We thank the Dictyostelium Stock Center for strains and reagents. This study was financed by grants from the Swiss National Science Foundation and the Canton of Basel.

<ol><li> Time-lapse mikroskobu ile Dictyostelium discoideum erken açlık yanıtı gözlemlemek. <ol><li> HPO 4 5 g proteoz pepton, 5g thiotone e pepton, 10 g glukoz 5 g maya özütü, 0.35 g 2 Na: 1 L için HL-5 ortamı (ihtiva eden bir Erlen bölgesi DH1.10 hücreleri ya da CORA-eksikli hücrelerin büyümesine * 7H 2 O, 0.35 gr KH 160 rpm&#39;lik bir rotasyon ile bir çalkalama inkübatöründe 22 ° C&#39;de 2 PO 4, 0.05 g dihidrostreptomisin-sülfat, pH 6.6). 0.01 x 10 6 hücre / ml ve 2 x 10 6 hücre / ml arasında bir yoğunlukta hücreler tutun. </li><li> Hasat log-faz büyüyen DH1.10 hücreleri 21 ya da 22 ° C &#39;de HL-5 orta kültür çalkalama yetiştirilen CORA-eksikli hücrelerin DH1.10 arka oluşturulan 10, hücre toplama inceleyin. Bunu yapmak için, 400 x g&#39;de 3 dakika boyunca hücreler (genellikle 10 ila 50 mi), santrifüj, uygun miktarda almak ve BSS (10 mM NaCI, 10 mM KCl hücreler iki kez yıkayın, 2.5 mM CaCl2, pH 6.5). </li><li> Bir hemasitometre kullanarak hücreleri saymak. Daha sonra, (5, 10, 20, ya da 40) bir yoğunlukta plaka hücreleri 24 oyuklu bir plaka içerisinde 10 4 hücre / cm2 x. bunları BSS 22 ° C de 1 saat yapışmaya izin verin. </li><li> Görüntüleri her 135 sn alarak, 10 daha önce açıklandığı gibi zaman atlamalı mikroskobu ile toplanmasını gözünüzde canlandırın. kurmak 5X objektif ve uygun yazılım tarafından otomatik bir elektron-çarparak şarj çiftli aygıt kamera ile donatılmış bir canlı hücre görüntüleme kullanarak (Malzeme Tablo). </li></ol></li><li> açlık sırasında Diktiyostelyum diskodeyum hücrelerinin cAMP nabız gibi atan. <ol><li> Hücrelerin hasat tarafından DH1.10 hücreleri 21 veya DH1.10 Cora -açıklı hücrelerin 10 gelişimine dışarıdan uygulanan cAMP darbelerinin etkisini incelemek. Bunu yapmak için, 400 x g&#39;de 3 dakika boyunca, uygun hücre miktarı (genellikle 10 ila 50 mi), santrifüj almak ve BSS içinde iki kez yıkayın. </li><li> couBSS 1 x 10 7 hücre / ml bir yoğunluğa hücreler bir hemasitometre kullanılarak ve tekrar süspansiyon nt hücreleri. bakliyat uygulamadan önce 2 saat boyunca 22 ° C &#39;de kültür (160 rpm) çalkalayın. Bir zamanlayıcı kontrollü peristaltik pompa kullanılarak cAMP darbeleri ekleyin. 5 saatlik bir süre boyunca 5 sn darbe 50 nM cAMP (son konsantrasyon) 15 ul, her 6.5 dakikada bir Pompayı programlayın. </li><li> Bir hemasitometre kullanarak hücreleri saymak. Daha sonra, (5, 10, 20, ya da 40) bir yoğunlukta hücreler plaka 24 oyuklu bir plaka içerisinde 10 4 hücre / cm 2 x. BSS 1 saat yapışmaya izin verin. </li><li> 5X objektif kullanılarak parlak alan mikroskopi ile 22 ° C de 16 saat sonra toplama görselleştirme. </li></ol></li><li> Durumunu ortama maruz kalma ile Dictyostelium discoideum gelişme indüksiyonu. <ol><li> 22 tarif edildiği gibi, taze koşullu ortam hazırlar. Kült sallayarak gelen log-faz DH1.10 hücreleri 21 veya DH1.10 Cora -açıklı hücreleri 10 topla400 x g&#39;de 3 dakika boyunca bir pipet, santrifüj kullanılarak 22 ° C&#39;de HL-5 ortamı ile etmiş olur ve PBM hücreleri üç defa yıkamak (0.02 M potasyum fosfat, 10 uM CaCl2, 1 mM MgCl2, pH 6.1). </li><li> Bir hemasitometre kullanarak hücreleri sayın mi, 1 x 10 7 hücre / yoğunluğunda PBM bu tekrar süspansiyon ve 110 rpm / 22 ° C&#39;de 20 saat süre ile çalkalanır. </li><li> 3 dakika boyunca 400 x g&#39;de santrifüj işleminden sonra koşullu ortam toplayın ve 4 ° C&#39;de 15 dakika boyunca 8000 x g&#39;da santrifüjleme ile belirler. </li><li> 0.45 um&#39;lik bir filtreden filtre koşullu ortam (Malzeme Tablo) ve PBM üç kat seyreltilir. </li><li> (5, 10, 20, ya da 40) bir yoğunlukta bir hemasitometre ve plaka kullanarak hücreleri sayın 24 oyuklu bir plaka içerisinde 10 4 hücre / cm2 x. bunları 22 ° C de 1 saat yapışmaya izin verin. </li><li> önceden hazırlanmış uygulanmış madde ile hücre değişimi yüzer. </li><li> toplama a görselleştirmek5X objektif kullanarak parlak alan mikroskobu ile fter 16 saat. </li></ol></li></ol>

