A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph

doi:10.1194/jlr.d070565

Cited by 23 publications

(20 citation statements)

References 54 publications

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“…PAC‐NAE contains a C15 fatty acid featuring diazirine and alkyne modifications (Fig 6B). After incubation with tissue explants, PAC‐NAE intracellular localization can be visualized by attaching azide‐modified fluorescent dyes using click chemistry (Haberkant et al , 2013; Gaebler et al , 2016; Höglinger et al , 2017; Müller et al , 2020). To study N‐acylethanolamide trafficking, we loaded PAC‐NAE onto preparations of Drosophila Lpp at levels comparable to those observed in circulation and applied these lipoproteins to explanted wing discs.…”

Section: Resultsmentioning

confidence: 99%

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

et al. 2020

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Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator—the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady‐state levels of ATP and stabilizes Smoothened (Smo), the 7‐pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full‐length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N‐acylethanolamides—lipoprotein‐borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo—a key step in pathway activation—in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.

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Section: Resultsmentioning

confidence: 99%

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

et al. 2020

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“…In this study, we addressed some fundamental questions about the cellular metabolism and localization of de-oxySLs, potentially leading to novel insights about their pathomechanism in diseases like diabetic neuropathy and HSAN1. To enable these experiments, we synthesized a novel traceable doxSA probe, alkyne-doxSA, with superior similarity to naturally formed doxSA and allowing for easy detection via click-chemistry methods (27)(28)(29)37).…”

Section: Discussionmentioning

confidence: 99%

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction

Alecu

Tedeschi

Behler

et al. 2017

Journal of Lipid Research

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1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

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“…To circumvent this, immune populations were isolated using magnetic beads or flow cytometry sorting prior to the chemical clicking method, to allow assessment of uptake within specific subsets without using additional fluorescent antibodies. However, copper-mediated click chemistry has been successfully combined with immunofluorescence by others ( 47 , 48 ). One study demonstrated that specific fluorophores are copper-resistant, such as td Tomato, allowing to combine conventional staining with copper-mediated chemistry ( 47 ).…”

Section: Discussionmentioning

confidence: 99%

Clickable Vitamins as a New Tool to Track Vitamin A and Retinoic Acid in Immune Cells

et al. 2021

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The vitamin A derivative, retinoid acid (RA) is key player in guiding adaptive mucosal immune responses. However, data on the uptake and metabolism of vitamin A within human immune cells has remained largely elusive because retinoids are small, lipophilic molecules which are difficult to detect. To overcome this problem and to be able to study the effect of vitamin A metabolism in human immune cell subsets, we have synthesized novel bio-orthogonal retinoid-based probes (clickable probes), which are structurally and functionally indistinguishable from vitamin A. The probes contain a functional group (an alkyne) to conjugate to a fluorogenic dye to monitor retinoid molecules in real-time in immune cells. We demonstrate, by using flow cytometry and microscopy, that multiple immune cells have the capacity to internalize retinoids to varying degrees, including human monocyte-derived dendritic cells (DCs) and naïve B lymphocytes. We observed that naïve B cells lack the enzymatic machinery to produce RA, but use exogenous retinoic acid to enhance CD38 expression. Furthermore, we showed that human DCs metabolize retinal into retinoic acid, which in co-culture with naïve B cells led to of the induction of CD38 expression. These data demonstrate that in humans, DCs can serve as an exogenous source of RA for naïve B cells. Taken together, through the use of clickable vitamins our data provide valuable insight in the mechanism of vitamin A metabolism and its importance for human adaptive immunity.

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A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins

Cited by 23 publications

References 54 publications

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction

Clickable Vitamins as a New Tool to Track Vitamin A and Retinoic Acid in Immune Cells

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