HaCaT keratinocytes exhibit a cholesterol and plasma membrane viscosity gradient during directed migration.

Keratinocyte migration plays an important role in cutaneous wound healing by supporting the process of reepithelialisation. During directional migration cells develop a polarised shape with an asymmetric distribution of a variety of signalling molecules in their plasma membrane. Here, we investigated front-to-back differences of the physical properties of the plasma membrane of migrating keratinocyte-like HaCaT cells. Using FRAP and fluorescence lifetime analysis, both under TIR illumination, we demonstrate a reduced viscosity of the plasma membrane in the lamellipodia of migrating HaCaT cells compared with the cell rears. This asymmetry is most likely caused by a reduced cholesterol content of the lamellipodia as demonstrated by filipin staining. siRNA-mediated silencing of the cholesterol transporter ABCA1, which is known to redistribute cholesterol from rafts to non-raft regions, as well as pharmacological inhibition of this transporter with glibenclamide, strongly diminished the viscosity gradient of the plasma membrane. In addition, HaCaT cell migration was inhibited by glibenclamide treatment. These data suggest a preferential role of non-raft cholesterol in the establishment of the asymmetric plasma membrane viscosity. Copyright Â