Bu çalışma, proje Z2-9229 ve program P2-0249 ile Slovenya Araştırma Ajansı tarafından desteklenmiştir. Bu video &quot;Elektroporasyon tabanlı Teknolojileri ve Tedaviler&quot;, Slovenya Ljubljana Üniversitesi Elektrik Mühendisliği Fakültesi tarafından yılda iki kez düzenlenen bilimsel atölye ve lisansüstü ders, ek malzeme temsil eder.

Bölüm I: Ön adımlar <ol><li> Bu deneyde, Çin hamsteri over hücre hattı (CHO-K1) kullanılır. Hücreler,% 8 fetal buzağı serumu ile takviye HAM-F12 kültür ortamında ~ 0.7x10 5 hücre / ml, 0.15 mg / ml Lab-Tek II odaları (2 kuyu, 4 cm 2 Her) (Nalge Nunc, Almanya) kaplanmıştır L-glutamin, 16 mg / ml gentamisin (Sigma-Aldrich, Steinheim, Almanya), 200 adet / ml Crystacillin (Pliva&#39;nın, Zagreb, Hırvatistan), ve 37% 5 CO 2 inkübe ° C Alternatif olarak, hücreler de böyle polylysine gibi hücresel bir yapıştırıcı ile kaplanmış # 1 cam kapak fişleri (kalın 0,13 ila 0,16 mm), kaplama olabilir. </li><li> Hücrelerin kendi kültür ortamında inkübe edin. Kuluçka hala kabaca küresel, ancak kendi zarının küçük bir kısmı ile yüzeye sıkıca bağlı 2 ila 4 saat verimi hücreleri süren. Alternatif olarak, inkübasyon 16 ila 20 saat sonra, hücreler tamamen yüzeye bağlı ve daha karmaşık şekiller var, ama çoğu hala bölünmesi değildir. </li><li> 843 ul DMSO (Sigma-Aldrich, Steinheim, Almanya), orjinal Invitrogen flakon boya, 5 mg ekleyerek di-8-ANEPPS (Invitrogen, Eugene, Oregon, ABD), 10 mM stok solüsyonu hazırlayın. Stok solüsyonu birkaç ay için 4 ° C&#39;de buzdolabında saklanabilir. DMSO kristallerin çözünmesi kadar deneyler başlamadan önce, çözüm ısınmak. </li><li> Bazı hücre dizilerinde hücre zarının içine boya dahil kolaylığı pluronic kullanımı gerekebilir. Pluronic% 20 DMSO (F-127, Invitrogen, Eugene, Oregon, ABD) stok solüsyonu veya DMSO pluronic eriterek tarafından hazırlanan aynı konsantrasyonda bir stok solüsyonu satın alınabilir. Pluronic stok solüsyonu oda sıcaklığında saklanabilir. </li></ol> Bölüm II: di-8-ANEPPS hücreleri yükleme <ol><li> 10 mM di-8-ANEPPS ve Minimum Temel Orta SMEM Spinner değişiklik 1 ml (kalsiyum-tükenmiş sürüm) (orta M8167 ve M4767, Sigma-Aldrich, Steinheim, Almanya% 20 pluronic 2.5 ul 3 ul Mix 1.5 ml Eppendorf tüp). Bu yaklaşık 30 mcM di-8-ANEPPS ve% 0.05 &#39;pluronic içeren bir &quot;yükleme çözüm&quot; ortaya çıkarır. Diğer hücre türleri için, doğdukları kültür ortamı yerine SMEM kullanılabilir. </li><li> Lab-Tek odasında yükleme çözümü ile kültür ortamı değiştirin. 10 dakika boyunca buzdolabında 4 odanın transfer ° C Bu sıcaklık, plazma zarı ile boya içselleştirilmesi büyük ölçüde inhibe edilir. </li><li> Boyama sonra, yavaşça saf SMEM fazla boya ile 2-3 kez yıkayın. </li><li> SMEM odasında 1,5 ml bırakın. </li></ol> Bölüm III: Deney ve görüntü alımı <ol><li> Hücreleri immersiyon yağı hedefi (X63, NA 1.4), monokromatör (Polychrome IV, Visitron, Almanya) ve soğutmalı bir CCD kamera (VisiCam ile donatılmış bir floresan mikroskobu (bizim durumumuzda Zeiss AxioVert 200, Zeiss, Almanya) kullanılarak gözlenmektedir 1280, Visitron, Almanya). MetaFluor 7.1.1 ile elde edilen görüntüleri ve (her ikisi de Moleküler Cihazlar, Downingtown, PA, USA) MetaMorph 7.1.1 işlenmiş, ancak diğer benzer satın alma