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Probing Cellular Responses to Extracellular Signals through Direct Writing of Proteins

American Society for Cell Biology, 2005

J. Werbin,1 C. Lemmon,2 G. Ledung,3 W. F. Heinz,1 L. H. Romer,4 J. H. Hoh1
1 Physiology, Johns Hopkins University School of Medicine, Baltimore, MD
2 Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD
3 Mlardalen University, Eskilstuna, Sweden
4 Anesthesiology & Crit Care Med, Johns Hopkins University School of Medicine, Baltimore, MD

Interactions between cells and the extracellular environment play a central role in a number of biological phenomena such as tissue growth, cell differentiation and migration. Patterning biomolecules at high resolution on solid supports is a powerful means to investigate how cells interpret and respond to spatially defined extracellular cues. We used a new technology based on microfabricated surface patterning tools, which delivers a protein solution (10-15 -10 -18 liters) to the surface via a microchannel in small cantilever (NanoArrayer, Bioforce Nanosciences). This direct writing process transfers proteins to the substrate by capillary action when the tool touches the surface for times ~100 ms. Proteins can be bound to the surface by adsorption, or via covalent chemistry. The resulting feature sizes range from submicrometer to tens of micrometers, and pattern dimensions are typically 0.5 x 0.5 mm. A series of patterning parameters and conditions have been optimized, and we have constructed glass substrates with a number of different proteins and peptides in complex patterns with multiple components. For example, patterns that are composed of fibronectin and vitronectin where the distance between the two molecules is subcellular, such that a single cell can contact both fibronectin and vitronectin at several well-defined points, have been produced. These patterns have been characterized by immunofluorescence, and are stable under cell culture conditions. In experiments with Mouse Embryo Fibroblasts plated on micropatterned fibronectin in serum-free medium, immunofluorescence labeling revealed that vinculin was localized to the patterned fibronectin foci in semilunar adhesions with concavities toward the cell center. These findings demonstrate spatial control over focal adhesion formation, and provide a foundation to investigate mechanisms by which different molecules contribute to the contacts these cells make with the extracellular environment.

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