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.

1. Expression of Fluorescently Tagged CME Components in Cultured Cells
HEK293 cells are useful model cells that have been used extensively to study GPCR biology and endocytosis, and therefore are used as models in this protocol. Use any transfection protocol providing uniform expression without overexpression and low cytotoxicity.
<ol>
	<li>Handle all cell culture in a sterile laminar flow hood. Fill 4 wells of a 12-well plate with 1 ml of Dulbecco&#8217;s Modified Essential Media (DMEM) wit...

<ol>
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Here we describe the use of TIRFM to visualize clathrin-mediated endocytosis (CME) at the level of individual CCPs in living cells in real time. CME is a rapid and highly dynamic event mediated by the cumulative effect of many spatially and temporally separate individual events. Most assays that are currently used, such as biochemical measurements of internalization using surface biotinylation or ligand binding, flow-cytometry or fixed cells assays measuring amount of internalized proteins, or electron microscopic locali...

The process of clathrin-mediated endocytosis (CME) is dependent upon the well-timed arrival of the many components of the clathrin-mediated endocytic machinery to gather cargo and manipulate the plasma membrane into the vesicles1-3. CME is initiated by membrane deforming and cargo-adaptor proteins that come together at nascent sites of endocytosis1. These proteins recruit the coat protein clathrin, which assembles into a cage-like structure that forms the clathrin-coated pit (CCP)4. Once the CCP is fully assembled into a spherical shape, membrane scission, primarily through action of the large GTPase, dynamin, generates free clathrin-coated vesicles (CCVs)5,6. This internalization triggers rapid disassembly of the clathrin coat, allowing components to be re-used for multiple rounds of CME.
The discovery and characterization of the proteins involved in CME has been rooted in traditional biochemical, genetic, and microscopy techniques4-6,8. These assays have elucidated the roles and interaction points of these endocytic components. Although very useful for defining essential components of trafficking machinery, these assays are highly limited in capturing the dynamic behavior of CME components or cargo concentration. This is a critical limitation, since CME is driven by the choreographed assembly of sets of protein modules in defined steps, and since small changes in the dynamics of individual endocytic events can have large cumulative consequences on endocytosis. Further, recent data indicate that individual CCPs might differ both in composition and in behavior, suggesting that the physiological regulation of this process is highly spatially and temporally constrained9-14. Visualizing individual endocytic events, therefore, is essential to understand why there are multiple redundant proteins involved in CME and how these proteins might be controlled by physiological signals to regulate cargo internalization.
Here we describe the use of Total Internal Reflection Fluorescence Microscopy (TIRFM) to observe CME at the level of the dynamics of individual CCPs in living cells. TIRFM relies on the difference in refractive index between the glass coverslip and the fluid environment of cells15,16. When the excitation light is directed towards the cells at more than the critical angle, it is internally reflected, creating an evanescent wave that maintains a thin field of illumination extending approximately 100 nm above the coverslip. This ensures that only the fluorescent molecules within this narrow field are excited. Practically, this allows the excitation of fluorescent molecules on or near the plasma membrane, and minimizes fluorescence from the interior parts of the cell. This provides a significantly higher signal-to-noise ratio and z-axis resolution to visualize events at the plasma membrane, compared to more commonly used modes such as conventional epifluorescence or confocal fluorescence microscopy. We also describe, at an introductory and practical level, the use of a commonly used image analysis platform to analyze and quantitate simple morphological features and dynamics of individual cargo endocytic events.

<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.

Using live-cell TIRF Microscopy we have recorded the endocytic dynamics of the &#181;-opioid-receptor (MOR), a G-protein coupled receptor (GPCR) and its endocytic adaptor protein &#946;-arrestin. The &#946;-arrestin construct was transiently transfected into a stably expressing MOR cell line using the protocol outlined in Figure 1, and imaged 96 hr later. The MOR in the stable cell line is N-terminally tagged with a pH-sensitive GFP. This fluorescent protein only fluoresces in the neutral extracellular f...

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.

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

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Biology

80.1K visualizações.  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. 