yazılımı da kullanılabilir. Dalgaboyu 490 nm kazanım yazılım ayarlayın ve örneğin ANEPPS uygun bir emisyon filtresi, 605 nm &#39;de merkezlenmiş bir bant geçiren filtre (605/55m, dikroik 565 DCXR, Chroma, Rockingham, VT, ABD) seçin. Monokromatör mevcut değilse, 490 nm &#39;de merkezlenmiş bir harekete geçirici filtre yerine kullanılabilir. </li><li> Odasının altındaki hücreleri mikroskop sahneye odasına yerleştirin, Elektrotların pozisyonu ve darbe jeneratörü bağlayın. Orta elektrotlar kapsamalıdır. </li><li> Hücre canlılığını korumak ve ısıtma azaltmak için, darbe yeterince düşük genlikli ve kısa süreli olmalıdır. Bu deneyde 35 V genlik ve 50 ms süresi (elektroporasyon önlemek için yeterince düşük) sahip bir kare dalga, bir DC gerilim kaynağı ve ısmarlama bir mikroişlemci kontrollü switcher cihaz kullanılarak oluşturulur. Alternatif olarak, 33210A (Agilent, Santa Clara, CA, USA) gibi ticari bir darbe jeneratörü, bir amplifikatör veya S88 (Çimen, West Warwick, RI, ABD) gibi ticari bir uyarıcıdır bağlı kullanılabilir. Darbe, bir elektrik alan elektrotlar ve indükleyici ΔΦ arasında yaklaşık 88 V / cm, 0.8 mm çapında ve 4 mm aralarındaki mesafe iki paralel Pt / Ir tel elektrotlar teslim edilir. Bu deneyde kullanılan elektrot arasındaki alan dağılımı tam olarak başka bir yerde [1] olarak tarif edilir. </li><li> Ilgi hücreleri bulun. Tek bir elektrik darbe veya darbe dizisi uygulayın. : Her darbe için, iki floresan görüntüler elde darbe (kontrol görüntü) hemen önce ve puls (darbe görüntü) sırasında biri. Di-8-ANEPPS düşük tepki nedeniyle, floresan değişiklikler çıplak gözle ayırt ve belirgin onl olmak zordur.işlendikten sonra y. Nabız teslimat kazanım yazılım bir özelliği olan görüntü satın alma, senkronize olmalıdır. Isıtma photobleaching ve olası önlemek için, aydınlatma, darbe süresi ile sınırlı olabilir. </li></ol> Bölüm IV: Görüntü işleme ve analiz <ol><li> MetaMorph yazılım görüntüleri açın. Her darbe için, kontrol ve nabız görüntü hem de arka plan çıkarmak. </li><li> Bir hücre seçin ve membran karşılık gelir, böylece faiz bölgeyi ayarlayın. Floresans şiddetleri kontrol ve nabız resmi bu bölgede boyunca ölçün ve bir elektronik tablo değerleri aktarmak. </li><li> Her darbe için nabız verileri kontrol veri toplama, çıkarma, ve göreli floresan değişiklikleri elde etmek için kontrol verileri sonucu bölmek. Darbe bir dizi uygulanırsa, her darbe için belirlenen göreceli floresan değişikliklerin daha güvenilir bir ölçüm değerleri elde etmek için ortalama olabilir. </li><li> ΔΦ içine bir kalibrasyon eğrisi kullanılarak göreceli floresan değişiklikler dönüştürün. Bu eğrinin literatürden elde edilen kaba bir tahmin, ama daha yüksek doğruluk için, her kurulum için ölçülebilir. Bu deneyde kullanılan hücreleri ve kurulum için floresan% 6&#39;lık bir azalma ΔΦ [2] 100 mV artışı karşılık gelir. </li><li> Son olarak, göreli yay uzunluğu Excel (Microsoft Corp, Redmond, WA, ABD), Sigma Arsa (Systat Software Inc, San Jose, CA, USA) gibi bir grafik yazılım paketi bir fonksiyonu olarak gerilim arsa, veya Kökeni (OriginLab Corp, Northampton, MA, ABD). Eğrisi de bu hareketli ortalama filtre olarak uygun bir filtre kullanılarak düzeltilmelidir. </li></ol>

