Biz Dr. Scott Henderson konfokal mikroskobu ile yardım için kabul etmek istiyoruz. Bu araştırma, WAB Mikroskopi Tıp Massey Kanser Merkezi ve Okulu (VCU) VCU-Bölümü yapıldı WAB için Ulusal Sağlık 1RO1CA127501 Enstitüleri hibe yanı sıra pilot proje finansmanı tarafından desteklenmiştir. Nörobiyoloji &amp; kısmen NIHNINDS Merkezi çekirdekli hibe 5P30NS047463 finansman ile desteklenen Anatomi Mikroskopi Tesisi,,.

Protokol üç önemli adımlar oluşur. Memeli bir ifade amino-terminal CFP ve / veya YFP monomerik sürümleri için vektör sadece kısa bir süre tartışılan olacaktır ilgi gen ilk adım klonlama. Ikinci ve üçüncü adımları EA.hy926 endotel hücreleri içine vektör DNA transfeksiyon ve FRET ile konfokal görüntüleme, daha derinlemesine aşağıda özetlenmiştir. 1. OBP ve YFP Kimerik Reseptörler İnşaatı Ilgi Reseptörler CFP ve YFP monomerik gelişmiş versiyonları için amino-terminal klonlanmış olmalıdır. FRET, belirlenmesi, deneysel ve eleştirel transmembran bölgeye yayılan ve fluorofor 1 başlaması arasında ilintileyici uzunluğu üzerine bağlıdır . Bu nedenle, birkaç farklı uzunluklarda ilintileyici soruşturma altında her yeni reseptör çifti için araştırılmalıdır. <ol><li> Monomerik C veya YFP (bu vektör laboratuarımızda elde edilebilir) içeren pcDNA3.1 rutubet R ya da neo R vektör içine NheI / EcoRI sitelerine ilgi reseptör klonu. * Not Gerekirse, NheI SpeI ve XbaI dahil uyumlu diğer birçok enzimler. </li><li> Uygun bir endA1 reca yetkili hücre hattı (rutin Mach1 kullanmak) ligasyon reaksiyonu dönüştürün. Ekran kolonilerin varlığı eklemek için DNA mutasyonlarının yokluğunda doğrulamak için sıralama tarafından izledi. </li><li> Başarılı moleküler klonlama konformasyon elde edilmesi üzerine, Qiagen, ya da benzer DNA kitleri kullanılarak steril su veya TE transfeksiyon dereceli vektör DNA hazırlamak. UV spektroskopisi kullanarak, konsantrasyon ve saflık değerlendirin. DNA tipik verim 100-200 mikrogram, rutin bir çalışma stok 1 mcg / ml &#39;ye seyreltilerek aralığında olmalıdır. </li></ol> 2. EA.hy 926 Hücreler, Transfeksiyon <ol><li> Mattek lamel (0) sayılı, 2 ml orta 35 mm yemekleri tam-DMEM yetişen EA.hy 926 hücreler 4, (% DMEM 10 fetal sığır serumu + Pen. / Strep.) Böl . Konservatif bir konfluent çanak 15 cm ~ 15 yemekleri (~% 90 konfluent ertesi gün) elde edebilirsiniz. Her bir reseptör ifade yanı sıra her alıcı çifti için bir çanak değerlendirmek için bir tabak hazırlayın. </li><li> Hücrelerin 37% 5 CO 2 atmosferde gece boyunca inkübe İzin ° C Ertesi gün, kültürleri, ~% 70-90 konfluent olmalıdır. </li><li> Transfeksiyon gün, Fugene6 8 mcL kimerik reseptör vektör DNA (2 mcL) 2 mg, önceden ısıtılmış OptiMEM 200 mcL karıştırılarak nucleolipid kompleksleri hazırlayacak. Reseptör