Bizim işimiz, VC NIH / NIGMS, NSF, USDA / NIFA ve BARD hibe tarafından desteklenmektedir.

Arkaplan Bitki hücrelerarası bağlantıları aracılığıyla makromoleküllerin Symplastic ulaşım, plasmodesmata, birçok bitki patologlar ve biyologlar ilgi. Örneğin, bazı viral proteinler, viral hareketi 1-3 etkinleştirmek için plasmodesmal boyut dışlama sınırlarını düzenlemek için bilinmektedir . Ayrıca, önemli gelişim düzenleyiciler arasında bazı endojen proteinler, 4-hücreli olmayan-özerk çalışması için, muhtemelen plasmodesmata aracılığıyla hücreden hücreye geçmek için varsayılır. Bu nedenle, bitki hücreleri arasındaki makromoleküler taşımacılığı tanımlamak ve görselleştirmek için güvenilir bir yöntem çok talep. 1) Hedef bitkiler büyümek Dönüşüm verimliliği yüksek, sağlıklı, sağlam bitkiler kullanılmalıdır. 1. Arabidopsis bitkiler Çevre kontrollü bir odasında kısa bir fotoperiyodun (130-150 μE m -2 s -1 ışık 8 saat 23 ° Grow bir tencerede Pro-Mix BX bir Arabidopsis bitkisi (10 cm x 10 cm x 10 cm ) C/16 saat karanlık 20 ° C) ve 6 ila 8 hafta 5 için% 40-65 bağıl nem. 5 açıklandığı gibi piyasada bulunan ürünleri ile zaman zaman onları Gübreleme . 15 mm x 35 mm (uzunluk ölçümü sapı dahil olmak üzere) daha büyük boyutu ile Yapraklar deneyler için seçilir. 2. N. benthamiana ve N. tabacum Pro-Mix BX bir bitki yetiştirmek uzun bir fotoperiyodun (23 ° C&#39;de 16 saat 130-150 μE m -2 s -1 ışık çevre kontrollü bir odasında bir pot (20 cm x 20 cm x 20 cm ) / 8 karanlık saat 20 ° C) ve 7 ila 10 hafta boyunca% 40-65 bağıl nem. Tarif olarak piyasada bulunan ürünleri ile zaman zaman onları gübreleyin. N. boyutu 50 mm daha büyük x 70 mm ile yaprak N. benthamiana, ya da 100 mm x 125 mm tabacum (bu uzunluk ölçümleri sapı dahil değildir) deneyler için seçilir. 2), DNA-kaplı Altın Gen Tabancası Kartuşu Hazırlık Mikroküreler Bu deneysel bir adım için protokol daha önce 6 ayrıntılı olarak tarif edilmiştir . Yüksek konsantrasyonlarda (~ 1 mcg / ml) Deneyin sonraki aşamalarında konfokal mikroskopi analiz kolaylığı açısından en yüksek dönüşüm verimi elde etmek için iyi arıtılmış plazmid DNA kullanımı çok önemlidir. Bu testte için, floresan sinyal kümesi ile ilişkilendirerek hücrelerinin sayıları symplastic taşıma ölçüde (yani bir göstergesi olarak kullanılması nedeniyle, hazırlanan kartuş, yüksek frekansta aynı anda iki veya daha fazla bitişik hücre dönüşümü yaptığı tespit etmek önemlidir. , çok hücreli sinyal kümesi) hareketi gösterir iken sinyal içeren tek bir hücre, herhangi bir hareketi gösterir. Her bir kartuş kaliteli protein 16-20 saat bombardımanı ifade analiz ederek kontrol edilebilir. Bizim protokol 6 sadece böylece bu deney için uygun olan bu zaman dilimi içinde bütün ifade olaylar &lt;% 3, çok hücreli ifade kümeleri kapasiteli kartuşlar üretir . 3) DNA kaplı Mikroküreler Biyolistik Teslimat <ol><li> Keskin bir jilet ile aynı gelişim aşamasında (yani aynı boyutta ve aynı yaş, adım 1) yaprakları çıkarın ve hemen eksen dışı tarafı yukarı bakacak şekilde, düz bir strafor yüzeye koyun. Yaprak eksen dışı tarafında nispeten küçük boyutu nedeniyle Arabidopsis yaprakları, pencere perde mesh bir parça ile kaplanmış olmalıdır. Alt trikom yoğunluğu ve ince kütikül nedeniyle bombardımanı için daha iyi bir substrat temsil eder ve sonra mesh pushpins ile güvence strafor yüzey. Bakımı düz parçacık teslim verimliliği artırır ve microbombardment sırasında doku hasarı en aza indirir bırakır. </li><li> Tabanca içine DNA kaplı mikro (adım 1 hazırlanan) kartuşu takın. Arabidopsis, çekim, 0.6 mikron mikro 1 mikron mikro ve 140-160 psi için 80-110 psi basınçta yapılır . N. benthamiana ve tütün 0.6 mikron mikro için, çekim 1 mikron mikro ve 160-180 psi 100-120 psi basınçta yapılır. Arabidopsis için her yaprak sadece bir atış mikro 10-12 mm çapında bir alana yayılır