The authors would like to thank Drs. C. Szalinski, H. Teng, and M. Bruchez for help with Imaris, R. Vistein, and D. Shiwarski for technical help and advice, and Dr. M von Zastrow, Dr. T Kirchhausen, Dr. D Drubin, and Dr. W Almers for reagents and helpful discussion. Funding provided by T32 grant NS007433 to SLB and NIH DA024698 and DA036086 to MAP.

<ol>
	<li>McMahon, H. T., Boucrot, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+and+physiological+functions+of+clathrin-mediated+endocytosis.">Molecular mechanism and physiological functions of clathrin-mediated endocytosis.</a> Nature Reviews Molecular Cell Biology. 12 (8), 517-533 (2011).</li><li>Kaksonen, M., Toret, C. P., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modular+Design+for+the+Clathrin-+and+Actin-Mediated+Endocytosis+Machinery.">A Modular Design for the Clathrin- and Actin-Mediated Endocytosis Machinery.</a> Cell. 123 (2), (2005).</li><li>Taylor, M. J., Perrais, D., Merrifield, 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=A+High+Precision+Survey+of+the+Molecular+Dynamics+of+Mammalian+Clathrin-Mediated+Endocytosis.">A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis.</a> PLoS Biology. 9 (3), (2011).</li><li>Ungewickell, E., Branton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+units+of+clathrin+coats.">Assembly units of clathrin coats.</a> Nature. 289 (5796), 420-422 (1981).</li><li>Bliek, A. M., Meyerowrtz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-like+protein+encoded+by+the+Drosophila+shibire+gene+associated+with+vesicular+traffic.">Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic.</a> Nature. 351 (6325), (1991).</li><li>Hinshaw, J. E., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin+self-assembles+into+rings+suggesting+a+mechanism+for+coated+vesicle+budding.">Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding.</a> Nature. 374 (6518), (1995).</li><li>Merrifield, C. J., Perrais, D., Zenisek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+between+Clathrin-Coated-Pit+Invagination,+Cortactin+Recruitment,+and+Membrane+Scission+Observed+in+Live+Cells.">Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells.</a> Cell. 121 (4), 593-606 (2005).</li><li>Grabs, D., Slepnev, V. I., 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=The+SH3+domain+of+amphiphysin+binds+the+proline-rich+domain+of+dynamin+at+a+single+site+that+defines+a+new+SH3+binding+consensus+sequence.">The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence.</a> The Journal of biological chemistry. 272 (20), 13419-13425 (1997).</li><li>Tosoni, D., Puri, 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=TTP+Specifically+Regulates+the+Internalization+of+the+Transferrin+Receptor.">TTP Specifically Regulates the Internalization of the Transferrin Receptor.</a> Cell. 123 (5), 875-888 (2005).</li><li>Puthenveedu, M. A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cargo+Regulates+Clathrin-Coated+Pit+Dynamics.">Cargo Regulates Clathrin-Coated Pit Dynamics.</a> Cell. 127 (1), 113-124 (2006).</li><li>Soohoo, A. L., Puthenveedu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+modes+for+cargo-mediated+control+of+clathrin-coated+pit+dynamics.">Divergent modes for cargo-mediated control of clathrin-coated pit dynamics.</a> Molecular Biology of the Cell. 24 (11), 1725-1734 (2013).</li><li>Posor, Y., Eichhorn-Gruenig, M., 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=Spatiotemporal+control+of+endocytosis+by+phosphatidylinositol-3,4-bisphosphate.">Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.</a> Nature. 10, (2013).</li><li>Mettlen, M., Stoeber, M., Loerke, D., Antonescu, C. N., Danuser, G., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+accessory+proteins+are+functionally+distinguished+by+their+differential+effects+on+the+maturation+of+clathrin-coated+pits.">Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits.</a> Molecular Biology of the Cell. 20 (14), 3251-3260 (2009).</li><li>Loerke, D., Mettlen, M., 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=Cargo+and+dynamin+regulate+clathrin-coated+pit+maturation.">Cargo and dynamin regulate clathrin-coated pit maturation.</a> PLoS Biology. 7 (3), (2009).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-substrate+contacts+illuminated+by+total+internal+reflection+fluorescence.">Cell-substrate contacts illuminated by total internal reflection fluorescence.