<ol>
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Eng. 34, 642-652 (2006).</li><li>Huang, C. J., Harootunian, A., Maher, M. P., Quan, C., Raj, C. D., McCormack, K., Numann, R., Negulescu, P. A., Gonzalez, 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=Characterization+of+voltage-gated+sodium-channel+blockers+by+electrical+stimulation+and+fluorescence+detection+of+membrane+potential.">Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential.</a> Nat. Biotechnol. 24, 439-446 (2006).</li><li>Bedlack, R. S., Wei, M., Fox, S. H., Gross, E., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+electric+potentials+in+soma+and+neurite+membranes.">Distinct electric potentials in soma and neurite membranes.</a> Neuron. 13, 1187-1193 (1994).</li><li>Cheng, D. K. L., Tung, L., Sobie, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonuniform+responses+of+transmembrane+potential+during+electric+field+stimulation+of+single+cardiac+cells.">Nonuniform responses of transmembrane potential during electric field stimulation of single cardiac cells.</a> Am. J. Physiol. 277, H351-H362 (1999).</li><li>Teissie, J., Eynard, N., Gabriel, B., Rols, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+cell+membranes.">Electropermeabilization of cell membranes.</a> Adv. Drug. Del. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+studies+of+cell+response+to+ultrashort,+high-intensity+electric+fields+-+Implications+for+intracellular+manipulation.">Modeling studies of cell response to ultrashort, high-intensity electric fields - Implications for intracellular manipulation.</a> IEEE Trans. Plasma Sci. 32, 1677-1686 (2004).</li><li>Gross, D., Loew, L. M., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cell+membrane+potential+changes+induced+by+applied+electric+fields.">Optical imaging of cell membrane potential changes induced by applied electric fields.</a> Biophys. J. 50, 339-348 (1986).</li><li>Fluhler, E., Burnham, V. G., Loew, L. M. 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M., Wei, M. D., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electric+properties+by+combined+patch+clamp+and+fluorescence+ratio+imaging+in+single+neurons.">Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons.</a> Biophys. J. 74, 48-53 (1998).</li><li>Montana, V., Farkas, D. L., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+ratiometric+fluorescence+measurements+of+membrane-potential.">Dual-wavelength ratiometric fluorescence measurements of membrane-potential.</a> Biochemistry. 28, 4536-4539 (1989).</li><li>Knisley, S. B., Justice, R. K., Kong, W., Johnson, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometry+of+transmembrane+voltage-sensitive+fluorescent+dye+emission+in+hearts.">Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts.</a> Am. J. Physiol. Heart Circ. Physiol. 279, H1421-H1433 (2000).</li><li>Pucihar, G., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-dependent+numerical+model+of+transmembrane+voltage+inducement+and+electroporation+of+irregularly+shaped+cells.">A time-dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells.</a> IEEE T. Biomed. Eng. 56, 1491-1501 (2009).</li></ol>

