Biology subjects

Deisl, C.

Publications and source records attributed to Deisl, C..

3 recordsLinked to original sources

bioRxiv2024

Constitutive Plasma Membrane Turnover in T-REx293 cells via Ordered Membrane Domain Endocytosis under Mitochondrial Control

Clathrin/dynamin-independent endocytosis of ordered plasma membrane domains (ordered membrane domain endocytosis, OMDE) can become massive in response to cytoplasmic Ca elevations, G protein activation by non-hydrolyzable GTP analogs, and enhanced oxidative metabolism. In patch-clamped murine bone marrow macrophages (BMMs), cytoplasmic succinate and pyruvate, but not {beta}-hydroxybutyrate, induce OMDE of 75% of the plasma membrane within 2 min. The responses require palmitoylation of membrane proteins, being decreased by 70% in BMMs lacking the acyltransferase, DHHC5, by treatment with carnitine to shift long-chain acyl groups from cytoplasmic to mitochondrial acyl-CoAs, by bromopalmitate/albumin complexes to block DHHCs, and by the mitochondria-specific cyclosporin, NIM811, to block permeability transition pores that may release mitochondrial coenzyme A into the cytoplasm. Using T-REx293 cells, OMDE amounts to 40% with succinate, pyruvate, or GTP{gamma}S, and it is inhibited by actin cytoskeleton disruption. Pyruvate-induced OMDE is blocked by the hydrophobic antioxidant, edaravone, which prevents permeability transition pore openings. Using fluorescent 3kD dextrans to monitor endocytosis, OMDE appears to be constitutively active in T-REx293 cells but not in BMMs. After 1 h without substrates or bicarbonate, pyruvate and hydroxybutyrate inhibit constitutive OMDE, as expected for a shift of CoA from long-chain acyl-CoAs to other CoA metabolites. In the presence of bicarbonate, pyruvate strongly enhances OMDE, which is then blocked by {beta}-hydroxybutyrate, bromopalmitate/albumin complexes, cyclosporines, or edaravone. After pyruvate responses, T-REx293 cells grow normally with no evidence for apoptosis. Fatty acid-free albumin (15 M) inhibits basal OMDE in T-REx293 cells, as do cyclosporines, carnitine, and RhoA blockade. Surprisingly, OMDE in the absence of substrates and bicarbonate is not inhibited by siRNA knockdown of the acyltransferases, DHHC5 or DHHC2, which are required for activated OMDE in patch clamp experiments. We verify biochemically that small CoA metabolites decrease long-chain acyl-CoAs. We verify also that palmitoylations of many PM-associated proteins decrease and increase when OMDE is inhibited and stimulated, respectively, by different metabolites. STED microscopy reveals that vesicles formed during constitutive OMDE in T-REX293 cells have 90 to 130 nm diameters. In summary, OMDE is likely a major G-protein-dependent endocytic mechanism that can be constitutively active in some cell types, albeit not BMMs. OMDE depends on different DHHC acyltransferases in different circumstances and can be limited by local supplies of fatty acids, CoA, and long-chain acyl-CoAs.

cell biology↗

bioRxiv2023

Pore-like diffusion barriers in murine cardiac myocytes

Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular weight increases from 30 to 2000, roughly as expected for pores with dimensions of cardiac porin channels. The Bodipy-FL ATP analogue diffuses [~]50-fold slower in BL6 cardiac cytoplasm than in free water. From several fluorophores analyzed, our estimates of bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. We estimate that diffusion coefficients of unbound fluorophores range from 0.5 to 8 x 10-7 cm2/s. Analysis of Na/K pump and veratridine-modified Na channel currents confirms that Na diffusion is nearly unrestricted (time constant for equilibration with the pipette tip, [~]20 s). Using three different approaches, we estimate that ATP diffuses 8 to 10-times slower in the cytoplasm of BL6 myocytes than in free water. To address whether restrictions are caused more by cytoplasmic protein or membrane networks, we verified first that a protein gel, 10 gram% gelatin, restricts solute diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is however less restricted than in intact myocytes. Notably, myofilaments from equivalently extracted skeletal (diaphragm) myocytes restrict diffusion less than cardiac myofilaments. Solute diffusion in myocytes with sarcolemma permeabilized by {beta}-escin (80 {micro}M) is similarly restricted as in intact myocytes. Diffusion restriction in cardiac myocytes is strain-dependent, being about two-fold greater in BL6 myocytes than in myocytes with a CD1/J6/129svJ background. Furthermore, diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, Vdac1, than in WT CD1/J6/129svJ myocytes. We conclude that both myofilaments and mitochondria networks restrict diffusion in cardiac myocytes. As a result, long-range solute diffusion may preferentially occur via passage through porin channels and intramembrane mitochondrial spaces, where diffusion is less restricted than in myofilament spaces.

physiology↗

bioRxiv2022

Thiazides induce glucose intolerance through inhibition of mitochondrial carbonic anhydrase 5b in β-cells

Thiazides are associated with glucose intolerance and new onset diabetes mellitus, but the molecular mechanisms remain elusive. The aim of this study was to decipher the molecular basis of thiazide-induced glucose intolerance. In mice, hydrochlorothiazide induced a pathological glucose tolerance, characterized by reduced first phase insulin secretion but normal insulin sensitivity. In vitro, thiazides inhibited glucose-and sulfonylurea-stimulated insulin secretion in islets and the murine {beta}-cell line Min6 at pharmacologically relevant concentrations. Inhibition of insulin secretion by thiazides was CO2/HCO3--dependent, not additive to unselective carbonic anhydrase (CA) inhibition with acetazolamide and independent of extracellular potassium. In contrast, insulin secretion was unaltered in islets of mice lacking the known molecular thiazide targets NCC (SLC12A3) or NDCBE (SLC4A8). CA expression profiling with subsequent knock-down of individual CA isoforms suggested mitochondrial CA5b as molecular target. In support of these findings, thiazides significantly attenuated Krebs cycle anaplerosis through reduction of mitochondrial oxalacetate synthesis. CA5b KO mice were resistant to thiazide-induced glucose intolerance, and insulin secretion of islets isolated from CA5b KO mice was unaffected by thiazides. In summary, our study reveals attenuated insulin secretion due to inhibition of the mitochondrial CA5b isoform in {beta}-cells as molecular mechanism of thiazide-induced glucose intolerance.

physiology↗