A Molecular Investigation of the Isoform-Specific Activities of Dictyostelium discoideum Class I PI3Ks During Macropinocytic Cup Formation

Class I phosphoinositide 3-kinases (PI3Ks) are central regulators of membrane dynamics and actin remodelling in most eukaryotic cells, yet the isoform-specific roles of these enzymes remain poorly understood. In Dictyostelium discoideum, the Class I PI3K isoforms PikA and PikF are both essential for macropinocytosis, but the molecular basis of their non-redundant activities has not been resolved. This thesis investigates their distinct contributions to macropinocytic cup formation through a multidisciplinary approach combining structural biology, genetic engineering, live-cell imaging, and proteomics.

We demonstrate that PikA and PikF share highly conserved catalytic domains but differ substantially in their N-terminal regions, which likely drive their isoform-specific localisation and function. Spinning Disk Confocal and Lattice Light Sheet Microscopy reveal that PikA is enriched at the base of macropinocytic cups, where it plays a critical role in initiating cup formation and coordinating early actin assembly. In contrast, PikF localises throughout the entire cup, extending into the rim, and plays a key role in restricting Rac1 activity, thereby facilitating proper cup extension and closure. Live-cell imaging further reveals divergent trafficking behaviours, with PikA undergoing vesicular recycling and PikF remaining cytosolic when not membrane-bound.

Affinity Purification-Mass Spectrometry (AP-MS) reveals largely distinct interactomes: PikA associates with microtubule motors, myosins, and actin nucleators, while PikF interacts with actin disassembly factors, IQGAP scaffolds, and GTPase regulators. These isoform-specific networks suggest that PI3K function is not solely determined by lipid kinase activity, but also by spatially organised protein-protein interactions.

Together, these findings support a revised model of D. discoideum Class I PI3Ks, where isoform specificity arises from domain-dependent localisation and scaffolding roles that coordinate actin dynamics at distinct stages of macropinocytosis – extending beyond the traditional view of Class I PI3Ks as mere PIP3 producers. This work advances our understanding of PI3K-driven large-scale endocytosis and offers broader insight into how spatial signalling architectures govern cellular behaviour in eukaryotes.