çiftleri için, her reseptör 2 mikrogram toplam 1 mcg transfect (aşağıya tartışma bakın). </li><li> Tersine çevirerek yavaşça karıştırın. </li><li> 30 dakika oda sıcaklığında inkübe devam edin. </li><li> Kuluçka dönemi sırasında, hücrelerin transfeksiyon için hazırlamak. Taze medya aspire ve antibiyotik olmadan önceden ısıtılmış 2 mL tam-DMEM (DMEM + FBS) ilave önce sıcak PBS ile hafifçe ve kısaca her plaka yıkayın. Fugene6 ile tedavi sırasında hücrelere çok daha fazla toksik olduğu, Penisilin ve Streptomisin varlığını önlemek için önemlidir. </li><li> Hücrelere bilge karmaşık damla ekleyin ve ek bir 20-24 saat boyunca inkübe 37 ° C,% 5 CO 2 atmosfer . </li><li> 24 saat sonra hücrelerin, dikkatlice medya aspirat ve antibiyotikler ile taze tam DMEM pipetle. Transfeksiyon sonra, hücre canlılığı nispeten yüksek kalmalıdır, yani hiçbir kayan hücreleri çok az dikkat edilmelidir. </li><li> Gen ifadesi, genellikle ek bir 48-60 saat sonrası transfeksiyon sonra maksimum. Ancak, görüntüleme, 24 saat gibi kısa bir tipik olarak uygulanabilir. En iyi gen ekspresyonu için zaman ders deneyler yapılmalıdır. </li></ol> 3. Konfokal Görüntüleme ve Analiz FRET Canlı hücre görüntüleme mavi diyot (405 nm), Argon (458, 476, 488, 514 nm) ile donatılmış bir Leica TCS SP2 Kelime Size Neyi Çağrıştırıyor konfokal lazer tarama mikroskobu yapılır. yeşil HeNe (543 nm), turuncu HeNe (594 nm), ve kırmızı HeNe (633 nm) lazerler, bir HCX PI Apo 63x/1.3 na gliserin daldırma objektif lens, bir motorize XY (Märzhäuser) ve çevre kontrollü ( sıcaklık, nem ve CO 2) evre inkübatör (Pecon). Deneysel kontrolleri fluorofor emisyon kanallarının yanı sıra, değerlendirmek ifade sorunları ve non-spesifik fluorofor multimerization arasında çapraz tartışma ortadan kaldırmak için kritik öneme sahiptir. Gibi, tek fluorofor transfections her görüntü oturumu kurmak için kullanılmaktadır. Negatif kontroller (bilinen noninteracting reseptörleri kullanarak), arka plan belirlemek amacıyla yapılacak gerekecektir FRET FRET aşırı ifade nedeniyle oluşur. <ol><li> CFP YFP için emisyon kanallar arasında crosstalk ortadan kaldırmak için, CFP ifade kontrol sahne kuvöz içine yerleştirin. Beyaz ışık kullanarak, odaklanmak için Z plaka ayarlamakhücrelere. </li><li> CFP 465-505 nm ve 525-600 nm YFP emisyon algılama SP pencere ayarları ayarlayın. </li><li> Hem de emisyon kanalları izleme, 458 nm&#39;de CFP heyecanlandırmak. AOTF kullanarak, YFP emisyon kanal içine CFP üzerinde kayda değer bir sızdırma ortadan kaldırmak için lazer güç ayarlayın. Bu ayarı kaydedin. </li><li> Sadece YFP ifade hücreleri için 514 nm uyarma gücü ayarlayarak CFP kanal YFP karışma (YFP ifade kontrolü kullanarak) aynı kontrol gerçekleştirin. Bu ayarı