Abstract
Apical-to-basal transcytosis by endothelial cells can be visualized and quantified using total internal reflection fluorescence (TIRF) microscopy of the basal membrane. Past techniques to study transcytosis including electron microscopy and transwells have several limitations such as confounding from paracellular leakage, low transfection efficiency, and the largely descriptive nature of electron microscopy. After the addition of a fluorescent ligand to the apical endothelial surface, using TIRF to measure exocytosis at the basal membrane bypasses these issues by studying transcytosis across a single cell of a confluent endothelial monolayer in real time. A major benefit of TIRF is that only a small volume of the cell is illuminated, thus greatly reducing background noise from the overlying cytosol in the images. This protocol outlines the steps to image and quantify exocytosis of apically applied fluorophore-tagged low-density lipoprotein (LDL) using TIRF microscopy and MATLAB. A similar approach can be used to study endothelial transcytosis of other ligands such as albumin or high-density lipoprotein.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mattheyses AL, Simon SM, Rappoport JZ (2010) Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123:3621–3628. https://doi.org/10.1242/jcs.056218
Axelrod D (2003) Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol 361:1–33. https://doi.org/10.1016/S0076-6879(03)61003-7
Poulter NS, Pitkeathly WTE, Smith PJ, Rappoport JZ (2015) The physical basis of total internal reflection fluorescence (TIRF) microscopy and its cellular applications. In: Verveer PJ (ed) Advanced fluorescence microscopy: methods and protocols. Springer, New York, NY, pp 1–23
Millis BA (2012) Evanescent-wave field imaging: an introduction to total internal reflection fluorescence microscopy. In: Espina V, Liotta LA (eds) Molecular profiling: methods and protocols. Humana Press, Totowa, NJ, pp 295–309
Fung KYY, Fairn GD, Lee WL (2018) Transcellular vesicular transport in epithelial and endothelial cells: challenges and opportunities. Traffic 19:5–18. https://doi.org/10.1111/tra.12533
Armstrong SM, Sugiyama MG, Fung KYY et al (2015) A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis. Cardiovasc Res 108:268–277. https://doi.org/10.1093/cvr/cvv218
Huang L, Chambliss KL, Gao X et al (2019) SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis. Nature 569:565–569. https://doi.org/10.1038/s41586-019-1140-4
Ghaffari S, Nabi FN, Sugiyama MG, Lee WL (2018) Estrogen inhibits LDL (low-density lipoprotein) transcytosis by human coronary artery endothelial cells via GPER (G-protein-coupled estrogen receptor) and SR-BI (scavenger receptor class B type 1). Arterioscler Thromb Vasc Biol 38:2283–2294. https://doi.org/10.1161/ATVBAHA.118.310792
Ghaffari S, Jang E, Nabi FN et al (2021) Endothelial HMGB1 is a critical regulator of LDL transcytosis via an SREBP2–SR-BI axis. Arterioscler Thromb Vasc Biol 41(1):200–216. https://doi.org/10.1161/atvbaha.120.314557
Palade GE (1961) Blood capillaries of the heart and other organs. Circulation 24:368–384. https://doi.org/10.1161/01.cir.24.2.368
Bruns RR, Palade GE (1968) Studies on blood capillaries. II. Transport of ferritin molecules across the wall of muscle capillaries. J Cell Biol 37:277–299. https://doi.org/10.1083/jcb.37.2.277
Simionescu M, Simionescu N, Palade GE (1974) Morphometric data on the endothelium of blood capillaries. J Cell Biol 60:128–152. https://doi.org/10.1083/jcb.60.1.128
Palade GE, Bruns RR (1968) Structural modulations of plasmalemmal vesicles. J Cell Biol 37:633–649. https://doi.org/10.1083/jcb.37.3.633
Simionescu N, Simionescu M, Palade GE (1975) Permeability of muscle capillaries to small heme-peptides. J Cell Biol 64:586–607. https://doi.org/10.1083/jcb.64.3.586
Armstrong SM, Khajoee V, Wang C et al (2012) Co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and Rac. Am J Pathol 180:1308–1323. https://doi.org/10.1016/j.ajpath.2011.12.002
Velagapudi S, Yalcinkaya M, Piemontese A et al (2017) VEGF-A regulates cellular localization of SR-BI as well as transendothelial transport of HDL but not LDL. Arterioscler Thromb Vasc Biol 37:794–803. https://doi.org/10.1161/ATVBAHA.117.309284
Raheel H, Ghaffari S, Khosraviani N et al (2019) CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells: role in fatty acid delivery. Am J Physiol Lung Cell Mol Physiol 316:L740–L750. https://doi.org/10.1152/ajplung.00127.2018
Fung KY, Wang C, Nyegaard S et al (2017) SR-BI mediated transcytosis of HDL in brain microvascular endothelial cells is independent of caveolin, clathrin, and PDZK1. Front Physiol 8:1–16. https://doi.org/10.3389/fphys.2017.00841
Rutledge JC (1992) Temperature and hydrostatic pressure-dependent pathways of low-density lipoprotein transport across microvascular barrier. Am J Physiol Heart Circ Physiol 262:H234–H245. https://doi.org/10.1152/ajpheart.1992.262.1.h234
Gong Q, Huntsman C, Ma D (2008) Clathrin-independent internalization and recycling. J Cell Mol Med 12:126–144. https://doi.org/10.1111/j.1582-4934.2007.00148.x
El-Sayed A, Harashima H (2013) Endocytosis of gene delivery vectors: from clathrin-dependent to lipid raft-mediated endocytosis. Mol Ther 21:1118–1130. https://doi.org/10.1038/mt.2013.54
Crocker JC, Grier DG (1996) Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 179:298–310. https://doi.org/10.1006/jcis.1996.0217
Steyer JA, Horstmann H, Almers W (1997) Transport, docking and exocytosis of single secretory granules in live chromaffin cells. Nature 388:474–478. https://doi.org/10.1038/41329
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
1 Electronic Supplementary Materials
TIRF Video—20 μg mL of DiI-LDL.avi. Video of DiI-LDL transcytosis by primary human coronary artery endothelial cells using TIRF microscopy. Cells on coverslips were treated apically with 20 μg/mL of DiI-LDL. After incubation at 4 °C for 10 min and rinsing to remove unbound ligand, exocytosis events were imaged and quantified by MATLAB. The white dots indicate vesicles containing DiI-LDL (MP4 3011 kb)
TIRF Video—Competition with 400 μg unlabeled LDL.avi. Video of DiI-LDL transcytosis by primary human coronary artery endothelial cells using TIRF microscopy. Cells on coverslips were treated apically with 400 μg of unlabeled (nonfluorescent) LDL and 20 μg/mL of DiI-LDL concomitantly. After incubation at 4 °C for 10 min and rinsing to remove unbound ligand, exocytosis events were imaged. The white dots indicate vesicles containing DiI-LDL. The appearance of fewer dots indicates competition between unlabeled LDL and DiI-LDL (MP4 2054 kb)
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Jang, E., Ghaffari, S., Lee, W.L. (2022). Quantifying Endothelial Transcytosis with Total Internal Reflection Fluorescence Microscopy (TIRF). In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2051-9_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2050-2
Online ISBN: 978-1-0716-2051-9
eBook Packages: Springer Protocols