Directed protrusion growth: communication and critical length-scales

Aldo Jesorkaa^a, Gavin D. M. Jeffriesa ^a, Shijun Xua^a, Haijiang Zhang^a, Anna A. Kim^a^,^b

^aChalmers University of Technology, Sweden, Fysikgränd, 3, Gothenburg, Sweden

^bDepartment of Physiology and Pharmacology, Karolinska Institutet, Stockholm

Proceedings of New Advances in Probing Cell-ECM Interactions (CellMatrix)

Berlin, Germany, 2016 October 20th - 21st

Organizers: Ovijit Chaudhuri, Allen Liu and Sapun Parekh

Poster, Anna A. Kim, 063
Publication date: 25th July 2016

Cellular protrusions were directed, emanating from adherent HEK 293 cells, across cytophobic microgaps. We investigated the ability of the protrusions to form intercellular connections, and their capability to communicate by means of molecular transport. Moreover, we evaluated the capability of cellular protrusions to bridge the microgaps, and established a length-scale, beyond which cells cannot probe free-space. We employed a photolithography-based surface fabrication strategy, to produce micro-patterned substrates composed of glass and the amorphous polymer Teflon® AF. This polymer was chosen for its cytophobic properties and very low autofluorescence. The micro-patterns were designed to include glass lanes, for directing protrusion growth, intersected by Teflon® AF lines to create microgaps of various sizes, between 2 and 16 µm. The surface topography of the micro-patterned substrates ensured that the cellular extensions formed over the cytophobic gaps would exist in free space. HEK 293 cells were incubated for 24 hours on the substrates, and the cellular protrusions were investigated for their ability to communicate over the gaps. We found that the frequency of bridging the Teflon® AF was strongly dependent on the gap size, with gaps greater than 4 µm appearing increasingly difficult to cross. Cellular extensions crossing the microgaps either appeared as nano-sized connections, in approximately 30% of the observations, or as micro-sized connections. Molecular transport within the established cell-to-cell connections across the microgaps was probed by activation of TRPM8 ion channels followed by an external supply of Ca2+, localized 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 cell-to-cell connections in both nano- and micro-sized cellular extensions.

Keywords: cellular protrusions, cell-to-cell communication, Teflon® AF, filopodia

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