Kaynaklı membran (transmembran) gerilim, ΔΦ, ölçümleri, membran voltaj kapılı kanallar, aksiyon potansiyeli yayılımı, kardiyak hücre stimülasyonu, hücre zarı elektroporasyon [3, 4, 5, 6 çalışmaları gibi çeşitli deneysel ayarları, önemli olabilir , 7]. Basit hücre şekilleri ile ΔΦ analitik olarak hesaplanabilir. Örneğin, küresel bir hücre için, ΔΦ gerilim alan şiddeti ve hücre büyüklüğü ile orantılı olduğunu belirten ve membran boyunca kosinüs fonksiyonu aşağıdaki Schwan denklemi ile verilir [8, 9]. Daha karmaşık hücre şekilleri için, ΔΦ kosinüsünü önemli ölçüde sapma ve potansiyometrik boya [12, 13, 14, 15], [2, 10, 11], ya da deneysel olarak bir bilgisayar kullanıyorsanız, ya da sayısal olarak tespit edilmelidir. Bu amaç için yaygın olarak kullanılan potansiyometrik boyaların Bir di-8-ANEPPS (di-8-butil-amino-naftil-piridinium-propil-sülfonat etilen), eksitasyon ve emisyon membran voltaj bağımlı spektrumları ile hızlı bir boya, hücre zarı ve değerini ölçmek için ΔΦ varyasyonları noninvaziv gözlemler sağlar. Bu video, di-8-ANEPPS kullanarak ΔΦ belirlenmesi için deneysel bir yaklaşım gösteriyor. Boya, Connecticut Üniversitesi Prof. Leslie Loew ve arkadaşları [13, 14] tarafından geliştirilen ve hızlı tepki boyalar sınıf aittir. di-8-ANEPPS su nonfluorescent ve hücre zarı çift katlı lipid içine içermektedir güçlü floresan olur. Intramoleküler yük dağılımı ve boya floresan spektral yoğunluk profili ve ilgili değişiklikler bir değişiklik ΔΦ sonuçlarında bir değişiklik. Di-8-ANEPPS ΔΦ bir değişiklik floresan yoğunluğu ile orantılı olarak değişir; boya yanıt -280 mV ile 250 arasında değişen mV [4, 16] gerilimler için lineer. Bu çabaların da bildirilmiştir [17] olmasına rağmen, boya, düzensiz membran boyanması floresan nispeten küçük değişiklikler, boya ve içselleştirilmesi, di-8-ANEPPS membran gerilimi, örneğin dinlenme bileşeni mutlak ölçümler için daha uygun hale getirmek. Ancak, Uyarılabilir hücreler [4, 5], dış elektrik alanlara maruz kalan nonexcitable hücrelerin kaynaklı membran gerilimi başlangıçlı [12, 13], ya da aksiyon potansiyelleri gibi büyük değişiklikler, membran gerilimi ölçmek için uygundur. Burada uygulanan olmamakla birlikte, di-8-ANEPPS ayrıca, floresan ikaz oranlı metrik ölçümleri ΔΦ belirlenmesi [18] veya emisyon yanıt duyarlılığını arttırır ve yukarıda belirtilen etkilerini azaltır [19]. Di-8-ANEPPS lekeleri membran gibi, aynı zamanda bir zar belirteci olarak sade kullanılan olabilir [2]. Bir boya sakıncaları photobleaching eğilimli olduğunu, bu nedenle güçlü ışık uzun süre maruz kalındığında kaçınılmalıdır. Kalibrasyon boyanın dış ortamda ya (i) potasyum İyonofor valinomycin ve bir dizi farklı potasyum konsantrasyonları ile yapılır [2,18], veya (ii) patch-klemp gerilim kelepçe modunda [17]. Son olarak, küresel hücreleri ve daha karmaşık şekiller hücreleri ΔΦ ölçümleri ile, video ΔΦ genlik ve mekansal dağılımının hücre şekli etkisi göstermektedir. Böylece küresel hücreler için ΔΦ daha karmaşık hücre şekiller için mekansal ΔΦ dağılımı daha karmaşık olmasına rağmen, Schwan denklemi ile anlaşma, bir kosinüs yakın [20] ...

