Demultiplexing overlapping signaling scaffold functions to probe lipid messenger coupling to cytoskeletal dynamics

Abstract

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific signaling and regulatory processes. While central to many aspects of cell physiology, this coupling often depends on the function of multi-domain scaffolds that orchestrate transient interactions and dynamic feedback among a spectrum of signaling intermediates and regulatory proteins on organelles. Understanding scaffold protein functions has remained challenging given this complexity. This work employs live-cell imaging and statistical analyses to deconvolve (demultiplex) how the multi-domain scaffold IQGAP1 coordinates phosphoinositide signaling with organelle-specific actin regulation and membrane processing events. Using actin-ensconced endosomes that localize to the basal cortex of polarized epithelial cells as a model system, we demonstrate abilities to dissect how IQGAP1 transitions between different actin and endosomal-membrane tethered states. We provide evidence IQGAP1 functions as a transient inhibitor of actin growth around the endosomes in at least one of these states. While not easily distilled via standard (static) colocalization analyses or traditional pathway perturbations methods, this negative regulation was revealed via a series of dynamic correlation and multiple regression analyses. These methods also uncovered that the negative actin regulation is linked to GTPase-dependent tethering to the endosomal membrane. Moreover, the scaffold transitions underlying this control are shown to depend on the production of PIP 3 lipid messengers by the lipid kinase PI3K. Overall, these methods and results provide new insights in to how IQGAP1 act as a signaling hub by orchestrating time-dependent membrane and cytoskeletal protein interactions and provide new routes to dissect scaffold-mediated pathway regulation in a variety of settings. INTRODUCTION:Phosphoinositides (PIs) are an important class of lipid messenger that drive the recruitment of cytoplasmic signaling proteins to the plasma membrane, organelles, and vesicles (1). PIs also participate in signal transduction by activating proteins locally and relaying signaling events between membrane receptors (2), ion channels (3), as well as downstream membrane and cytoskeletal regulatory proteins (4-6). This communication is at the center of mechanisms governing organelle development, and cell growth, polarization, death, proliferation and motility. Aberrant PI signaling has been associated with numerous human diseases (7). For example, multiple oncogenic mutations have been shown to cause hyperactivation of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) dependent signaling in the phosphoinositide 3-kinase (PI3K) / Akt pathway at the early stages of many common cancers (8). Unfortunately, efforts to target PIP 3 effector proteins are often confounded by unwanted side effects due to the extensive crosstalk and feedback that occurs within lipid signaling networks (9). While integral to mechanisms that coordinate signaling with membrane and c...