için yeterli yüzey alanına sahip olduğundan, genellikle, yaprak başına tek bir kartuş kullanmaktadır. Büyük N. ile benthamiana ve tütün yaprakları, aynı yaprak birden fazla bombardımanları mümkündür, ancak, onlar (Tartışma), aynı gelişimsel aşamada yaprak alanları hedef orta kaburga her iki tarafta simetrik pozisyonlarda yapılması gerekir . </li><li> Strafor yüzey yaprakları çıkarın ve onları i yerdeıslak Whatman filtre kağıdı 3 kat üzerinde bir Petri kabı, Parafilm Petri kabı mühür ve protein ürünleri teslim transgenlerin ve hücre-hücre hareketi potansiyel ifade izin oda sıcaklığında 36-48 saat süreyle inkübe . </li></ol> 4) Görüntüleme Protein Ekspresyonu Geçici olarak ifade etiketli proteinlerin floresan sinyal konfokal mikroskobu ile görüntülenmiştir. Bakım her test protein tespiti için en uygun mikroskopi ayarları bulmak için alınmalıdır. Örneğin, proteinler, Tütün mozaik virüsü hareket protein (TMV MP) 1-3,6 plasmodesmal yerelleştirme gibi zayıf sinyal intensitesi ile sınırlı intrasellüler birikimi, yüksek çözünürlük ve tanıyor 40X objektif lens altında gözlenen olmalıdır hassasiyet, oysa ücretsiz YFP gibi güçlü bir sinyal yoğunlukları, sitoplazmik dağılım gösterir proteinler daha hızlı görüntüleme için konfokal zoom fonksiyonu ile 10X objektif lens (bkz. Şekil 1) altında görüntülenmiştir olabilir. Symplastic ulaşım, floresan sinyal içeren çok hücreli kümeleri görünümünü anlaşılmaktadır. Her küme, kümeler ve hücrelerin sayısı sayısı, hücre-hücre taşıma ölçüde göstergesidir. Güvenilir veri elde etmek için, her bir deneysel sistem başına en az 100 ekspresyonu kümeleri kayıt altına alınmalıdır. Örneğin, bir proteinin hücre-hücre hareketi toplam 200 ifade kümeleri iki farklı genetik geçmişleri (örneğin, vahşi tip ve transgenik bitkiler), karşılaştırıldığında ise kayıt altına alınmalıdır. Önemlisi, deneyler,, birbirlerine göre hangi sonuçları eşzamanlı olarak yürütülen olmalıdır. 5. Temsilcisi Sonuçlar Aşağıdaki şekil, floresan etiketli proteinlerin symplastic ulaşım tespiti için temsili deneyler göstermektedir. Paneller A ve B bir N. aşağıdaki microbombardment elde edilen tipik konfokal resimleri gösterir. TMV MP-YFP-ifade benthamiana yaprak oluşturun. Panelinde TMV MP-YFP symplastic hareket YFP sinyal çok hücreli kümeleri görünümünü dayalı görülmektedir. Tüm geçici olarak ifade TMV MP-YFP bazı microbombardments (panel B) tek hücreli sinyal kanıtladığı gibi hareket etmek mümkün değildir. İstatistiksel olarak, içinde &lt;% 40 sayılır sinyal kümeleri, TMV MP-YFP ise hücreler arasında hareket edemez&gt; kümelerin% 60, protein, iki hücre yayılmasını en sık olmak üzere, 2-5 hücreler arasında hareket eder (yaklaşık ~ 50 Olguların%) (Şekil 1C). Doğuştan hareket aktivite olmadan, nispeten küçük moleküler boyutu ile proteinlerin hücre-hücre taşımak için PD yoluyla diffüz. Örneğin, ücretsiz YFP veya 1xYFP (yaklaşık 27 kDa, panel D) sayılır kümeleri (panel C)% 30 çeşitli hücreler arasında yayılır. Bu non-spesifik difüzyon TMV MP-YFP füzyon proteini (57 kDa) boyutu karşılaştırılabilir ve tamamen hücre özerk olduğu bir translasyonel YFP sönük veya 2xYFP (54 kDa, panel E) oluşmaz (Şekil 1C). <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="Şekil 1" /> N. symplastic taşıma testte elde edilen Şekil 1. Tipik sonuçlar benthamiana yaprak dokuları. (A, B) Görselleştirme TMV MP-YFP. (C) sinyal kümeleri kantitasyonu. 1xYFP ve 2xYFP, ücretsiz YFP ve sırasıyla translasyonel YFP dimer. (D) 1xYFP Görselleştirme. (E) 2xYFP Görselleştirme. Mikrograflar, sol paneller YFP sinyal göstermiyor ve sağ panelleri birleştirilmiş YFP görüntüleri (yeşil) ve kloroplast otofloresans (beyaz) sinyalleri gösteriyor. Görüntüler tek konfokal bölümleridir. Mikrograflar Asteriskler YFP sinyali gösteren epidermal hücreler göstermektedir. Barlar = 50 mm.

