Cellular communication via directed protrusion growth: Critical length-scales and membrane morphology

We investigated the growth of cell protrusions from adherent HEK 293 cells and their capability to bridge cytophobic Teflon®  AF microgaps, establishing a critical length scale, beyond which cells cannot probe free space. For this purpose, we employed a photolithography-based surface fabrication strategy for producing micropatterned substrates composed of glass and the amorphous polymer Teflon®  AF. Cell protrusions growing from HEK 293 cells on these substrates were confined to extend on 2 μm wide glass lanes, intersected by Teflon®  AF microgaps of various lengths between 2 and 16 μm. After 24 hours of incubation, the frequency of cell protrusions crossing the gap was found to be strongly dependent on the gap size. Gaps which are greater than 4 μm were found to be increasingly difficult to cross. Cell extensions crossing the microgaps either appeared as nanosized connections, in approximately 30% of all observed cases, or as microsized connections. Molecular transport in the established cell-to-cell connection across the microgap was investigated by activation of TRPM8 ion channels followed by supply of Ca2+ to one of the connected cells. The diffusion of the Ca2+ ions was visualized by means of a cell-permeant pre-fluorescent dye. We observed both open- and closed-ended intercellular connections in both nano- and microsized cell protrusions.