Loss of clathrin heavy chain enhances actin-dependent stiffness of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cell stiffness, and specific responses to features of the underlying substratum. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking the clathrin heavy chain (Cltc), display higher Young9s modulus, indicative of greater cellular stiffness, in comparison to WT mESCs. We have previously shown that mESCs lacking Cltc display a loss of pluripotency, and an initiation of differentiation. The increased stiffness observed in these cells was accompanied by the presence of actin stress fibres and accumulation of the inactive, phosphorylated, actin binding protein, Cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young9s modulus, to values similar to those obtained with WT mESCs. However, the expression profile of pluripotency factors was not rescued. This indicates that a restoration of mechanical properties, through modulation of the actin cytoskeleton, may not always be accompanied by a change in the expression of critical transcription factors that regulate the state of a stem cell, and that this may be dependent on the presence of active endocytosis in a cell.