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

Summary

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.

Abstract

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.

Introduction

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 vesicles^1-3. 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-c....

Protocol

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.

1. Handle all cell culture in a sterile laminar flow hood. Fill 4 wells of a 12-well plate with 1 ml of Dulbecco’s Modified Essential Media (DMEM) with 10% Fetal Bovine Serum (FBS). From a confluent T25 flask of HEK293 cells dilute 1:50, 1:25, 1:16, 1:10 respectively into each of the 4 wells.
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Results

Using live-cell TIRF Microscopy we have recorded the endocytic dynamics of the µ-opioid-receptor (MOR), a G-protein coupled receptor (GPCR) and its endocytic adaptor protein β-arrestin. The β-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 fluid. Also, the.......

Discussion

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.......

Materials

List of materials used in this article

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