Two factors could drive this partitioning: the difference in solvation between the dilute versus dense phase and intermolecular interactions between the client and scaffold proteins. The resulting distribution colocalizes molecular species to carry out a diversity of functions. #FUS PROTEIN SCAFFOLD AND CLIENT FREE#Other molecules, such as proteins and nucleic acids, will distribute between the cytoplasm and the liquid compartment in accordance with the thermodynamic drive to lower the free energy of the system. N.S., not significant.Membraneless organelles are cellular compartments that form by liquid–liquid phase separation of one or more components. Performed for individual comparisons against N2A WT cells. Were cultured and harvested for RNA isolation, followed by reverse transcriptionĪnd quantitative PCR using TaqMan probes for the indicated genes. T-test was performed for individual comparisons against WT N2A (E) Quantification of (D) from three independent KO cells were cultured and immunoblotting was performed using the indicatedĪntibodies. Independent t-test was performed for individual comparisonsĪgainst N2A WT cells. N2A and FUS KO cells were cultured and immunoblotting was performed Student’s t-test was performed for individualĬomparisons against FLAG-FUS WT. Results are shown in scatter plots with the bar representing the (F-G) Quantification of (E) from three independentĮxperiments. Transfections cells were collected and immunoblotting was performed with the Transfection were included as positive and negative controls. (E) FUS KO cells were transfected with FLAG-tagged LC3-positive autophagosomes from multiple random viewfields (N > 150Ĭells). (D) Quantification of the percentage of cells containing (C) Confocal imaging of FUS and LC3 in N2A and FUS KO cells. (B) N2A and FUS KOĬells were cultured and immunoblotting was performed using the indicatedĪntibodies. Immunoblotting was performed using the indicated antibodies. 48h after transfection cells were harvested and (A) N2A cells were transfected with FUS siRNA using © 2020 International Society for Neurochemistry. Our findings demonstrate a novel role of FUS in the autophagy pathway, that is, regulating the transcription of genes involved in early stages of autophagy such as the initiation and elongation of autophagosomes.įused in Sarcoma (FUS) amyotrophic lateral sclerosis (ALS) autophagy frontotemporal dementia (FTD). Re-expressing FUS in the KO cells restored the expression of FIP200 and ATG16L1. Moreover, using immunoblot and quantitative PCR techniques, we found that the mRNA and protein levels of the genes critical in the initial steps of the autophagy pathway (FIP200, ATG16L1 and ATG12) were significantly lower in FUS KO cells. Rapamycin and bafilomycin A1 treatment showed that FUS KO cells were not able to initiate autophagy as efficiently as wild-type cells, suggesting that the autophagosome formation is affected in the absence of FUS. Using immunoblot and confocal imaging techniques in this study, we found that FUS knockout (KO) cells showed a decreased basal autophagy level. However, the role of FUS in the autophagy pathway remains to be better understood. The protein- and RNA-containing inclusions are reported to be positive of autophagosome markers and degraded by the autophagy pathway. ALS-linked mutations cause the accumulation of the FUS protein in cytoplasm where it forms stress granule-like inclusions. FUS is mainly localized in the nucleus although it shuttles between the nucleus and the cytoplasm. Mutations in FUS have also been linked to a subset of familial ALS. FUS pathology has been reported in neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Fused in sarcoma (FUS) is a ubiquitously expressed RNA/DNA-binding protein that plays different roles in the cell.
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