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Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae

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Abstract

Membrane topology control is thought to involve peripheral membrane proteins of the F-BAR domain family including syndapins. These proteins are predestined to shape membranes by partial insertion and by imposing their curved shape onto the lipid bilayer. Direct observation of such functions on cellular membranes, however, was precluded by the difficulty to combine high-resolution imaging with visualization of membrane topology. Here, we report the ultrastructural visualization of endogenous syndapin II at the plasma membrane of NIH 3T3 cells using a combination of freeze-fracturing, immunogold labeling and transmission electron microscopy. Surprisingly, syndapin II was detected at flat and curved membrane areas. Ultrastructural colocalization with caveolin 1 identified syndapin II-positive invaginations as caveolae. Consistent with the syndapin II F-BAR domain interacting with caveolin 1, F-BAR overexpression affected caveolin 1 localization. Syndapin II knockdown did not alter caveolin 1 expression or plasma membrane recruitment. Instead, syndapin II knockdown reduced the density of caveolae and strongly increased the number of caveolin 1 molecules at flat membrane areas. Comparative immunoelectron microscopy and tilt series revealed that syndapin II was asymmetrically localized at the neck of caveolae. Double-immunogold labeling showed that the caveolae-shaping molecule PTRF/cavin 1 behaved similarly and that syndapin II and PTRF/cavin 1 colocalized. Visualization of a transiently membrane-binding F-BAR protein in direct relation to membrane topology of mammalian cells thereby revealed that syndapin II binds to both flat and curved membranes in vivo and that it plays an important role in caveolar shaping, a role that it shares with PTRF/cavin 1.

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Acknowledgments

We thank M. Öhler, B. Schade and R. Kaiser for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to BQ and MMK.

Author information

Correspondence to Britta Qualmann.

Additional information

D. Koch and M. Westermann contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Movie S1 Syndapin II is localized to the neck of caveolae. Tilt series of caveolae labeled with anti-syndapin II antibodies identified syndapin II at the neck of caveolae. A sequence of micrographs in steps of 4° was taken with a tiltable cryo holder. The image shift was calculated by cross-correlation using the software EMMENU V3.0 (AVI 41,130 kb)

Supplementary Movie S1 Syndapin II is localized to the neck of caveolae. Tilt series of caveolae labeled with anti-syndapin II antibodies identified syndapin II at the neck of caveolae. A sequence of micrographs in steps of 4° was taken with a tiltable cryo holder. The image shift was calculated by cross-correlation using the software EMMENU V3.0 (AVI 41,130 kb)

Fig. S2 PTRF/cavin 1 localizes at caveolae of NIH 3T3 cells. (a-c) Immunogold labeled plasma membrane of NIH 3T3 cells after freeze-fracture. PTRF/cavin 1 (indicated by 10 nm gold particles) localized at flat membrane sheets (a), but also at invaginated membranes with increasing curvature (b, c). Scale bar, 100 nm

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Koch, D., Westermann, M., Kessels, M.M. et al. Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae. Histochem Cell Biol 138, 215–230 (2012). https://doi.org/10.1007/s00418-012-0945-0

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Keywords

  • BAR domain proteins
  • Membrane curvature
  • Membrane topology visualization
  • Electron microscopy
  • Caveolar invagination
  • PACSIN