In addition to deregulating Rho GTPases and their effectors, KAI1/CD82 may also affect the trafficking of actin-binding proteins that are associated with TEMs to alter actin organization, based on the early observations that KAI1/CD82 proteins traffic between the plasma membrane and endosomes/lysosmes and regulate the trafficking of their associated integrins. As described earlier, KAI1/CD82 can initiate outside-in signaling. KAI1/CD82-initiated Mepiroxol signaling may intercept the promigration signaling derived from integrin and growth factor receptor. Alternatively, because KAI1/CD82 physically interacts with b1 integrins and growth factor receptors and downregulates their function, KAI1/CD82 can directly inhibit the promigration signaling at the very upstream. If the complexity of tetraspanin-enriched microdomain constituents is taken into consideration, multiple signaling pathways are susceptible to KAI1/CD82 inhibition. Indeed, we found in this study that KAI1/CD82-induced morphological and cytoskeletal changes could not simply be overridden or bypassed by one or two signaling mechanisms. For example, the signaling originating from b1 integrin, EGFR, c-Met, and CXCR4 promotes cell migration and actin reorganization through Rho small GTPases. Activation of these signaling pathways either alone or together, however, cannot reverse KAI1/CD82-induced morphological and cytoskeletal effects. This observation strongly suggests that KAI1/CD82 acts either on the signaling step at or after the converge point of multiple pathways, e.g., the signaling that immediately leads to actin reorganization, or on the very beginning of multiple signaling pathways, e.g., TEMs, in which integrins and growth factor receptors reside. In either case, KAI1/ CD82 likely acts directly at the plasma membrane where actin reorganization is triggered during cell migration and TEMs are located. The plasma membrane PIP2 is apparently perturbed by KAI1/ CD82 overexpression. Because the PIP2 mAb probes PIP2 after the fixation procedure, it can detect only free PIP2 at the plasma membrane. While GFP-PLCd PH domain fusion not only binds free PIP2 but also likely competes with endogenous PIP2-binding proteins in live cells to occupy PIP2. Thus, the readout of GFPPLCd PH domain fusion more likely reflects the level of free and occupied PIP2 at the plasma membrane. In either case, more PIP2 was found in the plasma membrane of Mock cells. PIP2 links the plasma membrane to actin cytoskeleton by either directly binding and/or activating actin-binding proteins such as b-spectrin, aactinin, vinculin, and ERM proteins or indirectly Gomisin-D inducing the cortical actin polymerization through profilin, cofilin, and NWASP. The level of PIP2 controls the connection of the membrane lipid bilayer to its underlying actin cytoskeleton. The reduced PIP2 at the plasma membrane upon KAI1/CD82 expression, especially at the membrane sites where actin actively undergoes reorganization, likely causes attenuated actin polymerization during cell spreading and migration. Moreover, the composition and distribution of membrane lipid also affect the functional status of Rho GTPases and their effectors because, to be functional, Rho GTPases and their effectors must translocate to the plasma membrane. Because KAI1/CD82 regulates the composition and distribution of PIP2 and other lipids at the plasma membrane, it likely alters the activities of Rho GTPases and their effectors through the translocation step. Interestingly and also surprisingly, cell-cell adhesion significantly alleviates the morphological and cytoskeletal effects of KAI1/ CD82.