Wymann, M. P. | bioRxiv Science

bioRxiv2024

An evolutionary-conserved VPS34-PIKfyve-TRPML1-Myosin II axis regulates the speed of amoeboid cell migration

Amoeboid cell migration is key to efficient T cell immunity. Spatial polarization of organelles within cells, including endo-lysosomes, is a prerequisite of migration. However, how ultrastructural polarization is linked to the signaling requirements governing T cell migration, remains unknown. Here we show that signaling molecules generated by endo-lysosome-localized kinases regulate velocity of amoeboid migration. Specifically, imaging of T cells identified accumulation of endo-lysosomes decorated with the lipid kinases VPS34-PIKfyve at the uropod of polarized cells. Activity of VPS34 and PIKfyve regulated speed, but not directedness, of migrating T cells. Mechanistically, PI(3,5)P2 generated by the sequential action of VPS34 and PIKfyve mediated Ca2+ efflux from lysosomes via the mucolipin TRP cation channel 1 (TRPML1), thus controlling activity of myosin IIA and hence the generation of propulsive force through retrograde actin flow. The VPS34-PIKfyve kinases also regulated velocity of myeloid cells, as well as of the amoeba Dictyostelium discoideum - establishing the axis as an evolutionary conserved speed control system of amoeboid cell migration. Graphical AbstractO_LIThe VPS34-PIKfyve axis is active on endo-lysosomes at the uropod of migrating T cells. C_LIO_LIVPS34 and PIKfyve promote myosin IIA activation and retrograde action flow. C_LIO_LIAmoeboid cell migration speed is controlled by VPS34 and PIKfyve via TRPML1. C_LIO_LIRegulation of amoeboid migration speed is a conserved function of the VPS34-PIKfyve axis. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/575998v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1b70e64org.highwire.dtl.DTLVardef@111d052org.highwire.dtl.DTLVardef@83bc24org.highwire.dtl.DTLVardef@b51931_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to selective inhibitors

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the p110{gamma} subunit playing a key role in immune signalling. PI3K{gamma} is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the c-terminus can inactivate or activate enzyme activity. Screening of small molecule inhibitors using HDX-MS revealed that activation loop binding inhibitors induce allosteric conformational changes that mimic those seen for the R1021C mutant. Structural analysis of clinically advanced PI3K inhibitors revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insight into the regulatory mechanisms that control PI3K{gamma} kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

biochemistry↗