Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Summary

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Abstract

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT_1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT_1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Introduction

The overall goal is to obtain quantitative evidence in time and space of receptor colocalization with specific subcellular organelles after treatment with a receptor agonist. Thus, the immediate goal here is to capture time-lapse confocal images of lysosomes and a transmembrane spanning receptor in human embryonic kidney cells (HEK) following transfection and to subsequently assemble and analyze the imaging data in 3D to quantitatively measure the delivery of receptor to lysosomes. Investigating the simultaneous trafficking of a transmembrane receptor with lysosomes or other organelles during endocytosis when activated by receptor ligand can help determine how the tra....

Protocol

Day One

1. Cell Seeding

1. Prepare complete Dulbecco's modified Eagle Medium (DMEM). Add 50 mL of fetal bovine serum, 5 mL of penicillin/streptomycin, 5 mL of L-glutamine to 450 mL of DMEM to make 500 mL of complete DMEM supplemented with fetal bovine serum (10%), penicillin/streptomycin (1%) and L-glutamine (1%).
2. Seed human embryonic kidney (HEK) cells, passage number 6-11 at 50,000/well in an 8 well chambered coverglass system in complete DMEM.
3. Maintain the chambered slide at 37 °C in a humidified incubator with 5% CO₂ for 24 h.

Day Two

Results

In this representative set of experiments, HEK cells were transfected with angiotensin type 1a receptor (AT_1aR-GFP) construct (E1,2,3-AT_1aR) cloned into the pEGFP-N2 plasmid¹³. Time-lapse images of HEK were acquired and played as a movie (See Live Cell Imaging 1 and Live Cell Imaging 2 movies). The movies and stills show that after ligand stimulation, AT_1aR-EGFPs are localized predominantly in the p.......

Discussion

Preliminary experiments presented here illustrate that over a rather short time course of 24 min, no appreciable change in delivery of AT_1aR-GFP to the lysosomes occurred. In general, delivery of internalized receptors to lysosomes could occur as early as 15-20 min. This experiment is consistent with the hypothesis that the bulk of the internalized AT_1aR is targeted to large, perinuclear endosomes. Evidence that at least some AT_1aR arrives in lysosomes was demonstrated by Li et al.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Angiotensin II (Ang II)	Sigma-Aldrich	A9525
Dulbecco's Modified Eagle medium (DMEM 1x)	Gibco -ThermoFisher Scientific	11960-044	Warm in 37 °C water bath before use
Fetal Bovine Serum (FBS)	Sigma-Aldrich	F2442	Warm in 37 °C water bath before use
Penicillin Streptomycin (Pen Strep)	Gibco -ThermoFisher Scientific	15140-122	Warm in 37 °C water bath before use
L-Glutamine 200 mM (100x)	Gibco (life technologies)	25030-081	Warm in 37 °C water bath before use
Human Embryonic Kidney (HEK)-293 cells	American Type Culture Collection (ATCC)	CRL-1573	Passage number 5 - 12
Nunc Lab-Tek II chambered 1.5 German Coverglass System	Sigma-Aldrich	155409
Lipofectamine 2000 Transfection Reagent	ThermoFisher Scientific	11668019	Store at 4 °C
Opti-MEM, Reduced Serum Medium	Gibco- ThermoFisher Scientific	31985-070	Warm in 37 °C water bath before use
PBS 10x	Fisher BioReagents	BP3994	Warm in 37 °C water bath before use
Dulbecco's Modified Eagle medium (DMEM 1x)	Gibco -ThermoFisher Scientific	31053-028	Warm in 37 °C water bath before use
LysoTracker Red DND-99	molecular probes -ThermoFisher Scientific	L7528	Store aliquotes in dark at -20 °C .
Volocity Ver. 6.3 Image Analysis software	PerkinElmer	Visualization and Quantitation Modules	For 3D image analysis of image stacks
Leica SP8 AOBS	Leica Microsystems	laser scanning confocal microscope	For image collection
Zeiss LSM 880	Carl Zeiss	laser scanning confocal microscope	For image collection