Articles by Whitney M. Cleghorn in JoVE
Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments Michelle M. Giarmarco1, Whitney M. Cleghorn1, James B. Hurley1,2, Susan E. Brockerhoff1,2 1Department of Biochemistry, University of Washington, 2Department of Ophthalmology, University of Washington Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.
Other articles by Whitney M. Cleghorn on PubMed
Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina The Journal of Biological Chemistry. | Pubmed ID: 26677218 Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5'-GMP, ribose-5-phosphate, ketone bodies, and purines.
Mitochondria Maintain Distinct Ca Pools in Cone Photoreceptors The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. | Pubmed ID: 28115482 Ca ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca domains are maintained by Ca uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment. The endoplasmic reticulum surrounds the basal and lateral surfaces of this cluster, but does not reach the apical surface or penetrate into the cluster. Using genetically encoded Ca sensors, we found that mitochondria take up Ca when it accumulates either in the cone cell body or outer segment. Blocking mitochondrial Ca uniporter activity compromises the ability of mitochondria to maintain distinct Ca domains. Together, our findings indicate that mitochondria can modulate subcellular functional specialization in photoreceptors. Ca homeostasis is essential for the survival and function of retinal photoreceptors. Separate pools of Ca regulate phototransduction in the outer segment, metabolism in the cell body, and neurotransmitter release at the synaptic terminal. We investigated the role of mitochondria in compartmentalization of Ca We found that mitochondria form a dense cluster that acts as a diffusion barrier between the outer segment and cell body. The cluster is surprisingly only partially surrounded by the endoplasmic reticulum, a key mediator of mitochondrial Ca uptake. Blocking the uptake of Ca by mitochondria causes redistribution of Ca throughout the cell. Our results show that mitochondrial Ca uptake in photoreceptors is complex and plays an essential role in normal function.
Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina The Journal of Biological Chemistry. Dec, 2013 | Pubmed ID: 24187136 Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.