doi: 10.1038/s41467-018-08178-7.
Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells
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- PMID:30664666
- PMCID: PMC6341166
- DOI: 10.1038/s41467-018-08178-7
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Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells
Anna Sartori-Rupp et al. Nat Commun..
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Abstract
The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.
Conflict of interest statement
The authors declare no competing interests.
Figures
Correlated light and cryo-electron microscopy strategies reveal individual TNTs.a A schematic diagram of the experimental workflow and approaches used to observe TNT-connected CAD cells by cryo-TEM.b Cryo-FM image of two CAD cells connected by a TNT stained with WGA (green).c Cells inb observed in cryo-TEM at low magnification. Yellow dashed squares inb,c are shown at intermediate magnification ind. Yellow dashed rectangles ind are shown at high magnification ine andf, respectively. The plasma membrane of individual TNTs (iTNTs) were drawn with dotted colored lines ine andf to show membrane boundaries. Enlargements of white dashed rectangles ine andf are shown ing andh, respectively.g,h Various vesicular compartments were observed within and between iTNTs: yellow arrowheads show single vesicles; pink arrowheads show vesicles surrounded by an outer membrane; turquoise arrowheads show vesicles enclosed by a double membrane. Red stars ine,f, andh show tips of iTNTs extending/retracting from neighboring cells. Green arrows indicate vesicles inside extending/retracting iTNT tips observed inh. Scale bars:b,c, 10 μm;d, 1 μm;e–h, 200 nm
Imaging iTNTs in 3D using correlated light and cryo-electron tomography.a iTNT diameter distribution in nm.b Distribution of the distance between parallel and non-parallel iTNTs in nm. CAD cells connected by a TNT stained with WGA (green) imaged by phase contrast (c), epifluorescence (d), and low-magnification TEM (e). Dashed squares over the TNT inc–e, denotedf–h, correspond to non-consecutive high-magnification cryo-ET slices (f–h, left, middle, and Supplementary Movies 1–3).f–h (right) Rendering of tomograms (Supplementary Movies 1–3, respectively). Turquoise arrows ing andh, left, middle show filaments connecting iTNTs. Vesicles within iTNTs (yellow arrowheads,g andh, left, middle) use thin filaments to connect to the plasma membrane on one side (orange arrows,g left, middle) and actin on the other (pink arrowheads,g left, middle). Additional example of cells connected by a TNT is shown ini (epifluorescence) andj (low-magnification TEM). Region denoted by a white dashed rectangle ini–j is shown at high-magnification cryo-TEM ink left.k right, rendering ofk, left (Supplementary Movie 5). Thin filaments connect the bundle of iTNTs by coiling around them (green arrows,g andk (rendering)). Source data fora,b are provided as a Source Data file. Scale bars:c–e,i–j, 5 μm;f–h,k, 100 nm
Ultrastructural analysis of N-cadherin and Myo10 in iTNTs.a Low-magnification electron micrograph displaying N-Cadherin immunogold-labeled CAD cells connected by iTNTs. (b-top) White dashed rectangle ina correspond to two high-magnification non-consecutive cryo-tomogram slices (25 nm in thickness) shown inb-top andb-bottom (Supplementary Movie 4). Green arrowheads inb and spheres inc indicate 10 nm gold particles attached to a polyclonal secondary antibody that binds to an N-Cadherin primary antibody. Red arrows indicate a long thin thread filament surrounding iTNTs anchored to the membrane of iTNTs by N-Cadherin molecules (green arrows).c Segmentation rendering of the tomogram described inb; N-Cadherin (green beads), thin thread surrounding iTNTs (green arrowheads).d Epifluorescence micrographs of CAD cells overexpressing GFP-Myo10 connected by TNTs stained with WGA (red). Yellow arrowheads indicate GFP-Myo10 signal in TNTs.e Low-magnification electron micrograph corresponding to TNTs shown ind.f,g High-magnification cryo-tomography slices corresponding to the yellow dashed squares ine. Yellow arrowheads ind mark GFP-Myo10 vesicles (Supplementary Movies 6 and 7). Orange arrows indicate vesicle-iTNT connections.h Rendering of tomogram shown ing (Supplementary Movie 7). Scale bars:a,e, 2 μm,b,f, 100 nm;d, 5 μm;g, h, 50 nm
