The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes

Abstract

Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca²⁺-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca²⁺ in regulation of TNT formation. Finally, we observed transport of various GM₁/GM₃⁺ vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.

Keywords: Fluorescence imaging; Intercellular matter transport; Membrane nanotubes; Membrane protrusion; Superresolution microscopy; Trogocytosis.

Fig. 1

Mature, but not immature, mouse and human B cells spontaneously form nanotubular connections under physiological conditions. a Representative live cell confocal images of immature 38C13 (left; monochrome DiO fluorescence) or mature A20 (middle and right) murine B cells stained with 1:1 mixtures of DiO (green) and DiI (red) dyes and incubated for 1 h. Tunneling nanotubes (TNTs) can grow unidirectionally (a , middle) or bidirectionally (a, right: dual color; see white arrow). b A20 B cells are often connected with multiple (thin and thick) TNTs (left), can form TNTs following cell division (middle; see midbody, white arrow), or can form even three way junction (right; see white arrow). c High-resolution AFM (left) and SEM (middle) images of A20 B cells show TNT morphology. Extremely long (≥100 μm) and thin TNTs can be detected in the culture of freshly isolated human tonsil B cells (right; see white arrows). d B cell TNTs form optimally at 37 °C (left) and on fibronectin or laminin coats (right). e B cell nanotubes are largely diverse in both their length (left) and width (right) with means (±SD) of 22 ± 10 μm and 650 ± 250 nm, respectively. Mean and SD values for TNT forming cell % were derived from at least five independent experiments, from ca. 500 cells/sample

Fig. 2

a Flow cytometric histograms show lack of α₅ (black isotype control antibody; grey anti-α₅ antibody) (left) or β₁ (black isotype control antibody; grey anti-β₁ antibody) (right) integrin chains on 38C13 immature murine B cells. b Both integrin chains were detected on mature A20 murine B cells, α₅ (black isotype control antibody; grey anti-α₅ antibody) (left) and β₁ (black isotype control antibody; grey anti-β₁ antibody) (right). c In A20 B cells NT growth frequency (left) and cell adhesion/spreading (right) were found to be dependent on the interaction between fibronectin and both of its integrin receptor subunits (α₅β₁), as evidenced by the lack of significant changes in these properties upon individual blocking of each integrin subunit but significant reduction upon simultaneous blockade of both subunits (left and right) (**p ≤ 0.01)

Fig. 3

B cell nanotubes may mediate bidirectional transport of membrane vesicles inside the tubes and also transport of molecules essential for T cell activation in the membrane of TNTs. a Representative superresolution (SIM; lateral resolution: 80–90 nm) image of TNTs connecting adjacent A20 B cells. The fluorescence on these images originates from extracellular labeling of cell membrane gangliosides with Alexa488-cholera toxin B (CTX-B) before a 1 h incubation for TNT formation at live cell imaging conditions (37 °C, 5 % CO₂). The image clearly shows existence of large and small vesicles in the cell bodies and the latter ones along the TNTs. (The average diameter of vesicles is 1.6 µm) (The intercellular transport of vesicles across TNTs and its bidirectional feature is also demonstrated by Supplementary Movies S1, S2.) b Representative confocal image of TNT-connected A20 B cells shows abundance of MHC-II/peptide complexes (green) along the TNTs (left), in a highly colocalized fashion with GM1/3 gangliosides (red), markers of lipid rafts (zoom: right). c B7-family costimulatory proteins (CD86) are also enriched along the TNTs (left), in a clustered fashion (zoom: right). CLSM images also demonstrate that both MHC-II/peptide (green) (d, e, white arrow) and B7-2/CD86 molecules (green) (f, g, white arrow) were able to reach the connected cell through the TNT membrane. Analysis of fluorescence intensity of the ‘acceptor’ cells shows that both molecules appear in their cell membrane close to the TNT, or even farther from the TNT connection (see white arrow). h, i In addition, lysosomes (Lysotracker, violet; h) and mitochondria (MitoTracker, violet; i) were also detected in B cell TNTs, suggesting the possibility of intercellular transport. (Movement of mitochondria within nanotubes is shown on Supplementary Movie S3.) Each representative image was derived from three independent experiments (≥100 TNT) (**p ≤ 0.01)

Fig. 4

TNT-growth of B cells is modulated by cellular activation and the cytoplasmic free Ca²⁺ level. a, b Representative DIC images show increased TNT number (white arrows) in LPS-treated (b) vs. untreated (a) A20 B cells. c Monoclonal (BCR-mediated) activation of B cells by anti-Ig antibody (10 μg/ml) slightly decreased, while polyclonal activation with LPS lipopolysaccharide (10 μg/ml) significantly enhanced TNT-formation. d Time lapse images (from left to right) show that Ca²⁺ influx induced by 1 µg/ml ionomycin Ca²⁺-ionophore resulted in rapid withdrawal (typically in 10–20 s) of TNT connecting two cells (position of the free end of the NT is shown by white arrow). (See also Supplementary Movie S4.) e–g Ionomycin or thapsigargin (an inhibitor of SER Ca²⁺-ATPase) both inhibited TNT-growth in a concentration-dependent fashion, if applied after TNT-growth reached saturation in the culture (e, f). Notably, ionomycin could also prevent TNT-growth if applied for 5 min, (and then washed out from the samples) before culturing for 1 h for TNT-growth (g). h EGTA (4 mM) applied extracellularly almost completely reversed the ionomycin-induced effect. The mean and SD values depicted on the panels were determined from more than three independent experiments (≥500 cells/sample)

Fig. 5

a Representative TIRF image of m-Cherry-actin transfected and b SIM image of Alexa488-phalloidin stained A20 B cells demonstrating (by white arrows) that the nanotubes always contain actin filaments. c Majority of the TNTs also contain microtubules as assessed by SIM images of Alexa488-anti-tubulin stained cells (white arrows). d Cytochalasin D (left) and latrunculin B (middle) as inhibitors of F-, and G-actin polymerization, respectively, efficiently blocked nanotube growth such as jasplakinolide (right) did, in a concentration dependent manner. e In contrast, nocodazole, inhibitor of microtubule polymerization (left) or paclitaxel (taxol) a stabilizer of microtubule structure (middle) left TNT growth unchanged. Latrunculin B (5 μM) could initiate a ca. fourfold reduction in nanotube number even in the presence of paclitaxel (10 μM) stabilizing microtubules (right). The mean TNT-frequencies and the SD values displayed on the panels were calculated from three independent experiments (≥300 cells in each) (**p ≤ 0.01)

Fig. 6

Non-muscle myosin 2 also controls B cell nanotube formation. a Flow cytometric histograms (white isotype control antibody; grey NM2A antibody; both detected with an Alexa488-secondary antibody) show high level of NM2A in A20 B cells. b Representative SIM image of anti-NM2A stained A20 B cells: the motor protein localized in both the cell bodies and in the nanotubes. c Suppressing myosin 2 activity through Rho-kinase inhibition with Y-27632, enhanced (ca. threefold) the TNT formation in a concentration-dependent manner. d, e Para-nitro-blebbistatin, an efficient inhibitor of myosin 2 also caused a more than twofold increase in TNT number (d) or branching degree (e) in the concentration range leaving cell viability unchanged. Ionomycin (1 µg/ml) could fully impair the effect of 25 µM para-nitro-blebbistatin (d, e). (The mean ± SD data were derived from at least three independent measurements with ca. 300 cells/sample)