<ol>
	<li>Weijer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+cell+movement+in+Dictyostelium.">Morphogenetic cell movement in Dictyostelium.</a> Semin Cell Dev Biol. 10, 609-619 (1999).</li><li>Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum:+a+model+system+for+cell-cell+interactions+in+development.">Dictyostelium discoideum: a model system for cell-cell interactions in development.</a> Science. 245, 1054-1058 (1989).</li><li>Kimmel, A. R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP+signal+transduction+pathways+regulating+development+of+Dictyostelium+discoideum.">cAMP signal transduction pathways regulating development of Dictyostelium discoideum.</a> Curr Opin Genet Dev. 1, 383-390 (1991).</li><li>Clarke, M., Gomer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSF+and+CMF,+autocrine+factors+that+regulate+gene+expression+during+growth+and+early+development+of+Dictyostelium.">PSF and CMF, autocrine factors that regulate gene expression during growth and early development of Dictyostelium.</a> Experientia. 51, 1124-1134 (1995).</li><li>Robertson, A., Drage, D. J., Cohen, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Aggregation+in+Dictyostelium+discoideum+by+an+External+Periodic+Pulse+of+Cyclic+Adenosine+Monophosphate.">Control of Aggregation in Dictyostelium discoideum by an External Periodic Pulse of Cyclic Adenosine Monophosphate.</a> Science. 175, 333-335 (1972).</li><li>Konijn, T. M., Chang, Y. Y., Bonner, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cyclic+AMP+in+Dictyostelium+discoideum+and+Polysphondylium+pallidum.">Synthesis of cyclic AMP in Dictyostelium discoideum and Polysphondylium pallidum.</a> Nature. 224, 1211-1212 (1969).</li><li>Iranfar, N., Fuller, D., Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+analyses+of+gene+regulation+during+early+development+of+Dictyostelium+discoideum.">Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum.</a> Eukaryot Cell. 2, 664-670 (2003).</li><li>Darmon, M., Brachet, P., Da Silva, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+signals+induce+cell+differentiation+in+Dictyostelium+discoideum.">Chemotactic signals induce cell differentiation in Dictyostelium discoideum.</a> Proc Natl Acad Sci U S A. 72, 3163-3166 (1975).</li><li>Mann, S. K., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+regulation+of+early+gene+expression+in+Dictyostelium+discoideum:+mediation+via+the+cell+surface+cyclic+AMP+receptor.">Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.</a> Mol Cell Biol. 7, 458-469 (1987).</li><li>Vinet, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+multicellular+differentiation+in+Dictyostelium+discoideum+is+regulated+by+coronin+A.">Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A.</a> Mol Biol Cell. 25, 688-701 (2014).</li><li>Eckert, C., Hammesfahr, B., Kollmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+holistic+phylogeny+of+the+coronin+gene+family+reveals+an+ancient+origin+of+the+tandem-coronin,+defines+a+new+subfamily,+and+predicts+protein+function.">A holistic phylogeny of the coronin gene family reveals an ancient origin of the tandem-coronin, defines a new subfamily, and predicts protein function.</a> BMC Evol Biol. 11, 268 (2011).</li><li>Pieters, J., Muller, P., Jayachandran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+guard:+coronin+proteins+in+innate+and+adaptive+immunity.">On guard: coronin proteins in innate and adaptive immunity.</a> Nat Rev Immunol. 13, 510-518 (2013).</li><li>Gatfield, J., Albrecht, I., Zanolari, B., Steinmetz, M. O., Pieters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+Leukocyte+Plasma+Membrane+with+the+Actin+Cytoskeleton+through+Coiled+Coil-mediated+Trimeric+Coronin+1+Molecules.">Association of the Leukocyte Plasma Membrane with the Actin Cytoskeleton through Coiled Coil-mediated Trimeric Coronin 1 Molecules.