University Of California Santa Barbara

NIH Research Projects · FY 2025 · 2005-02

During normal embryonic development, wound healing and cancer metastasis, cells move collectively, making and breaking cell-cell contacts as they squeeze between other cells and tissues. Individual cells can leave an epithelium by undergoing the epithelial to mesenchymal transition. However, the program(s) enabling cells to escape as a group are less clear. Even less studied are the mechanisms by which groups of cells establish new connections at their destination. We established an in vivo model, the border cells in the Drosophila ovary, to study collective cell movements. We deploy the powerful Drosophila genetics toolkit and organ culture methods we developed to carry out high-resolution live imaging and optogenetics. During the last funding period, we investigated mechanisms by which border cells separate from the follicular epithelium in the process of delamination and how they make new connections when they reach the oocyte, a process we call neolamination. We identified multiple steps of each process and proteins that control them. In Aim 1, we will address key open questions in the septin field and build on our discoveries that the septin cytoskeleton is essential for border cell delamination and that the GTPase Rho recruits septins to the cortex. We propose to use novel in vivo analysis of cell and cluster geometry that we developed to compare and classify Rho, myosin, and septin phenotypes to clarify their roles and relationships in delamination. We will identify the GEFs and GAPs that regulate Rho upstream of septins. In Aim 2, we will follow up on our discovery that the nucleus plays an essential role in collective delamination. As border cells delaminate, they squeeze into tiny spaces much smaller than even a single nucleus. The nucleus can impede cell movement through rigid plastic pores, but we found that the nucleus plays a role in promoting border cell delamination. We propose a new “nuclear wedge” model in which nuclei push substrate cells apart. In Aim 2, we will decipher the mechanisms that move nuclei within the lead cell to promote its wedge function. We will test the function of the LIS1/NudE/Dynein pathway, which moves nuclei on microtubules (MTs) during the migration of neural progenitors in the developing neocortex. We will also study myosin, which transiently accumulates near sites of nuclear deformation and movement. In Aim 3, we will pursue our discovery that innexin (Inx) gap junction proteins promote both delamination and neolamination. Inxs function in a MT-dependent but channel-independent manner. We propose to probe the unexplored relationship between Inxs and MTs. We recently overcame a major technical hurdle and can now image MT dynamics in vivo, which we will use to characterize innexin phenotypes. We will decipher the relationship between MT post translational modifications and dynamics in vivo. We will also test our integrative working model for how nuclear, and cortical cytoskeletal systems interact to accomplish collective delamination and neolamination in vivo.

NIH Research Projects · FY 2026 · 1995-01

Abstract The goal of this project is to break fundamentally new ground by investigating the molecular basis of vision in the daytime mosquito vector, Aedes (Ae.) aegypti. This mosquito is a major disease vector, which transmits the viruses that cause dengue, yellow fever, Zika and other diseases that affect many tens of millions of people each year. Due to climate change and travel, this invasive species is spreading to new locations, including multiple areas in the United States. Therefore, innovative strategies to control mosquito borne disease are urgently needed. Female Ae. aegypti detect and integrate multiple sensory cues to locate human hosts. Once they sense CO2 odor plumes emanating from human breath at distances of up to 10-15 meters, their visual attention to potential hosts is greatly increased. They then rely on visual information along with other human- derived cues, such as organic olfactory stimuli, to home in on people. Despite the important contribution of vision in promoting the ability of Ae. aegypti to find humans, there has been no comprehensive approach to apply molecular genetics to define the mechanisms underlying vision and odor-stimulated vision-guided host attraction. The objective of the proposed research is to address this gap. Aim 1 is to define the roles of the TRP and TRPL channels in Aedes vision. We have recently used CRISPR/Cas9 to generate mutations that disrupt the trp and trpl genes. We propose to examine the roles of these channels for the light response in photoreceptor cells. We also outline experiments to investigate potential roles for these channels in several vision- and light-driven behaviors, including CO2-stimulated vision-guided target attraction. The goal of aim 2 is to test the idea that the two phospholipase C genes expressed in the eyes of Ae. aegypti (NORPA and PLC21C) have distinct roles in visual transduction and in multiple vision- and light-driven behaviors, including circadian rhythms. We will also address the impact of mutations disrupting norpA and plc21C in combination with mutations that disrupt cryptochrome, which encodes a light sensitive protein in the insect brain. Ae. aegypti are most active and bite primarily after sunrise and before sunset. Therefore, understanding the regulation of circadian rhythms in this organism is important, and the contributions of retinal proteins to this behavior represents another major gap in our understanding of the biology of Ae. aegypti. Aim 3 focuses on testing an iconoclastic role for the Gaq in the light response. In addition to its classical role as an effector protein for rhodopsin, we will test the idea that Gaq directly regulates the TRP and TRPL channels. To accomplish our goals, we propose to employ a multidisciplinary approach, including electrophysiology, molecular genetics, biochemistry, cell biology and a wide diversity of behavioral assays. We propose that these studies will provide the conceptual framework for devising innovative strategies to limit the ability of these mosquitoes from efficiently locating hosts and spreading disease.

NIH Research Projects · FY 2025 · 1991-08

Normal tissue development and tumor metastasis require extensive cell movements, and border cell migration in the Drosophila ovary provides a powerful in vivo model. Border cells migrate as a group of two different cell types, a pair of non-migratory polar cells in the center that recruit 6-8 epithelial cells to surround and carry them between nurse cells to the developing oocyte, in a structure called an egg chamber. Using this system, we first discovered the in vivo role of the 21kD GTPase Rac in protrusion and migration, then showed that photoactivation of Rac in one cell could steer the entire cluster. For more than two decades though, we were puzzled that expression of constitutively active Rac in border cells seemed to destroy the entire egg chamber. During the current funding period, we solved this longstanding mystery. We discovered that border cells expressing active Rac kill the nurse cells. Anterior follicle cells normally engulf and kill nurse cells late in oogenesis, and we propose that active Rac prematurely activates this program. A similar mechanism may explain otherwise mysterious immune deficiencies in human patients. Here we propose to continue our exciting investigation of the spatiotemporal control of Rac-mediated cell migration and engulfment in Drosophila. In Aim 1 we propose to elucidate the mechanisms of Rac-mediated cell killing. Taking advantage of the border cell model, we will test the functional effects of each of the known activating Rac mutations that cause immunodeficiencies in patients. We will test the hypothesis that border cells expressing active Rac prematurely activate the normal developmental killing program, and we will define more precisely the role of Rac within the molecular pathway. We will investigate how just six cells can destroy an entire egg chamber, and we will identify the chemical, physical, and adhesive properties that govern target cell selection. In Aim 2, we propose to follow up on our discovery that a basally localized Rac activator is required for border cell cluster cohesion and migration. We will elucidate its relationship to basolateral complex proteins and test whether its primary function is to localize Rac activity to basal surfaces. We will test the hypothesis that basal Rac activity is required to generate basal protrusions that in turn coordinate collective cell behavior. In Aim 3, we will follow up on a screen in which we have identified Rho family activators and inhibitors required in border cells. We propose that border cells require elaborate spatiotemporal control of Rac due to their needs to: maintain apicobasal polarity despite being detached from basement membrane, extend and retract forward-directed protrusions, maintain cohesion under strain, and inhibit inappropriate protrusion. Our ultimate goal is the decipher the Rac regulatory network.

Other NSERC · FY 2024

topology, human movement, pose, deep learning, neural network, graph, higher order, geometry, transformer, action recognition