Abstract
Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.
Highlights
Phosphoinositides play important roles in numerous intracellular membrane pathways
To determine whether 3-phosphorylated phosphoinositides are dynamically regulated by light, we developed a highly sensitive assay based on phosphoinositide binding domains and ELISA with chemiluminescence detection that is capable of quantifying low-abundance phosphoinositides at the femtomolar level
A time course of the PI(3)P increase (Fig. 1A) reveals that the levels increase slowly over a time course of many hours. These changes likely do not play an important role in the characterized types of adaptation of photoresponses, which occur much more rapidly[16], implying that the PI(3)P surge is involved in regulation of cellular homeostasis in response to continuous light
Summary
Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps[34], resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. Phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2), another product of Class I PI-3 kinase, has been proposed to play important roles in cell regulation[4,5], but has not been studied in rods. The studies described here were designed to fill these gaps in understanding the functions and regulation of PI(3)P, PI(3,4)P2, and PIP3 in retinal rod cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