kaydedin. </li><li> Sonraki yerde hücrelerin ortak ifade, sahne kuvöz içine CFP ve YFP. Leica konfokal yazılımı için sıra ayarını kullanarak, CFP ve YFP uygun membran lokalizasyonu ile benzer seviyelerde ifade hücrelerin bulun. </li><li> Leica yazılım acceptor fotoğraf beyazlatma programı kullanarak, respectably, kaydedilmiş CFP ve YFP ayarları donör ve alıcı ayarlarını. </li><li> Zoomlama hücre, hücre zarının meydana gelen ve programa başlamak için YFP fotoğraf yok olduğu bir yatırım getirisi (faiz bölge) vurgulayın. </li><li> Acceptor fotoğraf imhası için% 100, 514 nm&#39;de AOTF ayarlamak ve YFP emisyonu% 70 tükendiği kadar devam beyazlatma lazer izin. Beyazlatma hızlandırmak için, ROI&#39;ler genellikle lazer tarama ekseni (X-ekseni) seçilir. </li><li> Foto-beyazlatma sırasında, bu ölçümler, sahte verimlilik FRET rapor gibi, hücresel hareketi izlemek ve XY düzleminde kayda değer bir hareketi ile elde edilen ölçümler reddetmek. </li><li> Donör (OBP) ve alıcı (YFP) için, öncesi ve sonrası-bleach görüntüleri kaydedilmiş ve verimlilik gibi hesaplanır FRET: Bütün D Mesaj&gt; D öncesi D ön Verim = (D post-D ön) / D post FRET ve D sonrası sırasıyla photobleaching önce ve sonra donör yoğunluğu. </li><li> JMP Yazılım (SAS), deneysel değişkenlik boyutunu belirlemek için kullanılır. </li></ol> 4. Temsilcisi Sonuçlar Transfeksiyon verimleri% 30 ila% 40 arasında genellikle. Başkaları tarafından önceki bulgulara rağmen, bir vektör ve ifade transfeksiyon diğer yana olduğunu görmedim. Gerçekten de, sık sık bir fluorofor chimera veya diğer özel ifade gözlemlemek. Tipik verimleri reseptör sistemleri arasında değişir FRET. Tie reseptör sistemi için tipik değerler için% 20-28 epitel hücreleri ve endotel hücreleri% 19-23. Negatif kontroller için tipik verimliliği% 2-3 altında. Değişkenlik ölçüde deneyimi ile büyük ölçüde azalacak. <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="Şekil 1" /> Şekil 1. Transfekte EA.hy 926 hücreler Temsilcisi görüntüler. EA.hy 926 hücre tek tabaka A) DIC görüntü. B) (A) görüntülenen hücreler Floresans görüntüsü. C) Overlay (A) ve (B) gösteren ~% 20-30 transfeksiyon verimliliği. <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="Şekil 2" /> Şekil 2. Temsilcisi acceptor CFP YFP arasında bir EA.hy926 hücrede meydana gelen FRET analizi fotoğraf beyazlatma CFP emisyon kanal (üst paneller) ve YFP emisyon kanal (alt iki panel) önce ayrı ayrı izlenir ve yazılan acceptor photobleaching . Photobleaching deneylere sınırlı ve yeşil kutu içinde bölge, hesaplanan değerler FRET edildi. FRET verimliliği sadece yönlendirme amaçlı görüntü birleştirilmiş bir CFP / YFP büyütülmüş bir kaplama düşük (mor-0.0) yüksek mutlak bir aralığı (kırmızı-1.0) olarak görüntülenir.

