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Histochemistry and Cell Biology

, Volume 145, Issue 6, pp 617–627 | Cite as

Megapinocytosis: a novel endocytic pathway

  • Andrea Bauer
  • Narmadha Subramanian
  • Clarissa Villinger
  • Giada Frascaroli
  • Thomas Mertens
  • Paul WaltherEmail author
Original Paper

Abstract

M2 macrophages showed large endocytotic structures, very different from classical macropinosomes that we named megapinosomes. As observed in the scanning electron microscope, megapinosome formation started with a large (diameter of several micrometers) invagination of the plasma membrane. When the invagination was almost completed, the remaining opening was closed by an actinomorphous centripetal arrangement of many (about 50–100) microvilli-like structures. In transmission electron microscopy using high-pressure freezing, we observed that the megapinosome was filled with a trabecular meshwork that originated from the highly structured plasma membrane. The trabecular meshwork was topologically part of the cytosol and separated from the extracellular fluid by a lipid bilayer. According to ultrastructural features, we could define different phases of megapinosome formation and decay. Megapinosomes became more frequent when M2 macrophages were inoculated with human cytomegalovirus. We did not find megapinosome formation in M1 macrophages.

Keywords

Macrophages Macropinocytosis Endocytosis Electron microscopy High-pressure freezing TEM SEM 

Notes

Acknowledgments

We thank Renate Kunz, Anke Lüske, Eberhard Schmid, and Reinhard Weih for technical assistance, and Dr. Rudolph Reimer, HPI Hamburg, and Gregor Neusser, Ulm University, for their help with the analysis of the stereo pairs and the height profiles.

Supplementary material

418_2015_1395_MOESM1_ESM.jpg (1.8 mb)
Figure S1 Anaglyph stereo pairs of Figs. 1 and 2 demonstrating the depressions in M2 macrophages. Figs S1A represents an early phase, B is a higher magnification of the boxed area, and C represents a later stage with an even deeper depression. Fig. S1D shows for comparison an M1 macrophage without depression. Fig S1E (F higher magnification) shows an M2 macrophage with a deep cavity most likely representing a late stage of megapinosome formation. (JPEG 1874 kb)
Movie 1

Animation of stereo images of a M1 macrophage without a surface depression, a M2 macrophage with an early phase megapinosome formation, a M2 macrophage with a later stage megapinosome. A M2 macrophage with a putative late stage megapinosome formation. 25 images were recorded with a tilt increment of 2°. Finally the megapinosome was closed by an actinomorphous ring of microvilli-like structures. Supplementary material 2 (M4 V 46585 kb)

418_2015_1395_MOESM3_ESM.m4v (33.4 mb)
Movie 2 3D reconstruction of a serial section analysis of a cell with a megapinosome (red) a megapinosome complex (red and orange) and a nucleus (beige). This cell shows a stage of megapinocytosis, where megapinosome and megapinosome complex are completely surrounded by cytoplasm in all sections. The reconstruction proves that megapinosomes exist without any connection to the extracellular space. Therefore, megapinocytosis is a real endocytotic process. (M4 V 34172 kb)
418_2015_1395_MOESM4_ESM.m4v (36.8 mb)
Movie 3 A polar large megapinosome complex. Also in this cell the megapinosome as well as the megapinosome complex are fully internalized into the cell without an opening to the cytoplasm. (M4 V 37636 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Andrea Bauer
    • 1
  • Narmadha Subramanian
    • 1
    • 2
  • Clarissa Villinger
    • 1
    • 2
  • Giada Frascaroli
    • 1
  • Thomas Mertens
    • 1
  • Paul Walther
    • 2
    Email author
  1. 1.Institute of VirologyUniversity Medical Center UlmUlmGermany
  2. 2.Central Facility for Electron MicroscopyUlm UniversityUlmGermany

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