</a> Mol Biol Cell. 16, 2786-2798 (2005).</li><li>Kammerer, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+trimerization+motif+controls+the+topology+of+short+coiled+coils.">A conserved trimerization motif controls the topology of short coiled coils.</a> Proc Natl Acad Sci U S A. 102, 13891-13896 (2005).</li><li>Uetrecht, A. C., Bear, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronins:+the+return+of+the+crown.">Coronins: the return of the crown.</a> Trends Cell Biol. 16, 421-426 (2006).</li><li>Jayachandran, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin+1+Regulates+Cognition+and+Behavior+through+Modulation+of+cAMP/Protein+Kinase+A+Signaling.">Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling.</a> PLoS Biol. 12, e1001820 (2014).</li><li>Suo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin-1+is+a+neurotrophin+endosomal+effector+that+is+required+for+developmental+competition+for+survival.">Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.</a> Nat Neurosci. 17, 36-45 (2014).</li><li>Mueller, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+survival+through+coronin-1-mediated+generation+of+inositol-1,4,5-trisphosphate+and+calcium+mobilization+after+T+cell+receptor+triggering.">Regulation of T cell survival through coronin-1-mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor triggering.</a> Nat Immunol. 9, 424-431 (2008).</li><li>de Hostos, E. L., Bradtke, B., Lottspeich, F., Guggenheim, R., Gerisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin,+an+actin+binding+protein+of+Dictyostelium+discoideum+localized+to+cell+surface+projections,+has+sequence+similarities+to+G+protein+beta+subunits.">Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.</a> EMBO J. 10, 4097-4104 (1991).</li><li>Shina, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundant+and+unique+roles+of+coronin+proteins+in+Dictyostelium.">Redundant and unique roles of coronin proteins in Dictyostelium.</a> Cell Mol Life Sci. 68, 303-313 (2011).</li><li>Cornillon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phg1p+is+a+nine-transmembrane+protein+superfamily+member+involved+in+dictyostelium+adhesion+and+phagocytosis.">Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis.</a> J Biol Chem. 275, 34287-34292 (2000).</li><li>Gomer, R. H., Yuen, I. S., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+80+x+10(3)+Mr+protein+mediates+sensing+of+cell+density+and+the+onset+of+development+in+Dictyostelium.">A secreted 80 x 10(3) Mr protein mediates sensing of cell density and the onset of development in Dictyostelium.</a> Development. (112), 269-278 (1991).</li><li>Iijima, N., Takagi, T., Maeda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteinous+factor+mediating+intercellular+communication+during+the+transition+of+Dictyostelium+cells+from+growth+to+differentiation.">A proteinous factor mediating intercellular communication during the transition of Dictyostelium cells from growth to differentiation.</a> Zoological science. 12, 61-69 (1995).</li><li>Mehdy, M. C., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+factor+and+cyclic+AMP+jointly+regulate+cell-type-specific+gene+expression+in+Dictyostelium+discoideum.">A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.</a> Mol Cell Biol. 5, 705-713 (1985).</li><li>Jain, R., Yuen, I. S., Taphouse, C. R., Gomer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+density-sensing+factor+controls+development+in+Dictyostelium.">A density-sensing factor controls development in Dictyostelium.</a> Genes Dev. 6, 390-400 (1992).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+during+development+of+Dictyostelium.">Cell signaling during development of Dictyostelium.</a> Dev Biol. 391, 1-16 (2014).</li></ol>