<ol>
	<li>Kim, M., Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+transmembrane+signaling+by+cytoplasmic+domain+separation+in+integrins.">Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.</a> Science. 301, 1720-1725 (2003).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipidmodified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Seegar, T. C. M., Eller, B., Tzvetkova-Robev, D., Kolev, M., Henderson, S. C., Nikolov, D. B., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie1-Tie2+interactions+mediate+functional+differences+between+angiopoietin+ligands.">Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.</a> Molecular Cell. 37 (5), 643-655 (2010).</li><li>Edgell, C. J., McDonald, C. C., Graham, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc Natl Acad Sci U S A. 80 (12), 3734-3737 (1983).</li></ol>

Başarısı için kritik olan birkaç adım vardır. Bunların arasında en belirgin iki kimerik reseptörleri arasında ifade göreli seviyeleri. Bu sorunu aşmak için, bir iki ilgi proteinleri ifade stabil hücre hatları yapmak veya eşdeğer ifade izin vektör DNA en iyi oranlar tespit edebilir. Benzer şekilde, bir yemek boyunca üniform olmayan transfeksiyon nedeniyle, protein düzeyleri nadiren hücreler arasındaki eşdeğer olacaktır. Bu nedenle, dikkat, &#39;düşük&#39;, &#39;yüksek&#39; expressors ayırt ödenmelidir. Ekran &#39;ortalama&#39; protein düzeyleri genellikle güvenilir ve tekrarlanabilir sonuç veren bu verimliliğin FRET. Laboratuarımızda bu konuda rahatlatmak için peşine düşmüş bir yöntem &#39;, hatta&#39; gen ekspresyonu adeno ve Lenti-virüs. Ayrıca, alternatif bir yöntem belirlemek için acceptor fotoğraf beyazlatma etkinlikleri sensitize emisyon FRET. Gerçek zamanlı olarak tek hücre izlemek için sensitize emisyon potansiyeline rağmen, biz acceptor fotoğraf beyazlatma daha duyarlı ve daha güvenilir olmak için bulduk rağmen. Son olarak, protein etkileşimleri izlemek için FRET kullanarak zor ve membran bağlı reseptörleri için linker uzunlukları dikkatli seçimi gerektirir. Ayrıca, C / YFP yanı sıra, sık sık büyük endoplazmik retikulum ve golgi aygıtı agregasyonunda protein ekspresyon düzeyleri ve sonuçları etkiler. Ancak, geçici, ya da dengesiz etkileşimleri, FRET kullanmak için ideal bir yöntem.

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<td>DMEM</td>
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<td>Invitrogen</td>
<td>11960-069</td>
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<td>Penicillin- Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15070-063</td>
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<td>&nbsp;</td>
<td>Hyclone</td>
<td>N/A</td>
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<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11058-021</td>
<td>&nbsp;</td>
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<td>FUGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11814443001</td>
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<td>Coverslip Dishes</td>
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<td>pcDNA3.1(+)</td>
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imaging protein-protein interactions in vivo

Protein-protein etkileşimleri, gerekli tüm hücresel süreçlerin bir işaretidir. Ancak, bu etkileşimlerin birçok co-immunoprecipitation gibi geleneksel biyokimyasal yöntemlerle belirlenmesi ve analiz önlenmesi, geçici ya da enerjik zayıf. Bu bağlamda, genetik encodable floresan proteinleri (GFP, TTT, vs) ve floresans spektrumu örtüşen ilişkili Förster rezonans enerji transferi (FRET) 1-3 kullanarak in vivo olarak zayıf etkileşimleri izlemek için yeteneğimizi devrim yarattı. Burada, endotel hücre yüzeyinde reseptör-reseptör etkileşimlerinde izlenmesi için bir FRET tabanlı yakınlık testinin detaylı kullanımı.

Watch this Scientific Journal Video about Imaging Protein-protein Interactions in vivo at JoVE.com

Imaging Protein-protein Interactions in vivo

Bu protokol bir FRET tabanlı yakınlık tahlil kullanarak nasıl görüntü protein-protein etkileşimleri açıklar.

Görüntüleme Protein-protein etkileşimleri In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

imaging-protein-protein-interactions-in-vivo

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

21.3K Görüntüleme.  Virginia Commonwealth University. Bu protokol bir FRET tabanlı yakınlık tahlil kullanarak nasıl görüntü protein-protein etkileşimleri açıklar.

Video: Imaging Protein-protein Interactions in vivo

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitin Dayalı Membran Maya İki Hybrid (MİT) Sistemi: protein-protein etkileşimleri belirlenmesi için güçlü bir araç

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Biyoloji

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Mikro desenli Yüzeyler, protein-protein etkileşimleri-vivo Algılama

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Biyoparlaklık Rezonans Enerji Transferi kullanma Canlı Hücrelerde Protein-protein etkileşimleri araştıran

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Yüksek yoğunluklu Fonksiyonel Protein mikrodizileri Probing Protein-protein Etkileşimleri Algılama

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Aracılığıyla DEĞİŞTİR Protein-protein etkileşimleri İnhibitörlerinin Geliştirilmesi: Tasarım ve Geliştirme Olmayan ATP Rekabetçi CDK inhibitörleri Uygulama

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Arabidopsis Protein-DNA Etkileşimleri Tespiti için Kromatin Immunoprecipitation Deneyi<em&gt; İn Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein etkileşimlerinin Autophagy araştırma çalışma

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Gelişmiş protein-protein etkileşimleri ve Kinetik DNA lezyonlar, çalışmaya Confocal mikroskobu teknikleri

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Görüntüleme Protein-protein etkileşimleri In vivo

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