Immunolocalization studies of vimentin and ACE2 on the surface of cells exposed to SARS-CoV-2 Spike proteins

  1. Vasiliki Lalioti
  2. Silvia González-Sanz
  3. Irene Lois-Bermejo
  4. Patricia González-Jiménez
  5. Álvaro Viedma-Poyatos
  6. Andrea Merino
  7. María A. Pajares
  8. Dolores Pérez-Sala

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

The Spike protein from SARS-CoV-2 mediates docking of the virus onto cells and contributes to viral invasion. Several cellular receptors are involved in SARS-CoV-2 Spike docking at the cell surface, including ACE2 and neuropilin. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Here we have explored the binding of Spike protein constructs to several cell types using low-temperature immunofluorescence approaches in live cells, to minimize internalization. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which showed scarce colocalization with a lipid raft marker, but consistent coincidence with ACE2. Under our conditions, vimentin immunoreactivity appeared as spots or patches unevenly distributed at the surface of diverse cell types. Remarkably, several observations including potential antibody internalization and adherence to cells of vimentin-positive structures present in the extracellular medium exposed the complexity of vimentin cell surface immunoreactivity, which requires careful assessment. Notably, overall colocalization of Spike and vimentin signals markedly varied with the cell type and the immunodetection sequence. In turn, vimentin-positive spots moderately colocalized with ACE2; however, a particular enrichment was detected at elongated structures positive for acetylated tubulin, consistent with primary cilia, which also showed Spike binding. Thus, these results suggest that vimentin-ACE2 interaction could occur at selective locations near the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.05.04.442648: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    (S1+S2; containing amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.
    Trp1212
    suggested: None
    Ser740
    suggested: (Antibodies-Online Cat# ABIN801498, RRID:AB_11187813)
    Biotechnology; mouse monoclonal anti-vimentin clone 13.2 (V5255) from Sigma; rabbit monoclonal SP20 anti-vimentin antibody from ThermoFisher Scientific; cell-surface vimentin (CSV) antibody, clone 84-1, from Abnova, and goat anti-vimentin antibody EB11207 from Everest Biotech.
    vimentin ( CSV )
    suggested: None
    anti-vimentin
    suggested: None
    Anti-ARL13B C-5 (sc-515784) was from Santa Cruz Biotechnology, anti-ACE2 rabbit antibodies were purchased from Invitrogen and Abcam, anti-human IgG-Alexa647 and anti-human IgG-Alexa568 were obtained from Invitrogen, anti-acetylated tubulin (T7451) was from Sigma and anti-SARS-CoV-2 Spike protein was a product of Sino Biological.
    Anti-ARL13B C-5
    suggested: (Santa Cruz Biotechnology Cat# sc-515784, RRID:AB_2890034)
    anti-ACE2
    suggested: None
    anti-human IgG-Alexa647
    suggested: None
    anti-human IgG-Alexa568
    suggested: None
    anti-acetylated tubulin
    suggested: (Sigma-Aldrich Cat# T7451, RRID:AB_609894)
    anti-SARS-CoV-2 Spike protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    (S1+S2; containing amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Human adrenal carcinoma SW13/cl.2 cells were the generous gift of Dr. A. Sarriá (University of Zaragoza, Spain) [41].
    SW13/cl.2
    suggested: RRID:CVCL_WY52)
    Human lung adenocarcinoma A549 cells (ATCC) were cultured in RPMI1640 with 10% (v/v)
    A549
    suggested: None
    HAP1 cells were cultured in DMEM-F12 with 10% (v/v)
    HAP1
    suggested: None
    For assessment of ACE2 levels, A549 and Vero cells were lysed in RIPA containing cOmplete™ Protease Inhibitor Cocktail (Sigma)
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Biotechnology; mouse monoclonal anti-vimentin clone 13.2 (V5255) from Sigma; rabbit monoclonal SP20 anti-vimentin antibody from ThermoFisher Scientific; cell-surface vimentin (CSV) antibody, clone 84-1, from Abnova, and goat anti-vimentin antibody EB11207 from Everest Biotech.
    ThermoFisher Scientific
    suggested: None
    Images were analyzed using LasX or ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analysis: Statistical analyses were performed with GraphPad Prism 5 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Antibody-based detection of cell surface antigens is widely used although it is not exempt from limitations. Many of the antibodies employed herein have been validated by various methods (see Suppl. Table 1), including the confirmation of lack of signal in vimentin-deficient or knockout cells. Nevertheless, most of them have not been validated for the specific detection of cell surface vimentin. Under our conditions, incubation of live, non-permeabilized cells with a variety of anti-vimentin antibodies gave a dotted pattern non-homogeneously distributed along the cell surface. In addition, the presence of adhered vimentin or of focal membrane permeabilization contributed to the heterogeneity of the pattern of cell surface vimentin staining. Of the antibodies used, the 84-1 clone gave the strongest signal, followed by the V9 antibody in cells subsequently fixed with 4% (w/v) PFA. Nevertheless, controls of specificity carried out in two vimentin-deficient cell lines, namely SW13/cl.2 adrenal carcinoma and HAP1 (vim −), with either the V9 or the 84-1 antibodies still yielded a non-negligible background. A putative source for this background could be vimentin present in serum, e.g., in exosomes [73]; however, it was not abolished by culturing SW13/cl.2 cells in the absence of serum. Alternatively, the possibility of a minor crossreactivity of the antibodies with other cellular structures needs to be considered. Indeed, certain anti-vimentin antibodies generated in patients show c...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32 and 33. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.05.04.442648 on bioRxiv