Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Purification of infection-associated macropinosomes by magnetic isolation for proteomic characterization

Nature Protocols volume 16, pages 5220–5249 (2021)Cite this article

Subjects

Abstract

Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Illustrative overview of the protocol for the magnetic purification of macropinosomes.
Fig. 2: Assembly of the magnetic isolation tubing.
Fig. 3: Overview of the HOKImag magnetic separation system for the purification of macropinosomes.
Fig. 4: Zoom-in of the HOKImag system for the magnetic purification of macropinosomes.
Fig. 5: Retrieval of the magnetic fraction upon magnetic fractionation.
Fig. 6: Expected results from quality controls.
Fig. 7: Expected results from mass spectrometric analysis.

Similar content being viewed by others

Data availability

Human proteome (Uniprot, v20150113) and Salmonella Typhimurium proteome (Uniprot, 99287) were used in the proteomic analysis. All data included in this protocol are openly available and can be found in the public data online repository PRIDE Archive (PXD012825)30. Source data are provided with this paper. The source data files for Fig. 6b–d are also available at online repository figshare (https://doi.org/10.6084/m9.figshare.14183024 and https://doi.org/10.6084/m9.figshare.14183090, respectively) and in the supporting primary research article30.

References

  1. Ha, K. D., Bidlingmaier, S. M. & Liu, B. Macropinocytosis exploitation by cancers and cancer therapeutics. Front. Physiol. 7, 381–381 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Kumari, S., Mg, S. & Mayor, S. Endocytosis unplugged: multiple ways to enter the cell. Cell Res. 20, 256–275 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Naslavsky, N. & Caplan, S. The enigmatic endosome—sorting the ins and outs of endocytic trafficking. J. Cell Sci. 131, jcs216499 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Garcia-Castillo, M. D., Chinnapen, D. J. & Lencer, W. I. Membrane transport across polarized epithelia. Cold Spring Harb. Perspect. Biol. 9 (2017).

  5. Joel, A. S. & Jason, S. K. The breadth of macropinocytosis research. Philos. Trans. R. Soc. Lond. B Biol. Sci. 374, 20180146 (2019).

    Article  CAS  Google Scholar 

  6. Lewis, W. H. Pinocytosis by malignant cells. Am. J. Cancer Res. 29, 666–679 (1937).

    Google Scholar 

  7. Egami, Y., Taguchi, T., Maekawa, M., Arai, H. & Araki, N. Small GTPases and phosphoinositides in the regulatory mechanisms of macropinosome formation and maturation. Front. Physiol. 5, 374 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Kühn, S., Lopez-Montero, N., Chang, Y. Y., Sartori-Rupp, A. & Enninga, J. Imaging macropinosomes during Shigella infections. Methods 127, 12–22 (2017).

    Article  PubMed  CAS  Google Scholar 

  9. Jayashankar, V. & Edinger, A. L. Macropinocytosis confers resistance to therapies targeting cancer anabolism. Nat. Commun. 11, 1121 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Redelman-Sidi, G. et al. The canonical Wnt pathway drives macropinocytosis in cancer. Cancer Res. 78, 4658–4670 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li, Y. et al. Macropinocytosis-mediated membrane recycling drives neural crest migration by delivering F-actin to the lamellipodium. Proc. Natl Acad. Sci. USA 117, 27400 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Freeman, S. A. et al. Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance. Science 367, 301–305 (2020).

    Article  CAS  PubMed  Google Scholar 

  13. Palm, W. Metabolic functions of macropinocytosis. Philos. Trans. R Soc. Lond. B Biol. Sci. 374, 20180285–20180285 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Canton, J. Macropinocytosis: new insights into its underappreciated role in innate immune cell surveillance. Front. Immunol. 9 (2018).

  15. Liu, Z. & Roche, P. A. Macropinocytosis in phagocytes: regulation of MHC class-II-restricted antigen presentation in dendritic cells. Front. Physiol. 6, 1 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kerr, M. C. & Teasdale, R. D. Defining macropinocytosis. Traffic 10, 364–371 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Jones, A. T., Gumbleton, M. & Duncan, R. Understanding endocytic pathways and intracellular trafficking: a prerequisite for effective design of advanced drug delivery systems. Adv. Drug Deliv. Rev. 55, 1353–1357 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Buckley, C. M. & King, J. S. Drinking problems: mechanisms of macropinosome formation and maturation. FEBS J. 284, 3778–3790 (2017).

    Article  CAS  PubMed  Google Scholar 

  19. Joel, A. S. & Sei, Y. Macropinosomes as units of signal transduction. Philos. Trans. R. Soc. Lond. BBiol. Sci. 374, 20180157 (2019).

