Study of the regulation of directional sensing during carcinoma cell chemotaxis

Abstract

Directional sensing, which is the initial step in chemotaxis, is characterized by the conversion of an extracellular stimulus gradient into an intracellular asymmetric response. Several models were proposed to explain sensing in chemotactic cells. In Dictyostelium, PIP3 accumulation at the leading edge is an important indicator of the sensing mechanism; this accumulation is accomplished through reciprocal regulation by PI3 kinase and PTEN activities. PI3K null Dictyostelium cells, nevertheless, are still chemotactic. In neutrophils the role of PTEN in sensing and chemotaxis is still debatable. This argues that the model with PIP3 as a key element in chemotaxis is not all-inclusive, especially in several cancer cell types where PTEN loss of function is associated with invasion. In carcinoma cells, the EGF-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium, occurs as two transients. Our results reveal that PLC is required for triggering the early barbed end transient, and PI3K selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either siRNA silencing or function blocking antibodies, selectively inhibits the early transient (Mouneimne et al., 2004). Overexpression of LIMK, which leads to the phosphorylation and inactivation of cofilin, inhibits the early barbed end transient and suppresses directional sensing in carcinoma cells. Overexpression of a kinase dead kinase domain of LIMK leads to an increase in barbed ends during the early transient and inhibits sensing as well. In conclusion, the early PLC and cofilin dependent barbed end transient is required for the initiation of stimulated protrusion and is involved in setting the direction of cell movement in response to EGF. The sharpening of the chemoattractant signal, moreover, is suggested to be achieved though cofilin activation by PLC, and inactivation by LIMK phosphorylation, and by binding G-actin, resulting in a transient and localized cofilin activity. The restrained spatio-temporal regulation of cofilin activity is required for directional sensing in carcinoma cells.