Convex hull as diagnostic tool in single-molecule localization microscopy

Ebert, Vincent; Eiring, Patrick; Helmerich, Dominic A.; Seifert, Rick; Sauer, Markus; Doose, Sören

doi:10.1093/bioinformatics/btac700

Cited by 5 publications

(4 citation statements)

References 29 publications

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“…To study the distribution of plasma membrane ACE2 receptors, we used a density‐based spatial clustering of applications with noise (DBSCAN) algorithm with customized localization analysis (LOCAN) [30] . After selecting basal membrane regions that do not show folded membrane areas, repeated localizations were grouped using the DBSCAN algorithm with appropriate parameters to ensure each cluster represents an isolated receptor [31] . Under the applied experimental conditions, we localized each AF647 antibody on average 9.2±0.2 times.…”

Section: Resultsmentioning

confidence: 99%

“…[30] After selecting basal membrane regions that do not show folded membrane areas, repeated localizations were grouped using the DBSCAN algorithm with appropriate parameters to ensure each cluster represents an isolated receptor. [31] Under the applied experimental conditions, we localized each AF647 antibody on average 9.2 � 0.2 times. Hence, each localization cluster with on average � 9 localizations corresponds to a labeled ACE2 receptor.…”

Section: Resultsmentioning

confidence: 99%

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Coronaviruses Use ACE2 Monomers as Entry‐Receptors

et al. 2023

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The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors μm À 2 . In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Coronaviruses Use ACE2 Monomers as Entry‐Receptors

et al. 2023

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“…To study the distribution of plasma membrane ACE2 receptors, we used a density-based spatial clustering of applications with noise (DBSCAN) algorithm with customized localization analysis (LOCAN) 30 . After selecting basal membrane regions that do not show folded membrane areas, repeated localizations were grouped using the DBSCAN algorithm with appropriate parameters to ensure each cluster represents an isolated receptor 31 . The Ripley-h function confirms that localization clusters only occur on the length scale of the localization precision (Extended Data Fig.…”

Section: Imaging and Quantification Of Plasma Membrane Ace2 Receptors...mentioning

confidence: 99%

Coronaviruses use ACE2 monomers as entry receptors

Eiring

Klein

Backes

et al. 2023

Preprint

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The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of preferably multimeric ACE2 receptors to achieve a higher binding and infection efficiency. However, our current knowledge about the influence of ACE2 expression and organization in the plasma membrane on SARS-CoV-2 infection efficiency remains elusive. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors um-2. In addition, binding of trimeric S proteins does not induce clustering of ACE2 receptors in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection which attests SARS-CoV-2 a high infectivity.

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“…The analysis parameters were selected by checking the clustering approach on simulated data 38 . The overall density of CARs in the plasma membrane was low enough to yield well separated nearest neighbors for all plasma membranes investigated.…”

Section: Cars Form Nanodomains In the Plasma Membrane Of T Cellsmentioning

confidence: 99%

CARs are organized in nanodomains in the plasma membrane of T cells that accumulate at tumor contact sites

Verbruggen

Gehrke

Banholzer

et al. 2023

Preprint

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Chimeric antigen receptors (CARs) are synthetic immune receptors that are expressed in T cells through genetic engineering. CAR-T cells have been successfully used to eradicate very advanced leukemias and lymphomas and their functional properties have been intensively studied. However, relatively little is known about the spatiotemporal expression and organization of CARs on the T-cell membrane and how this influences their efficacy. Here, we applied super-resolution microscopy to visualize CD19-, ROR1-, and ROR2-specific CARs in human CD4+ and CD8+ T cells that were engineered with lentiviral and transposon-mediated gene transfer. Our data show that the majority of CARs is organized in nanodomains virtually independent of the T cell type, CAR construct and expression level. Quantitative analyses revealed a slightly higher CAR density in transposon-engineered T cells correlating with higher antigen sensitivity and faster resolution of anti-tumor functions compared to lentivirally-engineered T cells. Live-cell fluorescence imaging revealed that both, CAR nanodomains and CAR monomers accumulate at tumor contact sites and form multifocal immunological synapses. Our study provides novel insights into the membrane organization of CARs with single-molecule resolution and illustrates the potential of advanced microscopy to inform the rational design of synthetic immune receptors for applications in immune cell therapy.

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Convex hull as diagnostic tool in single-molecule localization microscopy

Cited by 5 publications

References 29 publications

Coronaviruses Use ACE2 Monomers as Entry‐Receptors

Coronaviruses Use ACE2 Monomers as Entry‐Receptors

Coronaviruses use ACE2 monomers as entry receptors

CARs are organized in nanodomains in the plasma membrane of T cells that accumulate at tumor contact sites

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