    Article  CAS  Google Scholar 

  20. Ford, C., Nans, A., Boucrot, E. & Hayward, R. D. Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry. PLoS Pathog. 14, e1007051 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Lee, C. H. R., Mohamed Hussain, K. & Chu, J. J. H. Macropinocytosis dependent entry of Chikungunya virus into human muscle cells. PLoS Negl. Trop. Dis. 13, e0007610 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Kälin, S. et al. Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35. J. Virol. 84, 5336–5350 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Torriani, G. et al. Macropinocytosis contributes to hantavirus entry into human airway epithelial cells. Virology 531, 57–68 (2019).

    Article  CAS  PubMed  Google Scholar 

  24. Helenius, A. Virus entry: looking back and moving forward. J. Mol. Biol. 430, 1853–1862 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mercer, J. & Helenius, A. Virus entry by macropinocytosis. Nat. Cell Biol. 11, 510–520 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Hetzenecker, S., Helenius, A. & Krzyzaniak, M. A. HCMV induces macropinocytosis for host cell entry in fibroblasts. Traffic 17, 351–368 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Garcia-del Portillo, F. & Finlay, B. B. Salmonella invasion of nonphagocytic cells induces formation of macropinosomes in the host cell. Infect. Immun. 62, 4641–4645 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Weiner, A. et al. Macropinosomes are key players in early Shigella invasion and vacuolar escape in epithelial cells. PLoS Pathog. 12, e1005602 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Fredlund, J. et al. The entry of Salmonella in a distinct tight compartment revealed at high temporal and ultrastructural resolution. Cell. Microbiol. 20 (2018).

  30. Stévenin, V. et al. Dynamic growth and shrinkage of the Salmonella-containing vacuole determines the intracellular pathogen niche. Cell Rep 29, 3958–3973.e3957 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Chang, Y.-Y., Enninga, J. & Stévenin, V. New methods to decrypt emerging macropinosome functions during the host–pathogen crosstalk. Cell. Microbiol. 23, e13342 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chang, Y.-Y. et al. Shigella hijacks the exocyst to cluster macropinosomes for efficient vacuolar escape. PLoS Pathog 16, e1008822 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. de Araújo, M. E. G., Lamberti, G. & Huber, L. A. Isolation of early and late endosomes by density gradient centrifugation. Cold Spring Habor Protoc. 2015, pdb.prot083444 (2015).

    Article  Google Scholar 

  34. Kobayashi, T. et al. Separation and characterization of late endosomal membrane domains. J. Biol. Chem. 277, 32157–32164 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Tjelle, T. E., Brech, A., Juvet, L. K., Griffiths, G. & Berg, T. Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation. J. Cell Sci. 109, 2905–2914 (1996).

    Article  CAS  PubMed  Google Scholar 

  36. Walker, L. R., Hussein, H. A. & Akula, S. M. Subcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirus. Cell Biosci. 6, 1 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Plouffe, B. D., Murthy, S. K. & Lewis, L. H. Fundamentals and application of magnetic particles in cell isolation and enrichment: a review. Rep. Prog. Phys. 78, 016601–016601 (2015).

    Article  PubMed  CAS  Google Scholar 

  38. Wittrup, A. et al. Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery. Proc. Natl Acad. Sci. USA 107, 13342–13347 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Miltenyi, S., Muller, W., Weichel, W. & Radbruch, A. High gradient magnetic cell separation with MACS. Cytometry 11, 231–238 (1990).

    Article  CAS  PubMed  Google Scholar 

  40. Özbaykal, G. et al. The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli. eLife 9, e50629 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Vigouroux, A. et al. Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects. eLife 9, e51998 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Haigler, H. T., McKanna, J. A. & Cohen, S. Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor. J. Cell Biol. 83, 82–90 (1979).

    Article  CAS  PubMed  Google Scholar 

  43. Fujii, M., Kawai, K., Egami, Y. & Araki, N. Dissecting the roles of Rac1 activation and deactivation in macropinocytosis using microscopic photo-manipulation. Sci. Rep. 3, 2385 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Nakase, I. et al. Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol. Ther. 10, 1011–1022 (2004).

    Article  CAS  PubMed  Google Scholar 

  45. Al-Taei, S. et al. Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. implications for their utilization as drug delivery vectors. Bioconjug. Chem. 17, 90–100 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Nakase, I. et al. Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis. Biochemistry 46, 492–501 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. D’Astolfo, D. S. et al. Efficient intracellular delivery of native proteins. Cell 161, 674–690 (2015).

    Article  PubMed  CAS  Google Scholar 

  48. Hetrick, B. et al. Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem. Biol. 20, 701–712 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Koivusalo, M. et al. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J. Cell Biol. 188, 547–563 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ivanov, A.I. Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? in Exocytosis and Endocytosis (ed. Ivanov, A. I.) 15–33 (Humana Press, 2008).

  51. Addi, C. et al. The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis. Nat. Commun. 11, 1941 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Rizzo, J. et al. Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts. mSphere 5, e00476–00420 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Lelouard, H. et al. Pathogenic bacteria and dead cells are internalized by a unique subset of Peyer’s patch dendritic cells that express lysozyme. Gastroenterology 138, 173–184.e171-173 (2010).