Cryo-electron tomography reveal F-actin organization in iTNTs.a Segmentation rendering obtained from a tomogram showing iTNTs (Supplementary Movie 8). The average size and spacing between filaments of an iTNT shown inb were measured by computing the plot profile of actin bundles residing within.c–e Filament-to-filament distance measured by extracting peaks in the frequency domain using the fast Fourier transform (white arrowheads,e) and by computing plot profiles (c).f Model of the actin filaments displaying a parallel bundle in iTNTs obtained by retransforming peaks in the frequency domain shown ine. Insets inb andf show distances between the average measurements in nm obtained by the analysis shown inc andd.g High-magnification cryo-tomogram slice of two iTNTs (Fig. 3b and Supplementary Movie 4).h Cross-section of the cryo-electron tomogram ing displaying the actin arrangement within two iTNTs. Scale bars:a, 100 nm;b,f, 20 nm;g,h, 100 nm
Cryo-electron tomography reveal actin ultrastructure in filopodia. Representative electron micrographs of filopodial protrusions of untreated (a–c,j–m), GFP-VASP (d–f), and GFP (control) (g–i) transfected CAD cells imaged by cryo-TEM at low magnification (a, d, g, j).b,e,h Slices from volumes rendered inc,f, andi correspond to white dashed rectangles shown ina,d, andg, respectively. Red arrowheads inb andh indicate branched actin configuration.k–l slices corresponding to white dashed square inj.m–n Three-dimensional automated rendering ofk–l and cross-section of the filopodium (n). Segmentation of the plasma membrane (blue) and actin (pink).o High-magnification view of filaments corresponding to the white dashed rectangle ink, where filaments display a wavy periodic pattern (blue arrowhead,o). By computing its plot profile (r), the average filament size and spacing between filaments was measured.p Model of the parallel filament bundle obtained from the peak extraction in the fast Fourier transform (q).s Cross-section through the cryo-electron tomogram of the white dashed rectangle inj. Scale bars:a,d,g,j, 1 μm;b,c,e,f,h,i,k,l,m,n, 100 nm,o,p, 50 nm
Structural analysis and live imaging reveal mitochondrial transport via iTNTs.a Confocal micrograph of SH-SY5Y cells stained with WGA (green).a Lower andb upper optical slices show TNTs hovering over the substrate.c Low-magnification electron micrograph corresponding to white dashed rectangle inb.d–e High-magnification micrographs corresponding to yellow and white dashed squares inc. Yellow arrowheads indicate membranous compartments inside iTNTs.f–g Diameter distribution of iTNTs (f) and vesicles found within them (g) in nm.h Time-lapse sequence of two SH-SY5Y cells stained with WGA (green) and MitoTracker (red) reveal mitochondrial puncta (orange, cyan, pink, yellow, and blue arrowheads) traveling across a TNT (white arrowhead) entering the apposing cell, (cell #2).i–k SH-SY5Y cells stained with WGA (green) and mitotracker (red), (blue arrowheads indicate mitochondria inside the TNT).l Micrograph corresponding to blue dashed rectangles ini–k reveal mitochondria (blue arrowhead) observed by fluorescence (i–k).m Micrograph displays two iTNTs, one containing mitochondria.n Tomogram slice corresponding tom (blue dashed square) reveal mitochondrial cristae, actin, and membranous compartments found inside iTNTs.o Rendering ofn. Source data forf–g are provided as a Source Data file. Scale bars:a–b, 5 μm;c, 1 μm;d,e,l, n, 200 nm,h–i, 10 μm;m, 500 nm
FIB-SEM of TNTs in CAD and SH-SY5Y cells open-open contact sites.a,d Confocal micrographs of TNT-connected CAD (a) and SH-SY5Y (d) cells stained with WGA (green). Yellow dashed boxes ina andd (left) show the regions of interest chosen for FIB-SEM volume acquisition. A magnified view of these regions is shown ina andd (right) overlaid with their FIB-SEM segmented rendering counterpart. Segmented cell bodies and single open-ended connections are shown in cyan. Open-ended iTNTs are shown in blue, yellow, and magenta. Single close-ended protrusions are shown in red. Orange and pink arrowheads indicate contact sites with cells.b,e Renderings of FIB-SEM volumes produced by the Amira software are shown for CAD (b) and SH-SY5Y (e) cells.c,f Single frames obtained from the raw FIB-SEM data of CAD cells (c), obtained at a pixel size of 2.5 nm and displayed in an x-y orientation, and SH-SY5Y cells (f) obtained at a pixel size of 10 nm and displayed in anx-z orientation. Panels show TNT-to-cell contact sites at both ends of the connection: orange arrowhead (left); pink arrowhead (right). Scale bars:a,d, 10 μm;c,f, 500 nm
Schematic diagram depicting how cells connect via TNTs. TNT-connecting cells can either be single thick connections or a bundle of thin individual TNTs (iTNTs). iTNTs contain vesicles and mitochondria. Membranous compartments within iTNTs appear to be connected by thin filaments to actin on one side and to the inner side of the plasma membrane on the other. Thin membrane threads coil between and around several iTNTs. Cyan and magenta dashed squares show two types of contact sites: merging of iTNTs prior to fusion and fusion of iTNTs at separate locations, respectively
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