<ol>
	<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+non-destructive+cell-to-cell+and+systemic+movement+of+plant+viruses.+.">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. .</a> Crit Rev Plant Sci. 23, 195-250 (2004).</li><li>Lucas, 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=Plant+viral+movement+proteins:+agents+for+cell-to-cell+trafficking+of+viral+genomes.">Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes.</a> Virology. 344, 169-184 (2006).</li><li>Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+spread+by+diffusion+on+ER-associated+movement-protein-rafts+through+plasmodesmata+gated+by+viral+induced+host+beta-1,3-glucanases.">Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host beta-1,3-glucanases.</a> Semin Cell Dev Biol. 20, 1074-1081 (2009).</li><li>Zambryski, P. C., Crawford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+gatekeepers+for+cell-to-cell+transport+of+developmental+signals+in+plants.">Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants.</a> Annu Rev Cell Dev Biol. 16, 393-421 (2000).</li><li>Rivero-Lepinckas, L., Crist, D., Scholl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+plants+and+presercation+of+seeds.">Growth of plants and presercation of seeds.</a> Methods Mol Biol. 323, 3-12 (2006).</li><li>Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transient+genetic+transformation+of+Arabidopsis+leaves+by+biolistic+bombardment.">Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment.</a> Nat Protoc. 4, 71-77 (2009).</li><li>Oparka, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+but+not+branched,+plasmodesmata+allow+the+nonspecific+trafficking+of+proteins+in+developing+tobacco+leaves.">Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.</a> Cell. 97, 743-754 (1999).</li><li>Imlau, A., Truernit, E., Sauer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+and+long-distance+trafficking+of+the+green+fluorescent+protein+in+the+phloem+and+symplastic+unloading+of+the+protein+into+sink+tissues.">Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.</a> Plant Cell. 11, 309-322 (1999).</li><li>Gisel, A., Barella, S., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+regulation+of+symplastic+trafficking+during+development+in+Arabidopsis+thaliana+apices.">Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.</a> Development. 126, 1879-1889 (1999).</li><li>Kim, I., Cho, E., Crawford, K. M., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+movement+of+GFP+during+embryogenesis+and+early+seedling+development+in+Arabidopsis.">Cell-to-cell movement of GFP during embryogenesis and early seedling development in Arabidopsis.</a> Proc Natl Acad Sci USA. 102, 2227-2231 (2005).</li><li>Crawford, K. M., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+and+targeted+protein+movement+through+plasmodesmata+in+leaves+in+different+developmental+and+physiological+states.">Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.</a> Plant Physiol. 125, 1802-1812 (2001).</li><li>Roberts, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+unloading+in+sink+leaves+of+Nicotiana+benthamiana:+comparison+of+a+fluorescent+solute+with+a+fluorescent+virus.">Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus.</a> Plant Cell. 9, 1381-1396 (1997).</li><li>Guenoune-Gelbart, D., Elbaum, M., Sagi, G., Levy, A., Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+(TMV)+replicase+and+movement+protein+function+synergistically+in+facilitating+TMV+spread+by+lateral+diffusion+in+the+plasmodesmal+desmotubule+of+Nicotiana+benthamiana.">Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana.</a> Mol Plant Microbe Interact. 21, 335-345 (2008).</li></ol>

