Figure 1.
(A) High resolution 3D live-cell fluorescence image of a NT (white arrow) connecting two primary mesothelial cells one hour after plating on a collagen I coated glass cover slide. To facilitate detection, cell membranes were stained with WGA Alexa Fluor® 488. Scale bar: 20 µm. (B) Depiction of a NT (black arrow) between two cells with scanning electron microscopy one hour after cell plating. Scale bar: 10 µm. (C) F-actin staining by fluorescently labeled phalloidin showing actin being present in NTs between individual HPMCs (white arrow). Scale bar: 20 µm. (D) Scanning electron microscope picture of a substrate-associated filopodia-like extension as potential NT precursor (black arrowhead). The insert shows a fluorescence microscopic image of substrate associated filopodia-like protrusions approaching a neighboring cell (white arrowhead). Scale bar: 2 µm. (E) Quantitative analyses of the NTs/cells ratio from 4 different donors undergoing abdominal surgery. (F) Influence of dialysis solution and TNF-α on NT formation between cells from Donor I. For comparison, the NTs/cells ratio is shown for UPMCs from a patient undergoing peritoneal dialysis (Donor V). (G, H) Live-cell fluorescence microscopy showing an increased number of NTs between cells from Donor I after TNF-α treatment (G arrow) and between UPMCs from Donor V one hour after cell plating (H arrow). Scale bars: 20 µm. All data are means ± SEM. * p<0.05, ** p<0.01 (t-test in E, F).
Figure 2.
(A, B) Influence of TNF-α on actin cytoskeleton reorganization in HPMCs. (A) The fluorescence picture shows a predominant localization of F-actin at the rim of the cells (arrows) in absence of TNF-α. (B) The presence of TNF-α leads to reorganization of the actin cytoskeleton accompanied by the formation of actin stress fibers (arrowheads). Scale bars: 50 µm. (C, D) Spatial separation of HPMCs results in complete inhibition of NT formation. (C) Scanning electron and live-cell fluorescence microscopy pictures of HPMCs one hour after plating on homogeneously collagen I coated glass cover slides. The white arrows point to NTs spanned between the cells. Scale bars (left): 100 µm; (right): 20 µm. (D) HPMCs seeded on IKVAV functionalized (50 µM) microstructured surfaces with a gold disk diameter and spacing of 50 µm. Scanning electron microscopy (left panel) and live-cell imaging analyses (right panel) were performed one hour after plating. Note that cells exclusively attach to the biofunctionalized gold disk structures. The inserts in A, B and D represent higher magnified images. Scale bars (left): 100 µm; (right): 20 µm.
Figure 3.
Impact of MCD mediated cholesterol depletion on NT formation.
(A) Quantitative analyses of fluorescence intensities after cholesterol staining with Filipin III in HPMCs from different donors (left panel). Corresponding fluorescence images of stained cells one hour after cell plating are shown (right panel). (B) HPMCs were cultured in absence or presence of indicated MCD concentrations and Filipin III staining was performed one hour after plating. For quantification, fluorescence intensities based on representative microscopic images (top row) were determined as described. Please note that cholesterol depletion results in a decrease of fluorescence intensities (arrowheads, arrow). (C) Analysis of the NTs/cells ratio dependent on cholesterol depletion. HPMCs were treated with indicated MCD concentrations and NT numbers were assessed one hour after cell plating. The graph shows a significantly higher number of NTs when cells are treated with 2.5 mM MCD (arrow) as compared to the control or treatment with 1.25 or 3 mM MCD (arrowheads). (D) Quantitative analyses of NT lengths after cholesterol depletion. HPMCs were cultured in the presence of indicated MCD concentrations and NT lengths were analyzed one hour after cell seeding. Incubation of cells with 1.25 mM MCD led to comparatively short NT lengths (arrowhead) whereas incubation with 2.5 mM MCD led to extended NT lengths (arrow). All data are means ± SEM. * p<0.05, ** p<0.01 (t-test in A).
Figure 4.
Influence of MCD and simvastatin on NT formation between HPMCs.
(A) HPMCs from Donor VI were cultured in absence or presence of indicated MCD concentrations and Filipin III staining was performed one hour after plating. Cholesterol depletion results in a decrease of fluorescence intensities (arrowheads, arrow). (B) Analysis of the NTs/cells ratio dependent on cholesterol depletion. HPMCs were treated with indicated MCD concentrations and NT numbers were assessed one hour after cell plating. The graph shows a significantly higher number of NTs when cells are treated with 1.25 mM MCD (arrow) as compared to the control or treatment with 0.5 mM and 2.5 mM MCD (arrowheads). (C) Live-cell fluorescence microscopy showing NTs (arrow) between the cells under control conditions one hour after cell plating. Scale bar: 20 µm. (D) Quantitative analyses of fluorescence intensities after cholesterol staining with Filipin III. Filipin staining was performed in the presence of indicated concentrations of simvastatin as described. For quantification, fluorescence intensities based on representative microscopic images (top row) were determined as described. Increasing concentrations of simvastation resulted in a linear decrease of fluorescence intensities. (E) Live-cell fluorescence microscopy after simvastatin treatment revealed strongly elevated NT numbers between cells (arrows) as compared to the control condition. (F) Quantitative analyses of the NTs/cells ratio from Donor VI after treatment with rapamycin or simvastatin. Cells were cultured in absence (control) or presence of indicated rapamycin or simvastatin concentrations and NT numbers were analyzed one hour after cell seeding. Incubation of cells with 1 nM rapamycin led to a significant decrease of NT numbers (white bar) whereas incubation with 50 µM simvastatin resulted in a strong increase (black bar). All data are means ± SEM. ** p<0.01 (t-test in F).