimli Ekranlar için ER-Mitokondri Kontaklarını Ölçmek İçin Basit Bir Test

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>Yazar Spotlight: ER-Mitokondri Temaslarının Düzenlenmesi ve Düzensizliği — Nörodejeneratif Hastalık Patogenezi için Çıkarımlar

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.

Canlı Hücrelerde Endoplazmik Retikulum-Mitokondri Temaslarını Ölçmek için Split-Luciferase Yeniden Birleştirme Testi

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

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Kültür Hücrelerinin retikulum Subdomain&#39;ler Görselleştirme

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Biyoloji

Correlative Microscopy Reveals Stress-Induced Mitochondria-Lysosome Interactions

Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for ...

Endoplazmik retikulum ve mitokondri Etkileşimleri Çalışma ile

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Co-immunoprecipitation tarafından Nükleer ve Sitoplazmik Kesirler Protein-protein Etkileşim görselleştirme ve

Heterodimerization in Situ yakınlık ligasyonu tahlil kullanarak Protein izoformlarının tespiti

Göreli ışık ve hacim elektron mikroskobu ile mitokondri ve endoplazmik reticulum görüntüleme

Yakınlık Ligasyon Tayini kullanılarak C. elegans Germline Ribonükleoprotein Kompleksi Montaj Yerinde Algılama

Algılama

Canlı Hücrelerde Endoplazmik Retikulum-Mitokondri Temaslarını Ölçmek için Split-Luciferase Yeniden Birleştirme Testi

Canlı Hücrelerde Endoplazmik Retikulum-Mitokondri Temaslarını Ölçmek için Split-Luciferase Yeniden Birleştirme Testi

Canlı Hücrelerde Endoplazmik Retikulum-Mitokondri Temaslarını Ölçmek için Split-Luciferase Yeniden Birleştirme Testi

Kültür Hücrelerinin retikulum Subdomain'ler Görselleştirme

Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes