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Cell adhesion to agrin presented as a nanopatterned substrate is consistent with an interaction with the extracellular matrix and not transmembrane adhesion molecules.

Wolfram T, Spatz JP, Burgess RW - BMC Cell Biol. (2008)

Bottom Line: This has been demonstrated using nanopatterned RGD peptides, a canonical extracellular matrix ligand for integrin interactions.In contrast, adhesion to N-Cadherin decreased gradually over the entire range of distances tested (uniform, 30, 60, 90, and 160 nm).These results suggest nanopatterned substrates may provide a physiological presentation of adhesive substrates, and are consistent with cells adhering to agrin through a mechanism that more closely resembles an interaction with the extracellular matrix than a transmembrane adhesion molecule.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of New Materials and Biosystems, Max-Planck-Institute for Metals Research, University of Heidelberg, Stuttgart, Germany. wolfram@mf.mpg.de

ABSTRACT

Background: Molecular spacing is important for cell adhesion in a number of ways, ranging from the ordered arrangement of matrix polymers extracellularly, to steric hindrance of adhesion/signaling complexes intracellularly. This has been demonstrated using nanopatterned RGD peptides, a canonical extracellular matrix ligand for integrin interactions. Cell adhesion was greatly reduced when the RGD-coated nanoparticles were separated by more than 60 nm, indicating a sharp spacing-dependent threshold for this form of cell adhesion.

Results: Here we show a similar dependence of cell adhesion on the spacing of agrin, a protein that exists as both a secreted, matrix-bound form and a type-2 transmembrane form in vivo. Agrin was presented as a substrate for cell adhesion assays by anchoring recombinant protein to gold nanoparticles that were arrayed at tunable distances onto glass coverslips. Cells adhered well to nanopatterned agrin, and when presented as uniformly coated substrates, adhesion to agrin was comparable to other well-studied adhesion molecules, including N-Cadherin. Adhesion of both mouse primary cortical neurons and rat B35 neuroblastoma cells showed a spacing-dependent threshold, with a sharp drop in adhesion when the space between agrin-coated nanoparticles increased from 60 to 90 nm. In contrast, adhesion to N-Cadherin decreased gradually over the entire range of distances tested (uniform, 30, 60, 90, and 160 nm). The spacing of the agrin nanopattern also influenced cell motility, and peptide competition suggested adhesion was partially integrin dependent. Finally, differences in cell adhesion to C-terminal agrin fragments of different lengths were detected using nanopatterned substrates, and these differences were not evident using uniformly coated substrates.

Conclusion: These results suggest nanopatterned substrates may provide a physiological presentation of adhesive substrates, and are consistent with cells adhering to agrin through a mechanism that more closely resembles an interaction with the extracellular matrix than a transmembrane adhesion molecule.

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Cell adhesion on C100 Agrin nanopatterned substrates. Mouse primary cortical neurons (A) showed significantly higher adhesion to 30 nm and 60 nm spaced substrates than to 90 nm and 160 nm spaced substrates. Adhesion to more closely spaced substrates is comparable to homogenously coated agrin (first bar). Rat B35 neuroblastoma cells (B) showed a similar behaviour. Cell Adhesion on N-Cadherin nanopatterned substrates. Primary mouse cortical neurons (C) and Rat B35 neuroblastoma cells (D) do not show a significant decrease in cell adhesion with increasing spacing of the nanopatterned substrates, and adhesion is always below the level observed for uniformly coated N-Cadherin (first bar). This suggests a different mechanism for N-Cadherin mediated cell adhesion than for Agrin mediated cell adhesion.
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Figure 4: Cell adhesion on C100 Agrin nanopatterned substrates. Mouse primary cortical neurons (A) showed significantly higher adhesion to 30 nm and 60 nm spaced substrates than to 90 nm and 160 nm spaced substrates. Adhesion to more closely spaced substrates is comparable to homogenously coated agrin (first bar). Rat B35 neuroblastoma cells (B) showed a similar behaviour. Cell Adhesion on N-Cadherin nanopatterned substrates. Primary mouse cortical neurons (C) and Rat B35 neuroblastoma cells (D) do not show a significant decrease in cell adhesion with increasing spacing of the nanopatterned substrates, and adhesion is always below the level observed for uniformly coated N-Cadherin (first bar). This suggests a different mechanism for N-Cadherin mediated cell adhesion than for Agrin mediated cell adhesion.

Mentions: Agrin and N-Cadherin were next tested for their dependence on spacing of the nanopattern. This provided a comparison of agrin to a homophilic adhesion molecule where both provided good adhesion for a neuroblastoma cell line. In each case, both primary mouse cortical neurons and rat B35 neuroblastoma cells were tested (Figure 4). As above, results were obtained for three independent assays and adhesion was standardized to uniformly coated agrin or N-cadherin. For agrin, adhesion of both primary cortical neurons and B35 cells was comparable to uniformly coated protein at spacings of 30 and 60 nm (Figure 4A, B). Primary cortical neurons did show a small but significant reduction in adhesion to 60 nm spaced agrin (p = 0.004), but the difference was not significant compared to 30 nm spaced agrin (p = 0.8). However, adhesion dropped significantly with 90 and 160 nm spacing (p values = 0.0002). Adhesion to the 160 nm spaced agrin was comparable to the negative control for both cell types (p = 0.02 and 0.03). Negative controls were created using BSA coated gold nanoparticles in the same PEG passivated environment.


Cell adhesion to agrin presented as a nanopatterned substrate is consistent with an interaction with the extracellular matrix and not transmembrane adhesion molecules.

Wolfram T, Spatz JP, Burgess RW - BMC Cell Biol. (2008)

Cell adhesion on C100 Agrin nanopatterned substrates. Mouse primary cortical neurons (A) showed significantly higher adhesion to 30 nm and 60 nm spaced substrates than to 90 nm and 160 nm spaced substrates. Adhesion to more closely spaced substrates is comparable to homogenously coated agrin (first bar). Rat B35 neuroblastoma cells (B) showed a similar behaviour. Cell Adhesion on N-Cadherin nanopatterned substrates. Primary mouse cortical neurons (C) and Rat B35 neuroblastoma cells (D) do not show a significant decrease in cell adhesion with increasing spacing of the nanopatterned substrates, and adhesion is always below the level observed for uniformly coated N-Cadherin (first bar). This suggests a different mechanism for N-Cadherin mediated cell adhesion than for Agrin mediated cell adhesion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2612657&req=5

Figure 4: Cell adhesion on C100 Agrin nanopatterned substrates. Mouse primary cortical neurons (A) showed significantly higher adhesion to 30 nm and 60 nm spaced substrates than to 90 nm and 160 nm spaced substrates. Adhesion to more closely spaced substrates is comparable to homogenously coated agrin (first bar). Rat B35 neuroblastoma cells (B) showed a similar behaviour. Cell Adhesion on N-Cadherin nanopatterned substrates. Primary mouse cortical neurons (C) and Rat B35 neuroblastoma cells (D) do not show a significant decrease in cell adhesion with increasing spacing of the nanopatterned substrates, and adhesion is always below the level observed for uniformly coated N-Cadherin (first bar). This suggests a different mechanism for N-Cadherin mediated cell adhesion than for Agrin mediated cell adhesion.
Mentions: Agrin and N-Cadherin were next tested for their dependence on spacing of the nanopattern. This provided a comparison of agrin to a homophilic adhesion molecule where both provided good adhesion for a neuroblastoma cell line. In each case, both primary mouse cortical neurons and rat B35 neuroblastoma cells were tested (Figure 4). As above, results were obtained for three independent assays and adhesion was standardized to uniformly coated agrin or N-cadherin. For agrin, adhesion of both primary cortical neurons and B35 cells was comparable to uniformly coated protein at spacings of 30 and 60 nm (Figure 4A, B). Primary cortical neurons did show a small but significant reduction in adhesion to 60 nm spaced agrin (p = 0.004), but the difference was not significant compared to 30 nm spaced agrin (p = 0.8). However, adhesion dropped significantly with 90 and 160 nm spacing (p values = 0.0002). Adhesion to the 160 nm spaced agrin was comparable to the negative control for both cell types (p = 0.02 and 0.03). Negative controls were created using BSA coated gold nanoparticles in the same PEG passivated environment.

Bottom Line: This has been demonstrated using nanopatterned RGD peptides, a canonical extracellular matrix ligand for integrin interactions.In contrast, adhesion to N-Cadherin decreased gradually over the entire range of distances tested (uniform, 30, 60, 90, and 160 nm).These results suggest nanopatterned substrates may provide a physiological presentation of adhesive substrates, and are consistent with cells adhering to agrin through a mechanism that more closely resembles an interaction with the extracellular matrix than a transmembrane adhesion molecule.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of New Materials and Biosystems, Max-Planck-Institute for Metals Research, University of Heidelberg, Stuttgart, Germany. wolfram@mf.mpg.de

ABSTRACT

Background: Molecular spacing is important for cell adhesion in a number of ways, ranging from the ordered arrangement of matrix polymers extracellularly, to steric hindrance of adhesion/signaling complexes intracellularly. This has been demonstrated using nanopatterned RGD peptides, a canonical extracellular matrix ligand for integrin interactions. Cell adhesion was greatly reduced when the RGD-coated nanoparticles were separated by more than 60 nm, indicating a sharp spacing-dependent threshold for this form of cell adhesion.

Results: Here we show a similar dependence of cell adhesion on the spacing of agrin, a protein that exists as both a secreted, matrix-bound form and a type-2 transmembrane form in vivo. Agrin was presented as a substrate for cell adhesion assays by anchoring recombinant protein to gold nanoparticles that were arrayed at tunable distances onto glass coverslips. Cells adhered well to nanopatterned agrin, and when presented as uniformly coated substrates, adhesion to agrin was comparable to other well-studied adhesion molecules, including N-Cadherin. Adhesion of both mouse primary cortical neurons and rat B35 neuroblastoma cells showed a spacing-dependent threshold, with a sharp drop in adhesion when the space between agrin-coated nanoparticles increased from 60 to 90 nm. In contrast, adhesion to N-Cadherin decreased gradually over the entire range of distances tested (uniform, 30, 60, 90, and 160 nm). The spacing of the agrin nanopattern also influenced cell motility, and peptide competition suggested adhesion was partially integrin dependent. Finally, differences in cell adhesion to C-terminal agrin fragments of different lengths were detected using nanopatterned substrates, and these differences were not evident using uniformly coated substrates.

Conclusion: These results suggest nanopatterned substrates may provide a physiological presentation of adhesive substrates, and are consistent with cells adhering to agrin through a mechanism that more closely resembles an interaction with the extracellular matrix than a transmembrane adhesion molecule.

Show MeSH
Related in: MedlinePlus