Directional cell migration, chemotaxis, requires a polarized cell morphology in which the cell extends pseudopodia at the front and contracts the rear to move towards a stimulus. PI(4,5)P2 levels set up a threshold for the activity of signaling molecules at the rear and the leading-edge of a cell. To further demonstrate the importance of plasma membrane (PM) PI(4,5)P2 in maintaining cell morphology during chemotaxis, we used a mutant strain of the eukaryotic model system, Dictyostelium discoideum. This mutant strain lacks the type I PIP5 kinase, which is the main enzyme synthesizing PI(4,5)P2. These cells, designated pikI-, have highly reduced PI(4,5)P2 levels and higher Ras GTPase activity compared to wildtype cells. Leading-edge biosensors diffuse to the cytosol when the pikI- round-up and translocate back to the PM when the cells spread. These observations propose that PI(4,5)P2 levels elevate as cells round-up and decrease as cells spread. This interesting phenotype resembles the front and rear of a migratory cells. Interestingly, pikI- resemble similar cell morphology and biosensors dynamics observed when we use an inducible system to deplete PM PI(4,5)P2 levels. We, also, observed the dynamics of a biosensor for an F-actin polymerization protein called formin A (ForA). ForA has been shown to localize at a polarized cell’s rear and in the cleavage furrow of dividing cells. In addition, ForA have a PI(4,5)P2 binding motif and binds to PI(4,5)P2 preferentially in vitro. Our results support a role for PI(4,5)P2 in regulating ForA with the plasma membrane. Taken together, we proposed that local levels of PI(4,5)P2 contribute to the electrostatic interactions of regulatory proteins controlling actin dynamics and membrane protrusions. PM PI(4,5)P2 below a threshold activate regulatory proteins that excite the signaling that promotes protrusions, while below threshold levels would inhibit those proteins activity. The change in PI(4,5)P2 levels would be predicted to affect the membrane’s charge, which in turn changes the interaction and disassociation of many anionic regulatory proteins involved in the signaling pathway and cytoskeletal rearrangements. Additionally, we show for the first time, a correlation between the PM PI(4,5)P2 threshold and rates of phosphatidylserine (PS) exposure in cancer therapeutics. Receptor-mediated cell stimulation triggers PS exposure to the outer leaflet of the plasma membrane. Interestingly, pik1- and cells using an inducible system to deplete PM PI(4,5)P2 levels depicted the same responses. In addition to PI(4,5)P2, PS exposure affects the membrane’s charge which impacts the signaling molecules activity in the pathway. Altogether, PI(4,5)P2 and PS are proposed to be novel therapeutic targets in cancer treatments. Chemotaxis is a feature of metastatic cancer cells and is regulated by various regulators including actin cytoskeleton. Actin cytoskeleton reorganization during chemotaxis is regulated by actin-binding proteins including those that interact with the PM PI(4,5)P2. Energy production regulates cell migration as well, through glycolysis pathway. A previous study proposed that actin reorganization releases Aldo A enzyme which enters glycolysis, through activating PI3K signaling pathway. However, the mechanism of action remains unclear. We speculate that local PI(4,5)P2 levels regulate Aldo Activity through regulating actin-severing proteins activity including cofilin and gelsolin, and actin polymerizing protein including ForA. PI(4,5)P2 levels below a threshold release actin-severing proteins to the cytosol triggering the severing of actin and the release of Aldo A. while, above PI(4,5)P2 threshold activates and localizes for a on the PM promoting the F-actin polymerization and the sequester of Aldo A into F-actin. The goal of this work is to discover a new model for actin cytoskeleton regulation during migration, as its linkage to glycolysis and metabolism has important implications for cancer.

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