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, 9 (12), 4240-5

Nanoscale Arrangement of Apoptotic Ligands Reveals a Demand for a Minimal Lateral Distance for Efficient Death Receptor Activation

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Nanoscale Arrangement of Apoptotic Ligands Reveals a Demand for a Minimal Lateral Distance for Efficient Death Receptor Activation

Julia Ranzinger et al. Nano Lett.

Abstract

Cellular apoptosis, the prototype of programmed cell death, can be induced by activation of so-called death receptors. Interestingly, soluble and membrane-bound members of death receptor ligands can differentially activate their receptors. Using the death receptor ligand tumor necrosis factor (TNF) presented on a surface in a nanoscaled pattern with spacings between 58 and 290 nm, we investigated its requirements for spatial arrangement and motility to efficiently activate TNF receptor (TNFR)1 and TNFR2 as well as its chimeras TNFR1-Fas and TNFR2-Fas. We show that the mere mechanical fixation of TNF is insufficient to efficiently activate TNFR2 that is responsive to only the membrane bound form of TNF but not its soluble form. Rather, an additional stabilization of TNFR2(-Fas) by cluster formation seems to be mandatory for efficient activation. In contrast, TNFR1(-Fas) is strongly activated by TNF spaced within up to 200 nm distances, whereas larger spacings of 290 nm fails completely. Furthermore, unlike for TNFR2(-Fas) no dose-response relationship to increasing distances of nanostructured ligands could be observed for TNFR1-(Fas), suggesting that compartmentalization of the cell membrane in confinement zones of approximately 200 nm regulates TNFR1 activation.

Figures

Figure 1
Figure 1
Analyses of TNF-functionalized nanostructured surfaces by atomic force microscopy. Nanostructured surfaces of 200 nm spacings were left untreated (A) or were biofunctionalized with 500 ng/mL CysTNF (B). AFM images (top) and analyses of the surface topographies (botttom) generated using standard semicontact mode operation in air and a typical scanning speed of 1 line/s are shown. The black bars in the AFM images correspond to the surface analyzed in a height profile at the bottom. Both on the nonfunctionalized (A) and on the TNF-functionalized (B) surface the bare glass surface between the gold dots is visible. For the processing of the images only automatic linear background correction was applied for the individual scan lines.
Figure 2
Figure 2
Characterization of TNF-functionalized nanostructured surfaces by immunolabeling methods. Nanostructured surfaces with a gold dot distance of 58 nm were left untreated (A) or were TNF-biofunctionalized (B and C) using 500 ng/mL CysTNF. (A and B) Surfaces were analyzed by scanning electron microscopy. (B) Nanostructured surfaces were stained using monoclonal TNF-specific antibodies (clone T1; 2 μg/mL) and mouse IgG-specific antibodies coupled with gold particles of 9.8 nm diameter (dilution 1:20 in NaCl pH 6.0). The insert represents a closer view of the marked area (green arrow = additional gold dot conjugated to the secondary antibody, red arrow = gold dot most likely originating from the gold dot structure of the surface). (C) Immunofluorescence microscopy of a TNF-biofunctionalized nanostructured surface stained by monoclonal TNF-specific antibodies (clone T1; 2 μg/mL) and Alexa 488-coupled mouse anti-IgG antibodies.
Figure 3
Figure 3
Efficient apoptosis induction by TNF receptor 1 requires distinct ligand spacings. Nanostructured surfaces with gold dots spaced as indicated, i.e., (i) homogenously coated, (ii) 58 nm, (iii) 110 nm, (iv) 200 nm, (v) 260 nm, and (vi) 290 nm were TNF-functionalized with CysTNF (500 ng/mL). MF TNFR1-Fas (4 × 104 cells/surface; A–E) or the rhabdomyosarcoma cell line Kym-1 (F) were seeded and live cell imaging analyses were performed. Apoptotic cells were determined by their distinct morphological appearance and are given as percentages immediately after (0 h) and 1, 2, and 3 h after plating the cells (A, B, and F). (C–E) Phase contrast images of biofunctionalized nanostructured surfaces before (left, scanning electron microscopy) and 3 h after seeding the cells (right, phase contrast microscopy). The data represents the mean of three independent experiments; in each experiment three randomly chosen areas with appromximately 100 cells were analyzed. Error bars = standard deviation of the mean.
Figure 4
Figure 4
TNF receptor 2-Fas requires additional cross-linking for efficient TNF signaling. MF TNFR2-Fas (4 × 104) were plated on TNF-functionalized homogenously gold or surfaces spaced at 58–290 nm in the absence (A) or presence of 1 μg/mL mAb 80M2 (B); the number of apoptotic cells was determined by their distinct morphological appearance at different time points using live cell microscopy. (B) The number of apoptotic cells was determined as in panel A 3 h after seeding of the cells. The percentage of dead cells was markedly increased in the presence of mAb 80M2 on TNF spacings of 58 and 110 nm The data represents the mean of three independent experiments; in each experiment three randomly chosen areas with approximately 100 cells were analyzed. Error bars = standard deviation of the mean.

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