No conflicts of interest declared.

Koronin proteinler ökaryotik dalının en takson bulunurlar. Dictyostelium discoideum Koronin bir memeli Koronin 1 homologu hücreler erken gelişim sırasında çekiş merkezleri oluşturmak mümkün değildir bir eksikliği Koronin yana erken açlık yanıt olarak katılır döngüsü 10. nicel ve doğru bir suşları arasında gelişiminde gecikme değerlendirmek mümkün, otomatik sahne denetleyicisi ile bir mikroskop canlı hücre görüntüleme set-up önemli bir araçtır. <p class="jove_...

Proteinlerin Koronin aile çok ökaryotlarda boyunca muhafaza edilir. Bu proteinler, bir amino-terminali triptofan-aspartat (WD) tekrar-ihtiva eden bir karboksi-terminal halkalaştırılmış halka etki 13,14 bağlı eşsiz bir bölgesi takip bölge (Şekil 1) bulunması ile karakterize edilir. Coronins iskelet düzenlemesi ve sinyal iletimi 12 de dahil olmak üzere hücresel fonksiyonların çeşitli implike edilmiştir. Memelilerde, altı adede kadar kısa Koronin molekülleri (Koronin 1-6) ve 7 Koronin bir tandem, 12,15 birlikte ifade edilebilir. Koronin 1 üzerinde en çok çalışılan aile üyesi ve patojen yok, T hücre hayatta kalma ve nöronal sinyal dahil olduğu gösterilmiştir. Nasıl, tam, Koronin 1 bu faaliyetleri yürütmektedir belirsizliğini koruyor. 1 Koronin Ca 2+ ve cAMP bağımlı sinyal yanı sıra F-aktin olarak hücre iskeleti modülasyonu 16-18, potansiyel işbirliği düzenleyen gösterilmiştir ikenMemelilerde kadar 7 aile üyelerinin -Anlatım zorlu nedeniyle potansiyel işten çıkarmalara, bu sistemlerdeki coronins moleküler fonksiyonunu incelemek için yapmıştır. Memeli organizmalardan farklı, alt ökaryot Diktiyostelyum diskodeyum görünüşte olmayan gereksiz fonksiyonları 15,19,20 ile sadece iki Koronin aile üyelerini (Koronin A, memeli Koronin 1 ve Koronin B ortologunu, memeli Koronin 7 ortologu) ifade eder. Bu durum, Dictyostelium discoideum coronins fonksiyonu çalışma güçlü bir model haline getirir. Dictyostelium discoideum&#39;da Koronin A&#39;nın rolünü incelemek için, 10 Koronin barındırmayan vahşi tipli hücreler veya hücre kullanılarak dengelenmiş tuz çözeltisi (BSS) tampon içeren doku kültürü plakaları içinde açlık gelişim döngüsü neden olmuştur. Biz Koronin A eksik hücreler açlık üzerine çok hücreli agrega oluşturmak mümkün olduğunu bulmuşlardır. Bu fenotip doğru kantitatif bir değerlendirme içinBu protokol açıklanan otomatik canlı hücre görüntüleme önemli bir araçtır. Bir Koronin eksik hücrelerin erken açlık tepkisinin başlamasında kusur cAMP çağlayan yukarı bir eylemleri Koronin düşündüren, cAMP bakliyat tedarik tarafından kurtarıldı edilebilir. Gelişiminin başlaması simüle etmek için, cAMP darbelerinin egzojen uygulaması son 8,9 çeşitli laboratuarlar tarafından kullanılmıştır. Bununla birlikte, bu prosedür, aynı zamanda, hücre yoğunlukları ve zamanlaması büyük ölçüde bağlı olduğu bilinmektedir. Bu nedenle, burada açıklanan protokol tekrarlanabilirliği yüksek derecede garanti altına almak için, bu bir değişkenliğe azaltmayı amaçlamaktadır. Birlikte ele alındığında, bu çalışmalarda kullanılan teknikler Dictyostelium discoideum gelişimsel döngüsünün erken evrelerinde proteinlerin işlevleri araştırmak için güçlü araçlar sağlar ve yukarı tespit hem de bir işlevi Koronin aşağı doğru efektör potansiyeline sahiptir.

