Project Summary Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating high-tension contractile forces at the cell cortex before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be cellular processes to extract unidirectional changes that drive processive transformations in cell shape. In previous work (Jewett et al., 2017; Miao et al., 2019), we found that cytoskeletal force generation is coordinated with endocytic remodeling of the plasma membrane through Sbf- Rab35 tubular compartmental function to stabilize contracted cell surfaces and permit the shrinking of cell apices (apical constriction) or cell interfaces (cell intercalation). However, how this membranous cellular ratchet becomes engaged at particular cell surfaces remains unclear. In the proposed studies, we will examine the informational signals that engage ratcheting and direct Sbf/Rab35 compartmental behaviors to contracting interfaces or cell apices, and identify the fundamental changes in oscillatory durations, amplitudes, frequencies, and/or directionality that lead to contractile processivity. Our preliminary data indicates the PIP3 is a critical determinant for ratcheting engagement – through our proposed work we will perform the first characterization of phosphatidylinositol phosphates (PIPs) in providing lipid-based membrane cues for morphogenesis and gastrulation/ratcheting dynamics in the early Drosophila embryo. In the first aim, we will also analyze how the plasma membrane ultrastructure is remodeled by ratcheting processes and determine if PIP levels are developmentally patterned to drive apical constriction during mesoderm ingression. Our project then moves to a systematic identification of Sbf and Rab35 protein partners in directing ratcheting engagement, and examines the cell signaling pathways that direct a “switching” behavior of contractile force generation from the apical surface to cell interfaces. Our data indicates that, in the absence of JAK/STAT signaling, the Sbf-Rab35 ratchet becomes engaged on all apical surfaces in the embryo, resulting in global apical flattening and constriction. Further, our studies will define if a larger Upd-JAK-STAT-Pi3K-PIP3-Sbf- Rab35 pathway or if two independent pathways (PIP3 and JAK/STAT) have been coordinated to regulate ratcheting engagement. We also apply a new computational phase-based osculating circle approach to detect active periods of contraction and expansion displacements. Finally, we are developing a new mito-tag ectopic relocalization assay as a measure of “sufficiency” of recruiting factors in vivo, and examine if the Akt/mTOR pathway regulates cell ratcheting, potentially demonstrating a new, highly novel function of Akt/mTOR in controlling epithelial cell topologies. Thus, the planned project has the potential to elucidate a large, regulatory hierarchy of the mechanisms that guide engagement of cell ratcheting in epithelial tissu...