Probing ciliogenesis using micropatterned substrates.

Abstract

The primary cilium is a biomechanical sensor plugged in at the cell surface. It is implicated in the processing of extracellular signals and its absence or misfunctioning lead to a broad variety of serious defects known as ciliopathies. Unfortunately, the precise mechanisms underlying primary cilium assembly and operation are still poorly understood. Molecular dynamics and intracellular morphogenesis are easier to study in cell culture than in tissues. However, cultured cells are usually nonciliated and the empirical methods that are used to induce ciliogenesis in these cells have variable efficiencies. In addition, these methods require cells to be cultured at high density, which is not convenient for further automated image analysis. Here, we describe a method to induce and modulate ciliogenesis in mammalian cells in culture that is compatible with high-throughput imaging and analysis. Surface micropatterning is used to normalize cell shape and actin network architecture. In these conditions, the deprivation of growth factor induces ciliogenesis in individual single cells. The manipulation of cell-spreading area is used to modulate the proportion of ciliated cells. The manipulation of cell adhesion geometry is used to orient the position of the primary cilium. The spatial disposition of cells on a regular array offers a simple way to perform automated image acquisition. In addition, the regular cell shape is convenient to perform robust and automated image analysis to quantify the presence and location of primary cilia. This method offers a new way to study ciliogenesis in automated and high-throughput assays.