Symplastic taşıma testinin başarı için anahtar, yüksek dönüşüm verimliliği, istatistiksel olarak anlamlı ve kolayca detectible sinyal kümeleri üretimini sağlar elde etmektir. Bu, sağlıklı, sağlam bitkiler hasat yapraklar kullanılarak, saf ve yoğun bir DNA hazırlığı ile kaplı altın parçacıkları hazırlanması sağlanabilir. Aynı büyüme aşamasında yaprakları kullanarak da testin güvenilirliği için hayati önem taşımaktadır. Plasmodesmal diyafram farkl?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
<tr>
<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
<tr>
<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
<tr>
<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2420</td>
<td>&nbsp;</td>
<tr>
<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
<td>&nbsp;</td>
<tr>
<td>Helios gene gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
<tr>
<td>Helium gas regulator</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2413</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

Burada, planta içinde hücre-hücre makromoleküler taşıma kapsamını tespit etmek ve değerlendirmek için basit ve hızlı bir protokol mevcut. Bu protokol, geçici bir ilgi floresan etiketli protein kodlama DNA Biyolistik teslim inşa aşağıdaki bitki doku ifade edilir. Etiketli protein içi ve hücreler arası dağılımı sonra konfokal mikroskobu ile analiz edilir. Biz detaylı olarak tahlil için adım adım protokoller sağlayarak, bu teknoloji tanımlayan ve üç bitki türlerinin symplastic protein taşıma ölçüde, Arabidopsis thaliana, Nicotiana benthamiana ve N. değerlendirmek tabacum (tütün).

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Bitki hücreleri arasındaki makromoleküler ticareti, geçici bir ilgi floresan etiketli protein ifade ve konfokal mikroskobu ile içi ve hücreler arası dağılımı analiz değerlendirilebilir.

-Cell Hücre makromoleküler Ulaştırma Testi Planta Biyolistik Bombardıman kullanımı

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

a-cell-to-cell-macromolecular-transport-assay-planta-utilizing

Research

JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

11.8K Görüntüleme.  State University of New York at Stony Brook. Bitki hücreleri arasındaki makromoleküler ticareti, geçici bir ilgi floresan etiketli protein ifade ve konfokal mikroskobu ile içi ve hücreler arası dağılımı analiz değerlendirilebilir.

Video: A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Transgenik, Üretimi C. elegans Biyolistik Dönüşüm

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Biyoloji

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Makromoleküler Kristalografi tarafından Yapı Tayini için lipidik Mesophases Yetiştirilen Membran Proteinlerinin Hasat ve Cryo-soğutma Kristaller

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ bağımlı CFTR makromoleküler Sinyal Kompleksleri İn Vitro Analizi

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Kültürleme<em&gt; Caenorhabditis elegans</em &gt; Mikroparçacık bombardımanı ile aksenik Sıvı Medya ve Transgenik Worms Yaratılış

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Otomatik Flüoresans Ömrü Görüntüleme Mikroskopi kullanmak Open Source Yüksek İçerik Analizi

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

Nötron Makromoleküler Kristalografi için Kristal Büyümesinin Optimizasyonu

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Örnek Oda Bazlı Işık Tabakası Floresan Mikroskoplarında Birden Fazla Böcek Embriyosunun EşZamanlı Canlı Görüntülenmesi

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

Memeli Hücrelerinde Çinko Taşıma Mekanizmasının FluoZin-3 Testi ile İncelenmesi

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Yazar Spotlight: ZnT1 İşlevselliği ile Hücresel Çinko Düzenlemesini Keşfetmek 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

İn Vitro Çinko Taşıma Testi Kullanılarak Memeli Çinko Taşıyıcılarının Karakterize Edilmesi

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...