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>
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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 1L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4&#8727;2H2O, 0.35 g KH2PO4, 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>Na2HPO4&#8727;2H2O</td>
			<td>Fluka</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</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 &#956;M CaCl2, and l mM MgCl2, 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 CaCl2, pH 6.5)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>0.45-&#956;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><a href="https://www.fishersci.com/shop/products/falcon-tissue-culture-plates-23/087721h">08-772-1H</a></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&#8211;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 ...

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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

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Zebrafish görme fonksiyonu çalışma optokinetik yanıt

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Biyoloji

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

İyonlaştırıcı Radyasyon Cevabı γH2AX Foci Kantitasyonu

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

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 ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Erken Civciv Embriyo morfogenetik Doku deformasyonlar Takibi

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Na, K ve H, K-ATPaz bölgesindeki tarafından Katyon Taşıma Ölçüm<em&gt; Xenopus</emAtomik Absorpsiyon Spektrofotometresi ile&gt; Oosit: Radyoizotop Tahliller Alternatif Bir

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Erken Otofaji Etkinlikler Hücre Görüntüleme Canlı: Omegasomes ve Ötesi

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro toplama Deneyler hiperfosforile tau proteini kullanılarak

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Yetişkin Zebra balığı Böbrek Nefron Kompozisyon ve Fonksiyon Analizi

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

Fagositoz Erken Aşamaları görselleştirme

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...