<table><tbody><tr><td>HL-5 media (for 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O, 0.35 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)</td><td></td><td></td><td></td></tr><tr><td>Proteose peptone</td><td>BD Bioscience</td><td>211693</td><td></td></tr><tr><td>Thiotone E peptone</td><td>BD Bioscience</td><td>211684</td><td></td></tr><tr><td>Yeast extract</td><td>BD Bioscience</td><td>212750</td><td></td></tr><tr><td>Glucose</td><td>AppliChem</td><td>A3666</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fluka</td><td>71643</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1043</td><td></td></tr><tr><td>dihydrostreptomycin-sulfate</td><td>Sigma-Aldrich</td><td>D1954000</td><td></td></tr><tr><td>PBM (0.02 M potassium phosphate, 10 &amp;mu;M CaCl&lt;sub&gt;2&lt;/sub&gt;, and l mM MgCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.1)</td><td></td><td></td><td>self made</td></tr><tr><td>BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.5)</td><td></td><td></td><td>self made</td></tr><tr><td>0.45-&amp;mu;m Filtropure S filter</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>Falcon 24-well Tissue culture plate</td><td>Fisher Scientific</td><td>08-772-1H</td><td></td></tr><tr><td>Cellobserver microscope</td><td>Zeiss</td><td></td><td>custom built</td></tr><tr><td>AxioVision software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>IPC Microprocessor&amp;ndash;controlled dispensing pump</td><td>ISMATEC</td><td>ISM 931</td><td></td></tr><tr><td>Axiovert 135M microscope</td><td>Zeiss</td><td>491237-0001-000</td><td></td></tr><tr><td>Incubation Shaker</td><td>Inforst HT Minitron</td><td></td><td></td></tr></tbody></table>

investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays

Dictyostelium discoideum amiplerin beslenen toprak bulunur. Besin kaynakları kıt hale geldiğinde, tek hücre toplama merkezlerine 1-4 doğru chemotax sırasında bir çok hücreli geliştirme programını başlatmak için faktörleri salgılar. Bu işlem, siklik adenozin monofosfat (cAMP) 5 serbest bağlıdır. cAMP adenilat siklaz ve fosfodiesterazlarm birlikte hareket dalgasından üretilen ve G protein-eşli reseptör cAMP 6,7 ile bağlanır. Yaygın olarak kullanılan deney, daldırılmış koşullar 8,9 hücre toplanmasının gözlemine dayanır Alt düzey ökaryot Dictyostelium discoideum gelişimsel döngüsünde yer alan mekanizmaların analiz etmek. Bu protokol, dengelenmiş tuz çözeltisi (BSS) 10 içine daldırılmış doku kültürü plakaları içinde açlık gelişim döngüsünde Koronin A rolünün analizine açıklanmaktadır. Koronin bir yaygın korunmuş pr üyesidirfaaliyetleri 11,12 çeşitli suçlanmıştır coronins bir otein ailesi. A Koronin eksik Dictyostelium hücreleri çok hücreli agrega oluşturamamaktadırlar ve bu kusur düşündüren, cAMP bakliyat tedarik tarafından kurtarıldı olabilir üst baş eylemleri Koronin cAMP 10 basamakla. Bu çalışmalarda tarif edilen teknikler cAMP kaskadının yukan Dictyostelium discoideum gelişimsel döngüsünün ilk aşamalarında proteinlerin işlevleri araştırmak için güçlü araçlar sağlar. Bu nedenle, bu toplama tahlil kullanan bir fonksiyon Koronin ve daha fazla çalışma izin ve Koronin biyoloji anlayışımızı ilerleyebilir.