</a> The Journal of Cell Biology. 89 (1), 141-145 (1981).</li><li>Merrifield, C. J., Feldman, M. E., Wan, L., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+actin+and+dynamin+recruitment+during+invagination+of+single+clathrin-coated+pits.">Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.</a> Nature Cell Biology. 4 (9), 691-698 (2002).</li><li>Ehrlich, M., Boll, W., 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=Endocytosis+by+random+initiation+and+stabilization+of+clathrin-coated+pits.">Endocytosis by random initiation and stabilization of clathrin-coated pits.</a> Cell. 118 (5), 591-605 (2004).</li><li>Yarar, D., Waterman-Storer, C. M., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+actin+cytoskeleton+functions+at+multiple+stages+of+clathrin-mediated+endocytosis.">A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis.</a> Molecular Biology of the Cell. 16 (2), 964-975 (2005).</li><li>Rappoport, J. Z., Kemal, S., Benmerah, A., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+clathrin+and+adaptor+proteins+during+endocytosis.">Dynamics of clathrin and adaptor proteins during endocytosis.</a> American journal of physiology. Cell physiology. 291 (5), (2006).</li><li>Doyon, J. B., Zeitler, B., 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=Rapid+and+efficient+clathrin-mediated+endocytosis+revealed+in+genome-edited+mammalian+cells.">Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.</a> Nature Cell Biology. 13 (3), 331-337 (2011).</li><li>Hopp, T. P., Prickett, K. 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=A+Short+Polypeptide+Marker+Sequence+Useful+for+Recombinant+Protein+Identification+and+Purification.">A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification.</a> Bio/Technology. 6 (10), 1204-1210 (1988).</li><li>Miesenb&#246;ck, G., De Angelis, D. A., Rothman, 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=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Saffarian, S., Cocucci, E., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Dynamics+of+Endocytic+Clathrin-Coated+Pits+and+Coated+Plaques.">Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques.</a> PLoS Biology. 7 (9), (2009).</li><li>Yudowski, G., Puthenveedu, M., Henry, A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cargo-Mediated+Regulation+of+a+Rapid+Rab4-Dependent+Recycling+Pathway.">Cargo-Mediated Regulation of a Rapid Rab4-Dependent Recycling Pathway.</a> Molecular Biology of the Cell. 1, 1-11 (2009).</li><li>Batchelder, E. M., Yarar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Requirements+for+Clathrin-dependent+Endocytosis+at+Sites+of+Cell-Substrate+Adhesion.">Differential Requirements for Clathrin-dependent Endocytosis at Sites of Cell-Substrate Adhesion.</a> Molecular Biology of the Cell. 21 (17), 3070-3079 (2010).</li><li>Mattheyses, A. L., Atkinson, C. E., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Single+Endocytic+Events+Reveals+Diversity+in+Clathrin,+Dynamin+and+Vesicle+Dynamics.">Imaging Single Endocytic Events Reveals Diversity in Clathrin, Dynamin and Vesicle Dynamics.</a> Traffic. 12 (10), 1394-1406 (2011).</li><li>Aguet, F., Antonescu, C. N., Mettlen, M., Schmid, S. L., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+analysis+of+low+signal-to-noise+images+link+dynamin+and+AP2+to+the+functions+of+an+endocytic+checkpoint.">Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint.</a> Dev Cell. 26 (3), 279-291 (2013).</li><li>Boucrot, E., Howes, M. T., Kirchhausen, T., Parton, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+of+caveolae+during+mitosis.">Redistribution of caveolae during mitosis.</a> J Cell Sci. 124, 1965-1972 (2011).</li><li>Bhaya, D., Davison, M., Barrangou, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+in+bacteria+and+archaea:+versatile+small+RNAs+for+adaptive+defense+and+regulation.">CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.</a> Annual review of genetics. 45, 273-297 (2011).</li><li>Boch, J., Scholze, H., 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=Breaking+the+Code+of+DNA+Binding+Specificity+of+TAL-Type.">Breaking the Code of DNA Binding Specificity of TAL-Type.</a> III Effectors. Science. 326 (5959), 1509-1512 (2009).</li></ol>

