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.

Nous décrivons ici l&#39;utilisation de TIRFM de visualiser endocytose clathrine (CME) au niveau de l&#39;individu CCP dans les cellules vivantes en temps réel. CME est un événement rapide et très dynamique induite par l&#39;effet cumulatif de plusieurs spatialement et temporellement séparées épreuves individuelles. La plupart des tests qui sont actuellement utilisés, tels que les mesures biochimiques de l&#39;internalisation en utilisant biotinylation de surface ou de liaison ligand, la cytométrie de flux ou ...

Le processus de clathrine endocytose (CME) dépend de l&#39;arrivée au bon moment des nombreux composants de la machinerie d&#39;endocytose clathrine pour recueillir le fret et manipuler la membrane plasmique dans les vésicules 1-3. CME est initiée par la déformation de la membrane et de la cargaison à l&#39;adaptateur protéines qui se réunissent sur ​​les sites naissantes d&#39;endocytose 1. Ces protéines recrutent la clathrine de protéine d&#39;enveloppe, qui rassemble dans une structure en forme de cage qui forme la fosse recouverts de clathrine (PCC) 4. Une fois que le PCC est entièrement assemblé en une forme sphérique, la scission de la membrane, principalement grâce à l&#39;action de la grande GTPase dynamine, génère gratuits vésicules de clathrine (CCVS) 5,6. Cette internalisation déclenche démontage rapide de la couche de clathrine, permettant aux composants d&#39;être réutilisés pour de multiples séries de CME. La découverte et la caractérisation des protéines impliquées dans la FMC a été enracinée dans Bioch traditionnelleemical, génétiques, et des techniques de microscopie 4-6,8. Ces essais ont permis d&#39;élucider les rôles et les points d&#39;interaction de ces composants d&#39;endocytose. Bien que très utile pour définir des éléments essentiels de la machinerie de trafic, ces tests sont très limités à capturer le comportement dynamique des composants de FMC ou de la concentration de la cargaison. Il s&#39;agit d&#39;une limitation critique, puisque CME est entraîné par l&#39;ensemble chorégraphié des ensembles de modules de protéines dans les étapes définies, et depuis de petits changements dans la dynamique des événements d&#39;endocytose individuels peut avoir de grandes conséquences cumulatives sur l&#39;endocytose. En outre, des données récentes indiquent que les CCP individuelles peuvent différer à la fois dans la composition et dans le comportement, ce qui suggère que la régulation physiologique de ce processus est très spatialement et temporellement limitée 9-14. Visualisation des événements d&#39;endocytose individuels, par conséquent, il est essentiel de comprendre pourquoi il ya plusieurs protéines impliquées dans redondants CME et b comment ces protéines pourraient être contrôléssignaux physiologiques y pour réglementer cargaison internalisation. Nous décrivons ici l&#39;utilisation de la réflexion interne totale microscopie de fluorescence (TIRFM) pour observer CME au niveau de la dynamique des contreparties centrales individuelles dans les cellules vivantes. TIRFM repose sur la différence d&#39;indice de réfraction entre la lamelle de verre et l&#39;environnement de fluide de cellules 15,16. Lorsque la lumière d&#39;excitation est dirigée vers les cellules situées plus à l&#39;angle critique, elle est réfléchie à l&#39;intérieur, ce qui crée une onde évanescente qui maintient un champ d&#39;illumination mince s&#39;étendant à peu près 100 nm au-dessus de la lamelle. Cela garantit que seules les molécules fluorescentes dans ce domaine étroit sont excités. En pratique, cela permet à l&#39;excitation des molécules fluorescentes sur ou près de la membrane plasmique, et minimise la fluorescence à partir des parties intérieures de la cellule. Ceci permet d&#39;obtenir un rapport signal sur bruit et la résolution de l&#39;axe z sensiblement plus élevé pour visualiser les événements à la membrane plasmique, compared de modes plus couramment utilisés tels que épifluorescence classique ou microscopie par fluorescence confocale. Nous décrivons également, à une initiation et pratique, l&#39;utilisation d&#39;une image couramment utilisé la plate-forme d&#39;analyse pour analyser et quantifier les caractéristiques et la dynamique des événements individuels cargaison de endocytose morphologiques simples.

<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.

En utilisant des cellules vivantes microscopie TIRF nous avons enregistré la dynamique d&#39;endocytose de la μ-récepteurs opioïdes (MOR), un récepteur couplé aux protéines G (GPCR) et son endocytose protéine adaptatrice β-arrestine. La construction β-arrestine été transfecté de manière transitoire dans une lignée cellulaire exprimant de manière stable MOR en utilisant le protocole présenté sur la figure 1, et 96 heures plus tard imagée. Le MOR dans la lignée cellulaire stable est le...

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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.

Visualisation endocytose clathrine de G récepteurs couplés aux protéines à un seul événement résolution par microscopie TIRF

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

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JoVE Journal

Biology

80.1K vues.  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. 