Membrane Topography and the Overestimation of Protein Clustering in Single Molecule Localisation Microscopy – Identification and Correction-Reference-Cited by-同舟云学术

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

Published:2024-04-10 Issue: Volume: Page:
ISSN:
Container-title:
language:
Short-container-title:

Author:

Adler Jeremy¹,Bernhem Kristoffer²,Parmryd Ingela¹

Affiliation:

1. University of Gothenburg

2. Royal Institute of Technology

Abstract

Abstract According to single-molecule localisation microscopy (SMLM) 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 SMLM using the membrane probe DiI alongside either the transferrin receptor (TfR) or the GPI-anchored protein CD59, we found that pair correlation (PC) analysis reported both proteins and DiI as being clustered, as did its derivative PC-PALM 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 must be considered before concluding that membrane molecules cluster and present a method to this end.

Publisher

Research Square Platform LLC

Reference54 articles.

1. 1. Garcia-Parajo, M. F., Cambi, A., Torreno-Pina, J. A., Thompson, N. & Jacobson, K. Nanoclustering as a dominant feature of plasma membrane organization. J Cell Sci 127, 4995–5005, doi:10.1242/jcs.146340 (2014).

2. 2. Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U. & Radenovic, A. Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods 8, 527–528, doi:10.1038/nmeth.1627 (2011).

3. 3. van de Linde, S., Wolter, S., Heilemann, M. & Sauer, M. The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. J Biotechnol 149, 260–266, doi:10.1016/j.jbiotec.2010.02.010 (2010).

4. 4. van de Linde, S. & Sauer, M. How to switch a fluorophore: from undesired blinking to controlled photoswitching. Chem Soc Rev 43, 1076–1087, doi:10.1039/c3cs60195a (2014).

5. 5. Sengupta, P. et al. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat Methods 8, 969–975, doi:10.1038/nmeth.1704 (2011).