Adhesive Micropatterns to Study Intermediate Filament Function in Nuclear Positioning

Abstract

The nucleus is generally found near the cell center; however its position can vary in response to extracellular or intracellular signals, leading to a polarized intracellular organization. Nuclear movement is mediated by the cytoskeleton and its associated motors. While the role of actin and microtubule cytoskeletons in nuclear positioning has been assessed in various systems, the contribution of intermediate filaments is less established due in part to the lack of tools to study intermediate filament functions. The methods described here use micropatterned substrates to impose reproducible cell shape and nucleus position. Intermediate filament organization can be perturbed using gene downregulation or upregulation; intermediate filaments can also be visualized using fluorescent intermediate filament proteins. This protocol is valuable for characterizing the role of intermediate filaments in a variety of live or fixed adherent cells. © 2015 by John Wiley & Sons, Inc.

Regulation of microtubule-associated motors drives intermediate filament network polarization

“…The micropatterning technique was used to impose reproducible cell shapes and decipher cell morphogenesis and functions ( Théry, 2010 ). We have previously used astrocytes plated on micropatterns to study cell polarization ( Dupin et al, 2015 ). In brief, primary rat astrocytes were plated onto glass-bottomed tissue culture dishes coated with fibronectin after deep UV micropatterning of the surrounding polyethylene glycol.…”