We reasoned that while overexpression of Rab5A, as well as other isoforms, can elevate endocytosis and enhance Rac1 activation, Rab5A KD delays EGFR degradation and prolongs its signaling. Consequentially, KD of Rab5A may increase Ras-GTP levels that potentially mediate Rac1 activation through both PI3Kdependent and –independent mechanisms. Activated Ras can initiate a positive feedback loop by direct interaction with p110, thereby increasing PtdInsP3 levels at the leading edge. Activated PI3K may further enhance Ras activation through PtdInsP3-mediated stimulation of Gab1 phosphorylation and recruitment of Grb2/SOS. The PI3K-independent mechanism involves interaction of Ras-GTP and Tiam1, which subsequently activates Rac. For these reasons, we believe Rab5A-depleted cells have overall more stimulatory motogenic signals. Since Rab5C does not appear to regulate EGFR degradation, its loss of function migratory response is not skewed by the EGFR-Ras-Rac1 activation cascade. We explored the BMN673 in vivo possibility that endogenous Rab5C shows more specificity towards Rac-induced cell migration via the PI3K pathway. Rab5 not only interacts with both catalytic and regulatory subunits of PI3 kinase, but enhances the PI3K activity. It is unclear if the interaction between PI3K and Rab5 is isoform-specific, but the inhibition of pAkt and PIP3 production in response to Rab5C depletion does suggest that Rab5C preferentially modulates PI3K activity. One other possibility that could explain the differential effects on cell motility in response to individual Rab5 isoform depletion is an unbalanced endocytic trafficking of membrane adhesion proteins, such as cadherins and integrins. Cadherins that are internalized by several routes pass through Rab5- and EEA1-positive early endosomes, and the cell’s adhesive potential depends upon whether the adhesion molecules are sorted to lysosomes for degradation or recycled back to the cell surface. In zebrafish, prechordal plate progenitor cells exhibit active migratory behavior toward the animal pole of the gastrula using the overlying ectoderm as a substrate on which to migrate. E-cadherin is required for prechordal plate progenitor spreading at the interface between mesoderm and ectoderm and subsequent migration during later stages of gastrulation. Recently, the dynamics of E-cadherin turnover at the plasma membrane was found to be modulated by Rab5C-mediated endocytosis due to its sole expression at this developmental stage. Consistent with these findings, our data showed that Rab5C depletion significantly reduces the formation of cell focal adhesion, and the activity of focal adhesion kinase. A similar finding was recently reported by Mendoza et al. although Rab5C was not specifically tested. Though the current study did not address whether Rab5 isoforms differentially regulate the trafficking of adhesion molecules, recent reports indicate that Rab5C operates semi-independently from the other Rab5 isoforms by interacting directly and apparently selectively with AMAP1, thereby linking Rab5C to a growth factor-stimulated integrin recycling pathway that regulates cell invasion.