scholarly journals Interorganelle lipid flux revealed by enzymatic mass tagging in vivo

2021 ◽  
Author(s):  
Arun T John Peter ◽  
Matthias Peter ◽  
Benoit Kornmann
Keyword(s):  
Living Cells ◽  
Lipid Transport ◽  
Proper Function ◽  
Transport Activity ◽  
Transport Pathways ◽  
Lipid Trafficking ◽  
Lipid Exchange ◽  
Obvious Reason ◽  
Cellular Organelles

The distinct activities of cellular organelles are dependent on the proper function of their membranes. Coordinated membrane biogenesis of the different organelles necessitates interorganelle transport of lipids from their site of synthesis to their destination membranes. Several proteins and trafficking pathways have been proposed to participate in lipid distribution, but despite the basic importance of this process, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. An obvious reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside living cells. In this strategy, two enzymes, one directed to a "donor" and the other to an "acceptor" organelle, add two distinct mass tags to lipids. Mass spectrometry-based detection of lipids bearing the two mass tags is then used as a proxy for lipid exchange between the two organelles. By applying this approach to ER and mitochondria, we show that the ERMES and Vps13-Mcp1 complexes have lipid transport activity in vivo, and unravel their relative contributions to ER-mitochondria lipid exchange.

scholarly journals Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

2018 ◽  
Author(s):  
Maire F. Osborn ◽  
Andrew H. Coles ◽  
Annabelle Biscans ◽  
Reka A. Haraszti ◽  
Loic Roux ◽  
...  
Keyword(s):  
Sirna Delivery ◽  
Lipid Transport ◽  
Small Interfering Rnas ◽  
Transport Pathways ◽  
Efficient Delivery ◽  
Systemic Distribution ◽  
Lipoprotein Binding ◽  
The Impact ◽  
Plasma Half Life

AbstractEfficient delivery of therapeutic RNA is the fundamental obstacle preventing its clinical utility. Lipid conjugation improves plasma half-life, tissue accumulation, and cellular uptake of small interfering RNAs (siRNAs). However, the impact of conjugate structure and hydrophobicity on siRNA pharmacokinetics is unclear, impeding the design of clinically relevant lipid-siRNAs. Using a panel of biologically-occurring lipids, we show that lipid conjugation modulates siRNA hydrophobicity and governs spontaneous partitioning into distinct plasma lipoprotein classes in vivo. Lipoprotein binding influences siRNA distribution by delaying renal excretion and promoting uptake into lipoprotein receptor-enriched tissues. Lipid-siRNAs elicit mRNA silencing without causing toxicity in a tissue-specific manner. Lipid-siRNA internalization occurs independently of lipoprotein endocytosis, and is mediated by siRNA phosphorothioate modifications. Although biomimetic lipoprotein nanoparticles have been considered for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

scholarly journals Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids

2020 ◽  
Author(s):  
Mike F. Renne ◽  
Xue Bao ◽  
Margriet W.J. Hokken ◽  
Adolf S. Bierhuizen ◽  
Martin Hermansson ◽  
...  
Keyword(s):  
Molecular Species ◽  
Protein Complexes ◽  
Lipid Transport ◽  
Mass Spectrometry Analysis ◽  
Proper Function ◽  
Lipid Transfer ◽  
Spectrometry Analysis ◽  
Lipid Species ◽  
Donor And Acceptor

AbstractMitochondria depend on the import of phospholipid precursors for the biosynthesis of the non-bilayer lipids phosphatidylethanolamine (PE) and cardiolipin required for proper function, yet the mechanism of lipid import remains elusive. Pulse labeling yeast with stable isotope-labeled serine followed by mass spectrometry analysis revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-mitochondria complex (EMC) and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids through overexpression of the glycerol-3-phosphate acyltransferase Sct1p, conditions were created to investigate the mechanism of lipid transfer and the contribution of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or the vCLAMP component Vps39p exacerbated the lipid phenotype, indicating that ERMES and Vps39 contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. The results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species’ concentration gradient between donor and acceptor membrane.

Non-vesicular lipid trafficking at the endoplasmic reticulum–mitochondria interface

2018 ◽  
Vol 46 (2) ◽  
pp. 437-452 ◽  
Author(s):  
Francesca Giordano
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Transport ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Transport Pathways ◽  
Lipid Trafficking ◽  
Cellular Processes ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Mitochondria are highly dynamic organelles involved in various cellular processes such as energy production, regulation of calcium homeostasis, lipid trafficking, and apoptosis. To fulfill all these functions and preserve their morphology and dynamic behavior, mitochondria need to maintain a defined protein and lipid composition in both their membranes. The maintenance of mitochondrial membrane identity requires a selective and regulated transport of specific lipids from/to the endoplasmic reticulum (ER) and across the mitochondria outer and inner membranes. Since they are not integrated in the classical vesicular trafficking routes, mitochondria exchange lipids with the ER at sites of close apposition called membrane contact sites. Deregulation of such transport activities results in several pathologies including cancer and neurodegenerative disorders. However, we are just starting to understand the function of ER–mitochondria contact sites in lipid transport, what are the proteins involved and how they are regulated. In this review, we summarize recent insights into lipid transport pathways at the ER–mitochondria interface and discuss the implication of recently identified lipid transfer proteins in these processes.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

2021 ◽  
Author(s):  
Naresh Damuka ◽  
Miranda Orr ◽  
Paul W. Czoty ◽  
Jeffrey L. Weiner ◽  
Thomas J. Martin ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Ex Vivo ◽  
Neuroblastoma Cells ◽  
Proper Function ◽  
Brain Functions ◽  
Biodistribution Studies ◽  
Positron Emission ◽  
Vitro Binding

AbstractMicrotubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

scholarly journals In Vivo 17β-Estradiol Treatment Contributes to Podocyte Actin Stabilization in Female db/db Mice

Endocrinology ◽  
2012 ◽  
Vol 153 (12) ◽  
pp. 5888-5895 ◽  
Author(s):  
Paola Catanuto ◽  
Alessia Fornoni ◽  
Simone Pereira-Simon ◽  
Fayi Wu ◽  
Kerry L. Burnstein ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Transcriptional Activation ◽  
Cell Types ◽  
Proper Function ◽  
Estradiol Treatment ◽  
Diabetic Glomerulosclerosis ◽  
Podocyte Damage ◽  
Rac1 Activity ◽  
17Β Estradiol

Abstract We recently showed that 17β-estradiol (E2) treatment ameliorated type 2 diabetic glomerulosclerosis in mice in part by protecting podocyte structure and function. Progressive podocyte damage is characterized by foot process effacement, vacuolization, detachment of podocytes from the glomerular basement membrane, and apoptosis. In addition, podocytes are highly dependent on the preservation of their actin cytoskeleton to ensure proper function and survival. Because E2 administration prevented podocyte damage in our study on diabetic db/db mice and has been shown to regulate both actin cytoskeleton and apoptosis in other cell types and tissues, we investigated whether actin remodeling and apoptosis were prevented in podocytes isolated from E2-treated diabetic db/db mice. We performed G-actin/F-actin assays, Western analysis for Hsp25 expression, Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, and apoptosis assays on previously characterized podocytes isolated from both in vivo-treated placebo and E2 female db/db mice. We found that in vivo E2 protects against a phenotype change in the cultured podocytes characterized by a percent increase of F-actin vs. G-actin, suppression of Hsp25 expression and transcriptional activation, increase of Rac1 activity, and decreased apoptotic intermediates. We conclude from these studies that E2 treatment protects against podocyte damage and may prevent/reduce diabetes-induced kidney disease.

A near-infrared fluorescence off–on probe for sensitive imaging of hydrogen polysulfides in living cells and mice in vivo

2017 ◽  
Vol 53 (62) ◽  
pp. 8759-8762 ◽  
Author(s):  
Yu Fang ◽  
Wei Chen ◽  
Wen Shi ◽  
Hongyu Li ◽  
Ming Xian ◽  
...  
Keyword(s):  
Near Infrared ◽  
Living Cells ◽  
Near Infrared Fluorescence

A new near-infrared fluorescence off–on probe with phenyl 2-(benzoylthio)benzoate as the recognition moiety is developed and applied in imaging H2Sn in living cells and mice in vivo.

Generation of Slow Network Oscillations in the Developing Rat Hippocampus After Blockade of Glutamate Uptake

2007 ◽  
Vol 98 (4) ◽  
pp. 2324-2336 ◽  
Author(s):  
Adriano Augusto Cattani ◽  
Valérie Delphine Bonfardin ◽  
Alfonso Represa ◽  
Yehezkel Ben-Ari ◽  
Laurent Aniksztejn
Keyword(s):  
Nmda Receptors ◽  
Glutamate Uptake ◽  
Pyramidal Cells ◽  
Cell Types ◽  
Proper Function ◽  
Network Oscillations ◽  
Bath Application ◽  
Hippocampal Network

Cell-surface glutamate transporters are essential for the proper function of early cortical networks because their dysfunction induces seizures in the newborn rat in vivo. We have now analyzed the consequences of their inhibition by dl-TBOA on the activity of the developing CA1 rat hippocampal network in vitro. dl-TBOA generated a pattern of recurrent depolarization with an onset and decay of several seconds' duration in interneurons and pyramidal cells. These slow network oscillations (SNOs) were mostly mediated by γ-aminobutyric acid (GABA) in pyramidal cells and by GABA and N-methyl-d-aspartate (NMDA) receptors in interneurons. However, in both cell types SNOs were blocked by NMDA receptor antagonists, suggesting that their generation requires a glutamatergic drive. Moreover, in interneurons, SNOs were still generated after the blockade of NMDA-mediated synaptic currents with MK-801, suggesting that SNOs are expressed by the activation of extrasynaptic NMDA receptors. Long-lasting bath application of glutamate or NMDA failed to induce SNOs, indicating that they are generated by periodic but not sustained activation of NMDA receptors. In addition, SNOs were observed in interneurons recorded in slices with or without the strata pyramidale and oriens, suggesting that the glutamatergic drive may originate from the radiatum and pyramidale strata. We propose that in the absence of an efficient transport of glutamate, the transmitter diffuses in the extracellular space to activate extrasynaptic NMDA receptors preferentially present on interneurons that in turn activate other interneurons and pyramidal cells. This periodic neuronal coactivation may contribute to the generation of seizures when glutamate transport dysfunction is present.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

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