title: Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: Magalie
authors.initials: M
authors.lastname: Bénard
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Schapman
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: Alexis
authors.initials: A
authors.lastname: Lebon
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: Baptiste
authors.initials: B
authors.lastname: Monterroso
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: Marine
authors.initials: M
authors.lastname: Bellenger
authors.affiliation: UMR-I 02 INERIS-URCA-ULH SEBIO/Unité Stress Environnementaux et BIOsurveillance des milieux aquatiques, Université du Havre, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Le Foll
authors.affiliation: UMR-I 02 INERIS-URCA-ULH SEBIO/Unité Stress Environnementaux et BIOsurveillance des milieux aquatiques, Université du Havre, France.|Department of Genetic Medicine, Weill Cornell Medical College, New York, NY, USA.|Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha, Qatar.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pasquier
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: Hubert
authors.initials: H
authors.lastname: Vaudry
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: David
authors.initials: D
authors.lastname: Vaudry
authors.affiliation: Cell imaging platform of Normandy (PRIMACEN), Infrastructure en Biologie, Santé et Agronomie (IBiSA), Institut National de la Santé et de la Recherche Médicale (Inserm), Mont-Saint-Aignan, France.|Normandie University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Galas
mesh_terms: D000818:Animals; D002450:Cell Communication; D002462:Cell Membrane / Q000737:chemistry* / Q000502:physiology; D022081:Cell Surface Extensions / Q000737:chemistry* / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods*; D016716:PC12 Cells; D051381:Rats; D059008:Time-Lapse Imaging / Q000379:methods*
keywords: Deconvolution; PC12 cells; Time-gated Continuous Wave STED nanoscopy; Time-gated confocal microscopy; Tunneling nanotubes