The authors have nothing to disclose.

ここでは、リアルタイムで生きた細胞内の個別のCCPのレベルでクラスリン依存性エンドサイトーシス（CME）を可視化するTIRFMの使用を記載している。 CMEは、多くの空間的および時間的に独立した個別のイベントの累積効果により媒介される迅速かつ高ダイナミックなイベントです。そのような結合は、フローサイトメトリーまたは固定細胞アッセイは、内在化タンパク質の量、またはタンパ?...

クラスリン依存性エンドサイトーシス（CME）の過程は、小胞1-3に貨物を収集し、原形質膜を操作するクラスリン媒介エンドサイトーシス機構の多くのコンポーネントの時宜を得た到着時に依存している。 CMEは、膜が変形し、エンドサイトーシス1の発生期の現場で一緒になる貨物アダプタータンパク質によって開始されます。これらのタンパク質は、クラスリン被覆ピット（CCP）4を形成し 、かご状構造に組み立てるコートタンパク質クラスリンを募集。 CCPが完全に球状に組み立てられると、膜の切断は、主に大規模なGTPアーゼの作用を介して、ダイナミンは、無料のクラスリン被覆小胞（CCVS）5,6を生成します。この内在化は、コンポーネントが、CMEの複数のラウンドのために再利用することができるように、クラスリンコートの急激な分解をトリガします。 CMEに関与するタンパク質の発見および特徴は、伝統的なbiochに根付いてきたemical、遺伝的および顕微鏡技術4-6,8。これらのアッセイは、これらのエンドサイトーシスのコンポーネントの役割と相互作用点を解明した。売買機構の必須成分を定義するために非常に有用であるが、これらのアッセイは、高度CMEコンポーネントまたは貨物濃度の動的挙動を捉えることに限定されている。 CMEが定義されたステップにおけるタンパク質のモジュールのセットの振り付けアセンブリによって駆動されるので、これは重大な制限であり、そして個別のエンドサイトーシスイベントのダイナミクスの小さな変化は、エンドサイトーシスに大きな累積的な結果をもたらすことができるからである。さらに、最近のデータは、個別の清算機関がこのプロセスの生理学的調節が非常に空間的および時間的に9-14を拘束されていることを示唆している、組成物中にと行動の両方で異なる場合がありますことを示している。個別のエンドサイトーシス事象の可視化、したがって、複数の冗長CMEに関与するタンパク質とどのようにこれらのタンパク質は、Bを制御することかもしれないがある理由を理解することが不可欠であるyの生理学的信号は、貨物内在化を調節する。 ここでは、生きた細胞内の個別のCCPのダイナミクスのレベルでCMEを観測する全反射蛍光顕微鏡（TIRFM）の使用を記載している。 TIRFMは、カバーガラスとセル15,16の流体環境との間の屈折率の差に依存しています。励起光が臨界角以上で細胞に向けられるとき、それは内部カバースリップの上に、約100nmを拡張する照明の薄いフィールドを維持するエバネッセント波を作成し、反射される。これは、この狭いフィールド内の蛍光分子が励起されることを保証します。実際に、これは原形質膜上またはその近くの蛍光分子の励起を可能にし、細胞の内部部分からの蛍光を最小化する。これは、原形質膜、共同でイベントを視覚化するための有意に高い信号対雑音比、z軸の解像度を提供このような従来の落射蛍光または共焦点蛍光顕微鏡のようなより一般的に使用されるモードにmpared。また、入門、実用的なレベルで、個別の貨物エンドサイトーシスイベントの単純な形態学的特徴および動力学を分析し、定量するために一般に使用される画像解析プラットフォームの使用を記載している。

<table border="0" cellpadding="0" cellspacing="0" width="909">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:207px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">DMEM/High Glucose with L-glutamine and sodium pyruvate</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>SH3024301</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS), no calcium, no magnesium</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21600-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">EDTA Free Acid</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:157px;">0322-500G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Fetal Bovine Serum</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Opti-MEM</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">HEPES</td>
			<td style="width:180px;">CellPURE by Fisher Scientific&#160;</td>
			<td>BP2937-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Effectene&#160;</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:157px;">301425</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">25 millimeter coverglass</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>12-545-86&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Corning cell culture treated flasks, 25cm2</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>10-126-28</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Cell culture 6-well plate</td>
			<td style="width:180px;">Greiner Bio-One, by VWR</td>
			<td>82050-896</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Monoclonal ANTI-FLAG M1</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>F3040-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">[D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO)</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>E7384-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Alexa Fluor 647 Protein Labeling Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:157px;">A20173</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">ImageJ</td>
			<td style="width:180px;">NIH</td>
			<td style="width:157px;"></td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Imaris Image analysis software</td>
			<td style="width:180px;">BitPLane</td>
			<td style="width:157px;"></td>
			<td>http://www.bitplane.com/imaris/imaris, for automated analysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon Eclipse Ti inverted microscope and required accessories including filter cubes and filters</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon TIRF arm with required adapters for Nikon Eclipse Ti</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td>For adjusting angle of incidence</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">iXon+ EMCCD camera and adapters</td>
			<td style="width:180px;">Andor</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing clathrin-mediated endocytosis of g protein-coupled receptors at single-event resolution via tirf microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

生細胞TIRF顕微鏡を用いて、私たちは、Gタンパク質共役受容体（GPCR）とそのエンドサイトーシスアダプタータンパク質β-アレスチンをμ-オピオイド受容体（MOR）のエンドサイトーシスダイナミクスを記録している。 β-アレスチン構築物は一過性に、図1で概説したプロトコルを用いて安定に発現MOR細胞株にトランスフェクトし、96時間後に画像化した。安定した細胞株におけるMOR?...

Watch this Scientific Journal Video about Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy at JoVE.com

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF顕微鏡を経由してシングルイベント解像度のGタンパク質共役型受容体のクラスリン依存性エンドサイトーシスの可視化

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

visualizing-clathrin-mediated-endocytosis-g-protein-coupled-receptors

Research

JoVE Journal

Biology

80.1K ビュー.  Carnegie Mellon University. Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process. 

ビデオ: Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

全反射顕微鏡による網膜双極細胞におけるイメージングのエキソサイトーシス

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

蛍光顕微鏡による可視化単分子のDNAの複製

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

スペクトル的に解決された二光子顕微鏡を用いたGタンパク質共役受容体相互作用の in vivo定量で

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

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.

イメージングG -タンパク質共役受容体（GPCR）を介するシグナル伝達事象その制御走キイロタマホコリカビ</em

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

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

Gタンパク質共役受容体でアゴニスト活性を定量化

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

蛍光顕微鏡を用いて線虫の中の細菌の可視化

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

遺伝的にコードされた分子プローブは、Gタンパク質共役受容体を研究する

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

高速3D-構造化照明顕微鏡（F3D-SIM）を使用したライブ細菌における細胞質分裂のZリングの超解像イメージング

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...