Erken gelişme bir gösterisi bir kusur Koronin eksik hücreleri (Şekil 2). Bir hücre Koronin yokluğunda Dictyostelium discoideum gelişim devri sırasında ilk adımdır çok-agregatlar oluşturmak için mümkün değildir. Bu nedenle, Koronin erken açlık yanıtı ve / veya cAMP sinyali sırasında önemli bir rol oynadığı görülmektedir. Gerçekten de, Koronin A&#39;nın olmaması çok hücreli agrega oluşumunun olmaması, indirgenmiş cAMP 10</...

Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Erken Açlık Tepki Koronin A Fonksiyonu incelenmesi<em&gt; Dictyostelium discoideum</em&gt; Toplama Tahliller

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

investigating-function-coronin-early-starvation-response

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

7.2K Görüntüleme.  University of Basel. The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Video: Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Genetik Mühendisliği hücreleri Dictyostelium discoideum alan seçimi ve bakteri büyüme

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Genetik

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Caenorhabditis elegans'ta Otomatik Inklüzyon Sayımı Kullanarak Protein Agregasyon Kinetiğinin In Vivo'da İzlenmesi

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Biyokimya

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Görüntüleme G-protein Coupled Reseptör (GPCR) aracılı Sinyal Olaylar Kontrolü Kemotaksis Diktiyostelyum diskodeyum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Biyoloji

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

Heterolog Hücre Sisteminde Protein Toplama Yoluyla Hücrelerarası Etkileşimlerin Ölçülmesi

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

Nörobilim

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Protein Kalite Kontrol Sistemi incelenmesi<em&gt; D. diskodeyum</em&gt; Sıcaklık kontrollü Canlı Hücre Görüntüleme kullanarak

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Dictyostelium discoideum yanıt-e doğru akut mekanik stimülasyon değerlendirilmesi

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Dictyostelium discoideum Macropinocytosis akış sitometresi tarafından yüksek üretilen iş ölçümü

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Floresan Korelasyon Spektroskopisi Kullanılarak Protein Toplamanın Tespiti

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

Erken Açlık Tepki Koronin A Fonksiyonu incelenmesi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Genetik Mühendisliği hücreleri Dictyostelium discoideum alan seçimi ve bakteri büyüme

Caenorhabditis elegans'ta Otomatik Inklüzyon Sayımı Kullanarak Protein Agregasyon Kinetiğinin In Vivo'da İzlenmesi

Görüntüleme G-protein Coupled Reseptör (GPCR) aracılı Sinyal Olaylar Kontrolü Kemotaksis Diktiyostelyum diskodeyum

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Heterolog Hücre Sisteminde Protein Toplama Yoluyla Hücrelerarası Etkileşimlerin Ölçülmesi

Protein Kalite Kontrol Sistemi incelenmesi

Dictyostelium discoideum yanıt-e doğru akut mekanik stimülasyon değerlendirilmesi

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

Dictyostelium discoideum Macropinocytosis akış sitometresi tarafından yüksek üretilen iş ölçümü

Floresan Korelasyon Spektroskopisi Kullanılarak Protein Toplamanın Tespiti