Membrane Topography and the Overestimation of Protein Clustering in Single Molecule Localisation Microscopy - Identification and Correction

Overview

Journal Commun Biol

Specialty Biology

Date 2024 Jul 1

PMID 38951588

Authors

Jeremy Adler

Kristoffer Bernhem

Ingela Parmryd

Affiliations

Soon will be listed here.

Abstract

According to single-molecule localisation microscopy almost all plasma membrane proteins are clustered. We demonstrate that clusters can arise from variations in membrane topography where the local density of a randomly distributed membrane molecule to a degree matches the variations in the local amount of membrane. Further, we demonstrate that this false clustering can be differentiated from genuine clustering by using a membrane marker to report on local variations in the amount of membrane. In dual colour live cell single molecule localisation microscopy using the membrane probe DiI alongside either the transferrin receptor or the GPI-anchored protein CD59, we found that pair correlation analysis reported both proteins and DiI as being clustered, as did its derivative pair correlation-photoactivation localisation microscopy and nearest neighbour analyses. After converting the localisations into images and using the DiI image to factor out topography variations, no CD59 clusters were visible, suggesting that the clustering reported by the other methods is an artefact. However, the TfR clusters persisted after topography variations were factored out. We demonstrate that membrane topography variations can make membrane molecules appear clustered and present a straightforward remedy suitable as the first step in the cluster analysis pipeline.

References

Shim S, Xia C, Zhong G, Babcock H, Vaughan J, Huang B . Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc Natl Acad Sci U S A. 2012; 109(35):13978-83. PMC: 3435176. DOI: 10.1073/pnas.1201882109. View

van de Linde S, Sauer M . How to switch a fluorophore: from undesired blinking to controlled photoswitching. Chem Soc Rev. 2013; 43(4):1076-87. DOI: 10.1039/c3cs60195a. View

Cai E, Marchuk K, Beemiller P, Beppler C, Rubashkin M, Weaver V . Visualizing dynamic microvillar search and stabilization during ligand detection by T cells. Science. 2017; 356(6338). PMC: 6364556. DOI: 10.1126/science.aal3118. View

Lassalle H, Baumann H, Strauss W, Schneckenburger H . Cell-substrate topology upon ALA-PDT using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM). J Environ Pathol Toxicol Oncol. 2007; 26(2):83-8. DOI: 10.1615/jenvironpatholtoxicoloncol.v26.i2.30. View

Ponjavic A, McColl J, Carr A, Santos A, Kulenkampff K, Lippert A . Single-Molecule Light-Sheet Imaging of Suspended T Cells. Biophys J. 2018; 114(9):2200-2211. PMC: 5961759. DOI: 10.1016/j.bpj.2018.02.044. View

Martin-Fernandez M, Tynan C, Webb S . A 'pocket guide' to total internal reflection fluorescence. J Microsc. 2013; 252(1):16-22. PMC: 4285862. DOI: 10.1111/jmi.12070. View

Rossboth B, Arnold A, Ta H, Platzer R, Kellner F, Huppa J . TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells. Nat Immunol. 2018; 19(8):821-827. PMC: 6071872. DOI: 10.1038/s41590-018-0162-7. View

Nieves D, Owen D . Analysis methods for interrogating spatial organisation of single molecule localisation microscopy data. Int J Biochem Cell Biol. 2020; 123:105749. DOI: 10.1016/j.biocel.2020.105749. View

Magee A, Adler J, Parmryd I . Cold-induced coalescence of T-cell plasma membrane microdomains activates signalling pathways. J Cell Sci. 2005; 118(Pt 14):3141-51. DOI: 10.1242/jcs.02442. View

10.

Fujimoto T, Parmryd I . Interleaflet Coupling, Pinning, and Leaflet Asymmetry-Major Players in Plasma Membrane Nanodomain Formation. Front Cell Dev Biol. 2017; 4:155. PMC: 5222840. DOI: 10.3389/fcell.2016.00155. View

11.

Franke C, Sauer M, van de Linde S . Photometry unlocks 3D information from 2D localization microscopy data. Nat Methods. 2016; 14(1):41-44. DOI: 10.1038/nmeth.4073. View

12.

Durisic N, Cuervo L, Lakadamyali M . Quantitative super-resolution microscopy: pitfalls and strategies for image analysis. Curr Opin Chem Biol. 2014; 20:22-8. DOI: 10.1016/j.cbpa.2014.04.005. View

13.

Baumgart T, Hunt G, Farkas E, Webb W, Feigenson G . Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. Biochim Biophys Acta. 2007; 1768(9):2182-94. PMC: 2702987. DOI: 10.1016/j.bbamem.2007.05.012. View

14.

van Rheenen J, Jalink K . Agonist-induced PIP(2) hydrolysis inhibits cortical actin dynamics: regulation at a global but not at a micrometer scale. Mol Biol Cell. 2002; 13(9):3257-67. PMC: 124157. DOI: 10.1091/mbc.e02-04-0231. View

15.

Wallis T, Jiang A, Young K, Hou H, Kudo K, McCann A . Super-resolved trajectory-derived nanoclustering analysis using spatiotemporal indexing. Nat Commun. 2023; 14(1):3353. PMC: 10250379. DOI: 10.1038/s41467-023-38866-y. View

16.

Bohrer C, Yang X, Thakur S, Weng X, Tenner B, McQuillen R . A pairwise distance distribution correction (DDC) algorithm to eliminate blinking-caused artifacts in SMLM. Nat Methods. 2021; 18(6):669-677. PMC: 9040192. DOI: 10.1038/s41592-021-01154-y. View

17.

Tanaka K, Suzuki K, Shirai Y, Shibutani S, Miyahara M, Tsuboi H . Membrane molecules mobile even after chemical fixation. Nat Methods. 2010; 7(11):865-6. DOI: 10.1038/nmeth.f.314. View

18.

Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A . Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods. 2011; 8(7):527-8. DOI: 10.1038/nmeth.1627. View

19.

Jensen L, Hoh T, Williamson D, Griffie J, Sage D, Rubin-Delanchy P . Correction of multiple-blinking artifacts in photoactivated localization microscopy. Nat Methods. 2022; 19(5):594-602. DOI: 10.1038/s41592-022-01463-w. View

20.

Veatch S, Machta B, Shelby S, Chiang E, Holowka D, Baird B . Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting. PLoS One. 2012; 7(2):e31457. PMC: 3288038. DOI: 10.1371/journal.pone.0031457. View