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		<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>di-8-ANEPPS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3167</td>
<td>potentiometric fluorescent dye</td>
</tr>
<tr>
<td>pluronic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>potassium ionophore</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>SMEM</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M8167 or M4767</td>
<td>Spinner modification of the Minimum Essential Medium</td>
</tr>
<tr>
<td>Ham-F12</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N4888</td>
<td>culture medium</td>
</tr>
<tr>
<td>fetal calf serum</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>&nbsp;</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Lab-Tek II</td>
<td>&nbsp;</td>
<td>Nalge Nunc</td>
<td>155379</td>
<td>chamber</td>
</tr>
<tr>
<td>DC voltage supply</td>
<td>&nbsp;</td>
<td>Elektro-Automatik</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microprocessor-controlled switcher</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>electrodes</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
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measuring the induced membrane voltage with di-8-anepps

Yerleştirme harici bir elektrik alanı içine bir hücre zarından bir gerilim, yerel ücret yeniden dağıtılması, hücre zarı çevresindeki, hücre içi ve dışında neden olur. Bu gerilim, bağlı membran gerilimi olarak adlandırılan (de indüklenen transmembran gerilim, ya da uyarılan transmembran potansiyel farkı) ve ΔΦ ile başlar, sadece dış alanda mevcut olduğu sürece var. Membran dinlenme gerilim varsa, indüklenmiş voltaj (ekler) üzerine üst üste bindirir. Di-8-ANEPPS potansiyometrik floresan boyalar, birini kullanarak, hücre zarı ΔΦ varyasyonların gözlemlemek ve noninvaziv değerini ölçmek mümkündür. ΔΦ değişikliği ile orantılı floresan değişimi, hücre zarı çift katlı lipid bağlı di-8-ANEPPS güçlü floresan olur. Bu video ölçmek için protokol ΔΦ di-8-ANEPPS kullanarak gösterir ve aynı zamanda hücre şeklinin genlik ve ΔΦ mekansal dağılımının etkisini gösterir.

Watch this Scientific Journal Video about Measuring the Induced Membrane Voltage with Di-8-ANEPPS at JoVE.com

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Dış elektrik alan bir hücre zarı üzerinde bir gerilim indükler kaynaklı membran gerilimi (ΔΦ) olarak adlandırılır. Potansiyometrik boya di-8-ANEPPS kullanarak, noninvaziv ΔΦ ölçmek mümkündür. Bu video ölçmek için protokol gösterir ΔΦ di-8-ANEPPS kullanarak.

Di-8-ANEPPS ile İndüklenmiş Membran Gerilim Ölçme

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

measuring-the-induced-membrane-voltage-with-di-8-anepps

Research

JoVE Journal

Biology

17.3K Görüntüleme.  University of Ljubljana. Dış elektrik alan bir hücre zarı üzerinde bir gerilim indükler kaynaklı membran gerilimi (ΔΦ) olarak adlandırılır. Potansiyometrik boya di-8-ANEPPS kullanarak, noninvaziv ΔΦ ölçmek mümkündür. Bu video ölçmek için protokol gösterir ΔΦ di-8-ANEPPS kullanarak.

Video: Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Astrositler Plazma Membran ve Küresel Hücre içi kalsiyum Dynamics civarında Ölçüm

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biyoloji

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Tersinir biyotinilasyon Plazma Membran Protein endositik Oranları Ölçüm

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidik Mesophases kullanarak Yapı Tayini için Membran Proteinleri Kristalize

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Lipidik Mesophases Membran Proteinleri Kristallendirilmesi

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Membran Proteinleri Konformasyonel Dynamics incelenmesi In situ Site Floresan Etiketleme

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

<em&gt; Xenopus</emFluorometri ile&gt; Yumurta Kes-açık Vazelin Gap Gerilim-kelepçe Tekniği

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

PTIRFM ile görüntüleme Plazma Membran deformasyonlar

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra Glutatyon kaynaklı Besleme Tepki Ölçüm

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Şekilde, mikro Tekniği Sıçan mezenter mikrodamar geçirgenliği Yönetmelik Araştırma

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Memeli hücrelerinde devlet tükenmesi süper kararlılık düşsel yer

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...