A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on macropinosomes and phagosomes

Abstract

Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.

Phosphatidylinositol 3,5-bisphosphate facilitates axonal vesicle transport and presynapse assembly

“…Moreover, axonal PVs colocalized with the PI(3,5)P 2 -sensing engineered p85α-cSH2 domain in live iNs (Fig. 4G) and co-stained with recombinant SNXA, a selective PI(3,5)P 2 -binding protein ( 44 ), in fixed iNs (fig. S8C).…”

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

“…2e), likely due to the strong positive charge of this probe (with a pI of 9.7). The biosensor for PI(3,5)P2 is the recently described tandem repeat of a PX domain from Dictyostelium discoideum (Vines et al, 2023) and shows excellent specificity and affinity with almost undetectable binding to any of the other phosphoinositides (Fig. 2f).…”

“…Staining reveals hidden pools of PI(3,5)P2 following PIKfyve inhibition Multiplex staining of phosphoinositides enables investigation of conversion cascades in a straight-forward manner and avoids overexpression of multiple biosensors. To highlight this advantage, and to make use of a novel PI(3,5)P2 probe (Vines et al, 2023), we decided to focus on the conversion cascade leading to the generation of this rare phosphoinositide. Staining for PI(4)P, PI(3)P and PI(3,5)P2 in combination with Airyscan microscopy reveals the close relationship of these three phosphoinositide (Fig.…”

“…Through the combined effort of the community, these effector domains have been optimised to generate biosensors to detect phosphoinositides (Wills et al, 2018). With the recent addition of a PX domain from Dictyostelium discoideum that recognises PI(3,5)P2, (Vines et al, 2023), we now possess biosensors for each of the eight different phosphoinositides (albeit with uncertainties about PI(5)P). A common feature of many of these biosensors is the use of tandem and triple repeats to increase binding through an avidity effect, as is the case for the 3xPH domain of TAPP1, the 3xPHD domain from ING2, the 2xFYVE domain of Hrs and the 2xPX domain of SnxA to recognise PI(3,4)P2, PI(5)P, PI(3)P and PI(3,5)P2 respectively.…”

Molding a PI(3,5)P2 biosensor

“…Yet, the details of how this scarce inositol lipid functions in the pathway, and precisely at which membranes, has remained stubbornly inscrutable. That is, until this issue of the Journal of Cell Biology , where Vines and colleagues report a new genetically encoded lipid biosensor for this molecule ( 1 ). Already, the probe is shedding light on some of the mysteries that still shrouded this key regulator of membrane traffic.…”