Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Columbia University

Columbia College

Lipid rafts (LRs) are membrane realms characterized by high concentrations of cholesterol and sphingolipids. Often, they are portrayed as scaffolds on which many different signaling molecules can assemble their cascades. The idea of rafts as scaffolds is garnering significant attention as the consequences of LR disruption have been shown to be manifest in multiple signaling pathways. In this study, LRs in the brain of the twitcher (TWI) mouse, a bona-fide model for infant variants of human globoid cell leukodystrophy or Krabbe disease, were investigated. This mouse has deficient activity of GALC (beta-galactosylceramidase) that leads to a progressive accumulation of some galactosyl-sphingolipids in the brain. We hypothesized that the accumulation of psychosine (galactosyl-sphingosine) in the TWI CNS may result in the disruption of rafts in different cell populations such as neurons and oligodendrocytes, both cellular targets during disease. In this communication, we demonstrate that psychosine specifically accumulates in LRs in the TWI brain and sciatic nerve and in samples from brains of human Krabbe patients. It is also shown that this accumulation is accompanied by an increase in cholesterol in these domains and changes in the distribution of the LR markers flotillin-2 and caveolin-1. Finally, we show evidence that this phenomenon may provide a mechanism by which psychosine can exert its known inhibitory effect on protein kinase C. This study provides a previously undescribed biophysical aspect for the mechanism of pathogenesis in Krabbe disease.

Psychosine accumulates in membrane microdomains in the brain of krabbe patients, disrupting the raft architecture.

Radeaux lipidiques (LRs) sont des royaumes de membrane caract&eacute;ris&eacute;s par des concentrations &eacute;lev&eacute;es de cholest&eacute;rol et sphingolipides. Souvent, ils sont pr&eacute;sent&eacute;s comme des &eacute;chafaudages sur lequel de nombreuses mol&eacute;cules de signalisation diff&eacute;rents peuvent assembler leurs cascades. L&#39;id&eacute;e de radeaux comme &eacute;chafaudages est attirant beaucoup d&#39;attention comme les cons&eacute;quences de la perturbation de la LR ont &eacute;t&eacute; montr&eacute;s &agrave; se manifester dans plusieurs voies de signalisation. Dans cette &eacute;tude, LRs dans le cerveau de la souris twitcher (TWI), un mod&egrave;le de bonne foi pour des variantes infantiles de leucodystrophie &agrave; cellules globo&iuml;des humaine ou de la maladie de Krabbe, ont &eacute;t&eacute; &eacute;tudi&eacute;s. Cette souris a activit&eacute; d&eacute;ficiente de GALC (b&ecirc;ta-galactosylc&eacute;ramidase) qui m&egrave;ne &agrave; une accumulation progressive de certains galactosyl-sphingolipides dans le cerveau. Nous avons suppos&eacute; que l&#39;accumulation de la psychosine (galactosyl-sphingosine) dans le SNC de TWI peut entra&icirc;ner l&#39;interruption des radeaux dans diff&eacute;rentes populations cellulaires telles que les neurones et les oligodendrocytes, les deux cibles cellulaires au cours de la maladie. Dans cette communication, nous d&eacute;montrons que psychosine s&#39;accumule plus pr&eacute;cis&eacute;ment dans les LRs dans le cerveau de TWI et le nerf sciatique et dans des &eacute;chantillons de cerveaux de patients humains de Krabbe. On montre aussi que cette accumulation s&#39;accompagne d&#39;une augmentation du cholest&eacute;rol dans ces domaines et les changements dans la r&eacute;partition de la LR marqueurs flotillin-2 et de la cav&eacute;oline-1. Enfin, nous montrons la preuve que ce ph&eacute;nom&egrave;ne peut fournir un m&eacute;canisme par lequel la psychosine peut exercer son effet inhibiteur connu sur la prot&eacute;ine kinase C. Cette &eacute;tude fournit un aspect biophysique non d&eacute;crit le m&eacute;canisme de la pathogen&egrave;se de la maladie de Krabbe.

La psychosine s&#39;accumule dans les microdomaines membranaires dans le cerveau des patients de krabbe, perturbant l&#39;architecture du radeau.

Zattere lipidiche (LRs) sono i regni di membrana caratterizzati da elevate concentrazioni di colesterolo e sfingolipidi. Spesso, essi sono raffigurati come impalcature su cui molte molecole di segnalazione differenti possono assemblare loro cascades. L&#39;idea di zattere come ponteggi &egrave; raccogliere l&#39;attenzione significativa come le conseguenze della perturbazione LR hanno dimostrate di essere manifesto in molteplici vie di segnalazione. In questo studio, LRs nel cervello del mouse twitcher (TWI), un modello di bona-fide per neonato varianti di leucodistrofia a cellule globoidi umana o malattia di Krabbe, sono state studiate. Questo mouse &egrave; carente attivit&agrave; di GALC (beta-galattosilceramidasi) che porta ad un accumulo progressivo di alcuni galattosil-sfingolipidi nel cervello. Abbiamo ipotizzato che l&#39;accumulo di psicosina (galattosil-sfingosina) nel TWI SNC pu&ograve; causare l&#39;interruzione di zattere in popolazioni differenti delle cellule come i neuroni e oligodendrociti, entrambi bersagli cellulari durante la malattia. In questa comunicazione, dimostreremo che psicosina specificamente si accumula in LRs nel cervello di TWI e nervo sciatico e nei campioni dal cervello di pazienti umani Krabbe. Si &egrave; anche dimostrato che questo accumulo &egrave; accompagnato da un aumento del colesterolo in questi domini e cambiamenti nella distribuzione dei marcatori di LR flotillin-2 e caveolina-1. Infine, ci mostrano evidenza che questo fenomeno pu&ograve; fornire un meccanismo da cui psicosina pu&ograve; esercitare il suo noto effetto inibitorio sulla proteina chinasi C. Questo studio fornisce un aspetto biofisico precedentemente nuovo per il meccanismo della patogenesi della malattia di Krabbe.

Adam B. White

Herbert Irving Comprehensive Cancer Center

Columbia University

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Adam B. White

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Columbia University

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Herbert Irving Comprehensive Cancer Center