RESUMO
The primary cilia are known as biosensors that transduce signals through the ciliary membrane proteins in vertebrate cells. The ciliary membrane contains transmembrane proteins and membrane-associated proteins. Tubby-like protein 3 (TULP3), a member of the tubby family, has been shown to interact with the intraflagellar transport-A complex (IFT-A) and to be involved in the ciliary localization of transmembrane proteins, although its role in the ciliary entry of membrane-associated proteins has remained unclear. Here, to determine whether TULP3 is required for the localization of ciliary membrane-associated proteins, we generated and analyzed TULP3-knockout (KO) hTERT RPE-1 (RPE1) cells. Immunofluorescence analysis demonstrated that ciliary formation was downregulated in TULP3-KO cells and that membrane-associated proteins, ADP-ribosylation factor-like 13B (ARL13B) and inositol polyphosphate-5-phosphatase E (INPP5E), failed to localize to primary cilia in TULP3-KO cells. These defects in the localization of ARL13B and INPP5E in TULP3-KO cells were rescued by the exogenous expression of wild-type TULP3, but not that of mutant TULP3 lacking the ability to bind IFT-A. In addition, the expression of TUB protein, another member of the tubby family whose endogenous expression is absent in RPE1 cells, also rescued the defective ciliary localization of ARL13B and INPP5E in TULP3-KO cells, suggesting that there is functional redundancy between TULP3 and TUB. Our findings indicate that TULP3 participates in ciliogenesis, and targets membrane-associated proteins to primary cilia via binding to IFT-A.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/metabolismo , Fatores de Ribosilação do ADP/análise , Sistemas CRISPR-Cas , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Linhagem Celular , Cílios/genética , Cílios/ultraestrutura , Técnicas de Inativação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Monoéster Fosfórico Hidrolases/análise , Ligação Proteica , Proteínas/genéticaRESUMO
The positron emission tomography probes 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 2-tert-butyl-4-chloro-5-{6-[2-(2-[18F]fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ([18F]BCPP-EF) are designed to evaluate glycolysis and oxidative phosphorylation, respectively, and are both used to estimate neuronal activity. However, previous studies have shown a discrepancy in these probes' accumulation in the compromised region, possibly due to the presence of activated microglia acting like deleterious or neuroprotective phenotypes. Hence, we evaluated lipopolysaccharide (LPS)- and interleukin 4 (IL4)-stimulated microglial uptake of [14C]2DG and [18F]BCPP-EF to give a new insight into the hypothesis that different uptake of [18F]FDG and [18F]BCPP-EF can be ascribed to the different metabolic pathways activated during microglial activation. LPS or IL4 stimulation increased the proinflammatory or anti-inflammatory marker gene expression in microglial cells. In LPS-stimulated cells, [14C]2DG uptake and glycolysis related gene expression were elevated, and [18F]BCPP-EF uptake was reduced. In IL4-stimulated cells, [18F]BCPP-EF uptake was increased, and [14C]2DG uptake was decreased. The expression of genes involved in glycolysis and mitochondrial complex I subunits was not changed by IL4 stimulation. The uptake of [14C]2DG and [18F]BCPP-EF differs in LPS- and IL4-stimulated polarized microglial cells. The present results suggest that the in vivo accumulation of metabolic tracers [18F]FDG and [18F]BCPP-EF can be influenced by the different aspects of neuroinflammation.
Assuntos
Metabolismo Energético/fisiologia , Fluordesoxiglucose F18/metabolismo , Glucose/metabolismo , Interleucina-4/metabolismo , Microglia/metabolismo , Piridinas/metabolismo , Animais , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Glicólise/fisiologia , Lipopolissacarídeos/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/metabolismo , Tomografia Computadorizada por Raios X/métodosRESUMO
Arginine methylation is a common posttranslational modification that modulates protein function. SCY1-like pseudokinase 1 (SCYL1) is crucial for neuronal functions and interacts with γ2-COP to form coat protein complex I (COPI) vesicles that regulate Golgi morphology. However, the molecular mechanism by which SCYL1 is regulated remains unclear. Here, we report that the γ2-COP-binding site of SCYL1 is arginine-methylated by protein arginine methyltransferase 1 (PRMT1) and that SCYL1 arginine methylation is important for the interaction of SCYL1 with γ2-COP. PRMT1 was colocalized with SCYL1 in the Golgi fraction. Inhibition of PRMT1 suppressed axon outgrowth and dendrite complexity via abnormal Golgi morphology. Knockdown of SCYL1 by small interfering RNA (siRNA) inhibited axon outgrowth, and the inhibitory effect was rescued by siRNA-resistant SCYL1, but not SCYL1 mutant, in which the arginine methylation site was replaced. Thus, PRMT1 regulates Golgi morphogenesis via SCYL1 arginine methylation. We propose that SCYL1 arginine methylation by PRMT1 contributes to axon and dendrite morphogenesis in neurons.