UC San Diego

The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a pivotal role in cellular functions and its dysregulation is implicated in pathologies such as cancer and diabetes. Despite this, the specific spatial organization of key signaling activities, including the lipid substrates and products of PI3K as well as the serine-threonine protein kinase Akt, remains inadequately understood. To study Akt, we developed an excitation-ratiometric Akt activity reporter (ExRai-AktAR2) to precisely monitor subtle changes in Akt activity dynamics within subcellular compartments. Leveraging super-resolution expansion microscopy, our investigation uncovers that growth factor stimulation enhances Akt colocalization with lysosomes, resulting in the accumulation of lysosomal Akt activity. Furthermore, we observed the unexpected accumulation of 3-phosphoinositides (3-PIs), the lipid products of PI3K, on the lysosomal membrane, which is dependent on dynamin-mediated endocytosis. Crucially, lysosomal 3-PIs are imperative for growth-factor-induced Akt and mTORC1 activities on the lysosomal surface, as targeted 3-PI depletion induces deleterious effects. This localization of 3-PIs on the lysosomal membrane during growth factor stimulation directs the Akt kinase to govern lysosome-specific signaling. Our investigation further delves into the spatial regulation of specific 3-PI species, highlighting the significant accumulation of phosphatidylinositol (3,4,5) trisphosphate (PIP3) at the lysosome, challenging conventional assumptions of its exclusive presence in the plasma membrane. Our data suggests that PIP3, produced at the plasma membrane, is subsequently internalized to lysosomes through dynamin-dependent endocytosis, enabling spatially selective regulation of signaling within subcellular compartments. To facilitate the imaging of multiple phosphoinositide species, we engineered a suite of genetically encodable phosphoinositide biosensors, encompassing FRET-based and dimerization-dependent fluorescent protein (ddFP)-based biosensors to monitor dynamics of 3-PIs as well as phosphatidylinositol (4,5) bisphosphate (PIP2) levels in live cells. Moreover, a red fluorescent biosensor for 3-PIs allows simultaneous imaging with the green PIP2 indicator, unveiling the dynamic interplay between PIP2 and 3-PIs during EGF stimulation.