    Article  CAS  PubMed  Google Scholar 

  54. Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).

    Article  CAS  PubMed  Google Scholar 

  55. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. Wieczorek, S. et al. DAPAR & ProStaR: software to perform statistical analyses in quantitative discovery proteomics. Bioinformatics 33, 135–136 (2017).

    Article  CAS  PubMed  Google Scholar 

  57. Giai Gianetto, Q., Wieczorek, S., Couté, Y. & Burger, T. A peptide-level multiple imputation strategy accounting for the different natures of missing values in proteomics data. Preprint at bioRxiv https://doi.org/10.1101/2020.05.29.122770 (2020).

  58. Giai Gianetto, Q. et al. Calibration plot for proteomics: a graphical tool to visually check the assumptions underlying FDR control in quantitative experiments. Proteomics 16, 29–32 (2016).

    Article  CAS  PubMed  Google Scholar 

  59. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Mi, H., Muruganujan, A., Ebert, D., Huang, X. & Thomas, P. D. PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools. Nucleic Acids Res. 47, D419–D426 (2018).

    Article  PubMed Central  CAS  Google Scholar 

  61. Santos, J. C. et al. The COPII complex and lysosomal VAMP7 determine intracellular Salmonella localization and growth. Cell. Microbiol. 17, 1699–1720 (2015).

    Article  CAS  PubMed  Google Scholar 

  62. Ibarra, J. A. et al. Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella-host cell interactions in vitro. Microbiology 156, 1120–1133 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Steele-Mortimer, O. Infection of epithelial cells with Salmonella enterica. in Bacterial Pathogenesis: Methods and Protocols (eds. DeLeo, F. R. & Otto, M.) 201–211 (Humana Press, 2008).

  64. Li, L. et al. The effect of the size of fluorescent dextran on its endocytic pathway. Cell Biol. Int. 39, 531–539 (2015).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank V. Sohst and N. Reiling (Research Center, Borstel, Germany) for the enthusiasm and great help at the initial stages of this project. Y-Y.C was supported by a fellowship from the Fondation pour la Recherche Médicale (FRM; SPF20160936275). V.S. was supported by a PhD fellowship from the University Paris Diderot (now Université de Paris) allocated by the ENS Paris-Saclay, a grant from the FRM (FRM; FDT20170436843), and an extension grant from the BCI Department of Institut Pasteur. J.E. is a member of the LabEx consortia IBEID and MilieuInterieur. J.E. also acknowledges support from the ANR (grant StopBugEntry and AutoHostPath) and the ERC (CoG EndoSubvert).

Author information

Authors and Affiliations

  1. Institut Pasteur, Dynamics of Host-Pathogen Interactions Unit and CNRS UMR 3691, Paris, France

    Virginie Stévenin, Jost Enninga & Yuen-Yan Chang

  2. Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands

    Virginie Stévenin

  3. Université de Paris, Ecole Doctorale BioSPC, Paris, France

    Virginie Stévenin

  4. Proteomics Platform, Mass Spectrometry for Biology Unit, Institut Pasteur, USR 2000 CNRS, Paris, France

    Quentin Giai Gianetto, Magalie Duchateau & Mariette Matondo

  5. Hub Bioinformatics et Biostatistics, Computational Biology Department, USR CNRS, Institut Pasteur, Paris, France

    Quentin Giai Gianetto

  6. Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

    Yuen-Yan Chang

Authors
  1. Virginie Stévenin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Quentin Giai Gianetto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Magalie Duchateau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mariette Matondo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jost Enninga
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuen-Yan Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.S. and Y-Y.C. developed the protocol with input from J.E. V.S. and Y-Y.C. performed the experiments and wrote the manuscript. M.D. and Q.G.G. acquired data, performed analysis and wrote the manuscript. M.M. and J.E. supervised the study, provided feedback on the manuscript and secured funding.

Corresponding authors

Correspondence to Virginie Stévenin or Yuen-Yan Chang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Stévenin, V. et al. Cell Rep. 19, 3958–3973.e7 (2019): https://doi.org/10.1016/j.celrep.2019.11.049

Chang, Y. Y. et al. PLoS Pathog. 16, e1008822 (2020): https://doi.org/10.1371/journal.ppat.1008822

Chang, Y. Y. et al. Cell. Microbiol. 23, e13342 (2021): https://doi.org/10.1111/cmi.13342

Supplementary information

Source data

Source Data Fig. 6

Unprocessed gel (stained with Coomassie Blue)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Stévenin, V., Giai Gianetto, Q., Duchateau, M. et al. Purification of infection-associated macropinosomes by magnetic isolation for proteomic characterization. Nat Protoc 16, 5220–5249 (2021). https://doi.org/10.1038/s41596-021-00610-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00610-5

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research