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Updated: Jun 22, 2025
Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Published on: June 30, 2018
Membrane topography and the overestimation of protein clustering in single molecule localisation microscopy -
Jeremy Adler1, Kristoffer Bernhem2, Ingela Parmryd3
1Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
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.
Insights
Plasma membrane protein clusters may be artifacts of membrane topography. Researchers developed a method using a membrane marker to distinguish true protein clusters from false ones, revealing topography
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Single-molecule localization microscopy (SMLM) suggests most plasma membrane proteins form clusters.
- Interpreting SMLM data requires distinguishing genuine protein aggregation from artifacts.
- Membrane topography variations can potentially create false positive clustering signals.
Purpose of the Study:
- To investigate whether variations in membrane topography can cause apparent clustering of membrane proteins.
- To develop and validate a method to differentiate true protein clusters from topography-induced artifacts.
- To re-evaluate the clustering of specific membrane proteins, transferrin receptor (TfR) and CD59, considering membrane topography.
Main Methods:
- Dual-color live-cell SMLM was employed.
- The membrane probe DiI was used alongside TfR and CD59.
- Analysis included pair correlation, pair correlation-photoactivation localization microscopy, and nearest neighbor analyses.
- Image analysis incorporated a DiI-based membrane topography correction.
Main Results:
- Standard analyses indicated clustering for TfR, CD59, and DiI.
- After correcting for membrane topography using DiI signals, CD59 clusters were no longer detected, suggesting they were artifacts.
- TfR clusters remained detectable even after topography correction, indicating genuine clustering.
Conclusions:
- Membrane topography variations can lead to false positive results in standard cluster analysis methods.
- A topography correction method using a membrane marker is effective in distinguishing true from false protein clusters.
- While CD59 clustering appears to be an artifact of membrane topography, TfR exhibits genuine clustering.
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