Direct stochastic optical reconstruction microscopy combined with different labeling approaches for the visualization and quantification of ACE2 on different cells

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.Jan 30 2023

In a recent study posted to the bioRxiv* preprint server, researchers performed dSTORM (direct stochastic optical reconstruction microscopy) with several labeling techniques for visualizing and quantifying angiotensin-converting enzyme 2 (ACE2) receptor expression on various cells.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Background

Studies have reported that ACE2 receptors are critical for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) entry into host cells, and SARS-CoV-2 S  (spike) glycoproteins have demonstrated binding interactions with the host cell plasma membranes exhibiting increased ACE2 expression. However, data on the impact of ACE2 receptor expression and organization within plasma membranes of SARS-CoV-2 infectivity are limited.

About the study

In the present study, researchers visualized ACE2 receptors, investigated their oligomeric states, and quantified endogenous ACE2 expression within cellular plasma membranes of Vero cells, Vero E6 cells, U2-OS cells, COS-7 cells, HEK293T (human embryonic kidney 293 cells), and ACE2 overexpressing-HEK293T cells by high-resolution fluorescence imaging at a molecular level.

Vero cells, Vero E6 cells, and HEK293T cells were used for reference in the cell-culture experiments, and ACE2-overexpressing HEK293T cells were used to assess the impact of elevated ACE2 receptor expression on the efficiency of host cellular infection by SARS-CoV-2. U2-OS cells and COS-7 cells were used as controls since they reportedly have minimal SARS-CoV-2 infection efficiency due to low ACE2 levels.

TIRF (total internal reflection fluorescence) analysis and dSTORM analysis were performed to visualize and quantify ACE2 receptors in the plasma membranes, respectively. For SARS-CoV-2 S-ACE2 binding evaluation, the monoclonal antibody of the A20069I clone with known anti-ACE2 activity was selected. The team verified the impact of endogenous ACE2 expression within plasma membranes on SARS-CoV-2 infectivity using proliferation-defective VSV (vesicular stomatitis virus) particles encoding coronavirus spike proteins.

The ability of dSTORM analysis to discriminate between the endogenous monomer from and dimeric forms of plasma membrane ACE2 proteins was investigated by imaging the monomeric cluster of differentiation (CD18), homo-dimeric CD69 and hetero-dimeric CD11a/CD18 using Jurkat cells and prime AF647-labelled antibodies for cellular immunostaining.

Results

Endogenous ACE2 receptor expression was observed in monomeric form within the cellular plasma membranes with one to two receptors per µm². Additionally, trimeric spike protein binding did not induce ACE2 receptor clustering in the cellular plasma membranes. The findings were underpinned by infection efficiency analyses performed utilizing VSV particles encoding CoV spike proteins.

The dSTORM analysis showed homogeneous ACE2 distribution within plasma membranes of Vero cells, Vero E6 cells, HEK293T cells, and the HEK293T cells that overexpressed ACE2. Every cluster of ACE2 with a mean of nine localizations corresponded to labeled ACE2 receptor expression. In Vero cells and Vero E6 cells, a comparable quantity of angiotensin-converting enzyme 2 receptors were observed (1.4 ACE2 receptors and 2.0 ACE2 receptors per µm², respectively). ACE2 receptor expression was marginally lesser among the human embryonic kidney 293T cells having one ACE2 receptor per µm^-2, whereas ACE2 overexpression-HEK293T showed 17.0 ACE2 receptors per µm².

The findings translated into mean distances between the ACE2 receptors among plasma membranes of 500.0 nm. ACE2 receptor levels are comparable among Vero cells and Vero E6 cells; however, VSV infection was observed only among Vero E6 cells and the green fluorescent protein (GFP) expression among Vero E6 cells was six percent.

Of interest, the efficiency of host cell infection by SARS-CoV-2 among Vero E6 cells was five-fold greater than that observed among HEK293T cells, albeit the ACE2 receptor expression levels differed slightly. HEK293T cells overexpressing ACE2 exhibited the greatest efficiency of SARS-CoV-2 infection (28.0%) concordant with the high ACE2 receptor expression.

The findings indicated that the angiotensin-converting enzyme 2 receptor expression among plasma membranes of host cells is critical for the entry of SARS-CoV-2 into the host, and the efficiency of host cellular infection by SARS-CoV-2 infection is proportional to the number of ACE2 receptors within the plasma membranes of the cells. However, the efficiency could strongly differ among different cells, indicating that various factors contribute to the efficiency of SARS-CoV-2 infections.

The average numbers for ACE2 receptor localization in each cluster were 7.0, 14, and 16 for the monomer form of CD18, the dimer form of CD69, and the hetero-dimer form of CD11a/CD18, respectively. The dSTORM analysis performed using prime AF647-labelled anti-ACE2 receptor antibodies demonstrated identical results, as obtained for CD18, and dSTORM analysis utilizing anti-neuropilin-1 antibodies verified the presence of dimers of neuropilin-1 receptors among HEK293T cells. Taken together, ACE2 localization analysis findings indicated that treatment with the trimer form of the S protein did not induce multimeric ACE2 formation.

Conclusion

Overall, the study findings showed that a single spike protein interaction per SARS-CoV-2 particle with monomer forms of ACE2 receptors is adequate for efficient infection of host cells by SARS-CoV-2. Endogenous ACE2 was present in monomeric form within the plasma membranes, irrespective of spike protein presence, and did not show dimer formation or greater aggregate formation.

Additionally, the findings indicated that the efficiency of SARS-CoV-2 infections could be determined by various factors, inclusive of the lipid component on plasma membranes and probable other types of molecules involved in the spike protein-ACE2 receptor binding interactions.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources