Biology subjects

Verhoeven, N.

Publications and source records attributed to Verhoeven, N..

3 recordsLinked to original sources

bioRxiv2026

HAPI Cells are SIM-A9-related Mouse Microglial Cells Useful for In Vitro Modeling of Microglial Immunometabolism

Highly aggressively proliferating immortalized (HAPI) cells were initially described as a spontaneously immortalized rat cell line isolated from a mixed neonatal rat glial population. It was demonstrated that HAPI cells are phagocytic, stain for macrophage-/microglia-specific markers like CD11b and GLUT5, and exhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-) release. These characteristics led to their widespread use as a rat microglial cell line. Here, we report that HAPI cells are mouse cells, not rat cells, but further establish that they have a microglia-like identity and properties useful for in vitro modeling. Cell line authentication by short tandem repeat (STR) profiling, a method that detects identifying DNA signatures, indicates that HAPI cells are a 100% match for SIM-A9 cells, a mouse microglial cell line reported to be spontaneously immortalized from primary cell culture. We find that both HAPI cells and SIM-A9 cells express the microglia-selective gene Tmem119, as well as the microglia-/macrophage-selective marker Cx3cr1, supporting a microglial origin. Like primary rodent microglia or macrophages, HAPI cells respond to combined stimulation with LPS and the Type II interferon, interferon-gamma (IFN-{gamma}), with a pro-inflammatory morphology, NO production, NO-dependent suppression of mitochondrial oxygen consumption, and increased extracellular acidification (an indicator of glycolysis). The Type I interferon, interferon-alpha (IFN-), also reduces mitochondrial oxygen consumption when administered alone or in combination with LPS. Overall, results indicate that HAPI cells are SIM-A9-related mouse cells of microglial origin and support their continued use to study microglial behavior in vitro, including immunometabolism.

neuroscience↗

bioRxiv2023

Na+/K+ATPase - Cav1.2 nanodomain differentially regulates intracellular and local adrenergic signaling in cardiac myocytes

BackgroundThe intracellular Na+ concentration ([Na+]i) is a crucial but understudied regulator of cardiac myocyte function. The Na+/K+ ATPase (NKA) controls the steady-state [Na+]i and thereby determines the set-point for intracellular Ca2+. Here, we investigate the nanoscopic organization and local adrenergic regulation of the NKA macromolecular complex and how it differentially regulates the intracellular Na+ and Ca2+ homeostases in atrial and ventricular myocytes. MethodsMulticolor STORM super-resolution microscopy, Western Blot analyses, and in vivo examination of adrenergic regulation are employed to examine the organization and function of Na+ nanodomains in cardiac myocytes. Quantitative fluorescence microscopy at high spatiotemporal resolution is used in conjunction with cellular electrophysiology to investigate intracellular Na+ homeostasis in atrial and ventricular myocytes. ResultsThe NKA1 (NKA1) and the L-type Ca2+-channel (Cav1.2) form a nanodomain with a center-to center distance of [~]65 nm in both ventricular and atrial myocytes. NKA1 protein expression levels are [~]3 fold higher in atria compared to ventricle. 100% higher atrial INKA, produced by large NKA "superclusters", underlies the substantially lower Na+concentration in atrial myocytes compared to the benchmark values set in ventricular myocytes. The NKAs regulatory protein phospholemman (PLM) has similar expression levels across atria and ventricle resulting in a much lower PLM/NKA1 ratio for atrial compared to ventricular tissue. In addition, a huge PLM phosphorylation reserve in atrial tissue produces a high {beta}-adrenergic sensitivity of INKA in atrial myocytes. {beta}-adrenergic regulation of INKA is locally mediated in the NKA1-Cav1.2 nanodomain via A-kinase anchoring proteins. ConclusionsNKA1, Cav1.2 and their accessory proteins form a structural and regulatory nanodomain at the cardiac dyad. The tissue-specific composition and local adrenergic regulation of this "signaling cloud" is a main regulator of the distinct global intracellular Na+ and Ca2+ concentrations in atrial and ventricular myocytes.

physiology↗

bioRxiv2023

Outer mitochondrial membrane E3 Ub ligase MARCH5 controls mitochondrial steps in peroxisome biogenesis.

Peroxisome de novo biogenesis requires yet unidentified mitochondrial proteins. We report that the outer mitochondrial membrane (OMM)-associated E3 Ub ligase MARCH5 is vital for generating mitochondria-derived pre-peroxisomes. MARCH5 knockout results in accumulation of immature peroxisomes and lower expression of various peroxisomal proteins. Upon fatty acid-induced peroxisomal biogenesis, MARCH5 redistributes to newly formed peroxisomes; the peroxisomal biogenesis under these conditions is inhibited in MARCH5 knockout cells. MARCH5 activity-deficient mutants are stalled on peroxisomes and induce accumulation of peroxisomes containing high levels of the OMM protein Tom20 (mitochondria-derived pre-peroxisomes). Furthermore, depletion of peroxisome biogenesis factor Pex14 leads to the formation of MARCH5- and Tom20-positive peroxisomes, while no peroxisomes are detected in Pex14/MARCH5 dko cells. Reexpression of WT, but not MARCH5 mutants, restores Tom20-positive pre-peroxisomes in Pex14/MARCH5 dko cells. Thus, MARCH5 acts upstream of Pex14 in mitochondrial steps of peroxisome biogenesis. Our data validate the hybrid, mitochondria-dependent model of peroxisome biogenesis and reveal that MARCH5 is an essential mitochondrial protein in this process. SummaryThe authors found that mitochondrial E3 Ub ligase MARCH5 controls the formation of mitochondria-derived pre-peroxisomes. The data support the hybrid, mitochondria-dependent model of peroxisome biogenesis and reveal that MARCH5 is an essential mitochondrial protein in this process.

cell biology↗