Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches

Magalie Bénard, Damien Schapman, Alexis Lebon, Baptiste Monterroso, Marine Bellenger, Frank Le Foll, Jennifer Pasquier, Hubert Vaudry, David Vaudry, Ludovic Galas

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Background information: Tunneling nanotubes (TnTs) are thin plasma membrane bridges mediating transfers of materials and signals between cells. Heterogeneity of heterocellular and homocellular TnTs is largely described but ultrafine imaging of these light-sensitive floating nanometric structures represents a real challenge in microscopy. We propose here imaging strategies designed to dissect structural and dynamic aspects of TnT formation and function in fixed or living PC12 cells. Results: Through time-gated Continuous Wave STimulated Emission Depletion (gCW STED) nanoscopy associated with deconvolution, we provided nanoscale details of membrane and cytoskeleton organisations in two subtypes of TnTs, namely type 1 TnT (TnT1) and type 2 TnT (TnT2). In fixed PC12 cells, TnT1 (length, several tens of micrometres; diameter, 100-650 nm) exhibited a large trumpet-shaped origin, a clear cytosolic tunnel and different bud-shaped connections from closed-ended to open-ended tips. TnT1 contained both actin and tubulin. TnT2 (length, max 20 μm, diameter, 70-200 nm) only contained actin without clear cytosolic tunnel. In living PC12 cells, we observed through gCW STED additional details, unrevealed so far, including a filament spindle emerging from an organising centre at the origin of TnT1 and branched or bulbous attachments of TnT2. However, the power of depletion laser in STED nanoscopy was deleterious for TnTs and prolonged time-lapse experiments were almost prohibited. By circumventing the hazard of photoxicity, we were able to monitor dynamics of bud-shaped tips and intercellular transfer of wheat germ agglutinin labelled cellular elements through time-gated confocal microscopy. Conclusions: Our work identified new structural characteristics of two subtypes of TnTs in PC12 cells as well as dynamics of formation and transfer through complementary imaging methods combined with image processing. Therefore, we could achieve maximum lateral resolution and sample preservation during acquisitions to reveal new insights into TnT studies. Significance: Due to large disparity of TnT-like structures in neuronal, immune, cancer or epithelial cells, high- and superresolution approaches can be utilised for full characterisation of these yet poorly understood routes of cell-to-cell communication.

Original languageEnglish
Pages (from-to)419-425
Number of pages7
JournalBiology of the Cell
Volume107
Issue number11
DOIs
Publication statusPublished - 1 Nov 2015

Fingerprint

Nanotubes
PC12 Cells
Actins
Wheat Germ Agglutinins
Tubulin
Cytoskeleton
Confocal Microscopy
Cell Communication
Microscopy
Lasers
Epithelial Cells
Cell Membrane
Light
Membranes
Neoplasms

Keywords

  • Deconvolution
  • PC12 cells
  • Time-gated confocal microscopy
  • Time-gated Continuous Wave STED nanoscopy
  • Tunneling nanotubes

ASJC Scopus subject areas

  • Cell Biology
  • Medicine(all)

Cite this

Bénard, M., Schapman, D., Lebon, A., Monterroso, B., Bellenger, M., Le Foll, F., ... Galas, L. (2015). Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches. Biology of the Cell, 107(11), 419-425. https://doi.org/10.1111/boc.201500004

Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches. / Bénard, Magalie; Schapman, Damien; Lebon, Alexis; Monterroso, Baptiste; Bellenger, Marine; Le Foll, Frank; Pasquier, Jennifer; Vaudry, Hubert; Vaudry, David; Galas, Ludovic.

In: Biology of the Cell, Vol. 107, No. 11, 01.11.2015, p. 419-425.

Research output: Contribution to journalArticle

Bénard, M, Schapman, D, Lebon, A, Monterroso, B, Bellenger, M, Le Foll, F, Pasquier, J, Vaudry, H, Vaudry, D & Galas, L 2015, 'Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches', Biology of the Cell, vol. 107, no. 11, pp. 419-425. https://doi.org/10.1111/boc.201500004
Bénard M, Schapman D, Lebon A, Monterroso B, Bellenger M, Le Foll F et al. Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches. Biology of the Cell. 2015 Nov 1;107(11):419-425. https://doi.org/10.1111/boc.201500004
Bénard, Magalie ; Schapman, Damien ; Lebon, Alexis ; Monterroso, Baptiste ; Bellenger, Marine ; Le Foll, Frank ; Pasquier, Jennifer ; Vaudry, Hubert ; Vaudry, David ; Galas, Ludovic. / Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches. In: Biology of the Cell. 2015 ; Vol. 107, No. 11. pp. 419-425.
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