How membrane lipids mediate ciliary signaling

The National Institute of Diabetes and Digestive and Kidney Diseases awarded The Regents of the University of California, San Francisco $191,700 on January 11, 2026, under the Diabetes, Digestive, and Kidney Diseases Extramural Research program (CFDA 93.847) to investigate how membrane lipids mediate ciliary signaling in polycystic kidney disease.

The research addresses the role of phosphatidylserine, a membrane lipid, in regulating the polycystin complex within the primary cilium. Mutations in polycystin proteins cause autosomal-dominant polycystic kidney disease (ADPKD), the most common form of polycystic kidney disease in humans. The project deploys a tool called CILIA-PISD—a fusion protein encoding a ciliary localization sequence, mCherry fluorescent protein, and phosphatidylserine decarboxylase—to deplete ciliary phosphatidylserine in vivo and in vitro. The first aim assesses polycystin signaling in developing zebrafish embryos by injecting CILIA-PISD mRNA into embryo yolks and observing tissue development phenotypes including pronephros cysts and craniofacial defects. The second aim evaluates polycystin signaling in cultured cells using the same depletion approach. Work is performed in San Francisco, California, with a period of performance from January 11, 2026, through August 31, 2028. This is a Project Grant assistance type.

Abstract

Mutations of the polycystin complex, consisting of a 1:3 ratio of membrane-spanning proteins polycystin (PC) 1 and 2, result in autosomal-dominant polycystic kidney disease (adpkd) in humans. Adpkd is monogenic and the most common form of polycystic kidney disease (PKD). The polycystin complex localizes to the ciliary membrane, where IT performs unique roles in cell signaling and maintenance of kidney function. While the role of these membrane proteins at the primary cilium has begun to be identified, relatively little is known about the membrane lipid composition of the primary cilium or its role in ciliary polycystin signaling. Cellular membranes are well established as essential signaling components within and between cells, but there is significant unexplored area to cover in the field of ciliary membrane biology. We aim to discover the relationship between membrane lipid phosphatidylserine and the polycystin complex within the primary cilium. I propose to utilize a tool i developed to deplete ciliary phosphatidylserine in vivo and in vitro to test a novel parameter that may impact polycystin channel localization and signaling. This tool, named cilia-pisd, encodes a fusion protein that includes the ciliary localization sequence of ARL13B, mcherry, and phosphatidylserine decarboxylase, an enzyme that depletes phosphatidylserine in the cell. In aim 1, i propose to assess the role of ciliary phosphatidylserine in polycystin signaling in vivo. I will achieve this aim by injecting cilia-pisd mrna into the yolk of developing zebrafish embryos to observe tissue development consequences during the early stages of development. I hypothesize that i will observe phenotypes consistent with previously described zebrafish polycystin mutations, including pronephros cysts and craniofacial defects. In aim 2, i propose to assess the role of ciliary phosphatidylserine in polycystin signaling in vitro. I propose to investigate the role of ciliary membrane phosphatidylserine in the localization of the polycystin complex and cilia electrophysiology. I will stably express cilia-pisd in IMCD3 cells, and i will assess changes in polycystin localization with immunofluorescent staining for polycystin proteins PC1, visible with a surface HA tag, and PC2. I hypothesize that ciliary PC1 localization will be destabilized in the absence of phosphatidylserine because of recent work describing that the plat domain of PC1, a single subunit of the polycystin tetramer, has binding specificity for negatively charged phospholipids including phosphatidylserine. I will also assess changes in ION channel function in IMCD3 cells stably expressing cilia-pisd via ciliary patch clamping. I hypothesize that loss of ciliary PS will increase the open probability of ION channels in the primary cilium. Completion of the proposed experiments will apply a novel tool for ciliary lipid perturbation and analysis and provide key insight into the role of membrane lipids in polycystin localization and signaling. Together, these findings will also be a foundation for clinical understanding of diseases of ciliary origin that may arise from membrane lipid MIS localization.