Mansour, F. | bioRxiv Science

bioRxiv2022

The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation towards the kidney lineage

BackgroundDuring embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while remaining LPM forms cardiovascular, hematopoietic, mesothelial and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human induced pluripotent stem cells (hiPSCs) towards intermediate mesoderm. MethodsWe induced inactivating deletions of CEP83 in hiPSCs and applied a 7 day in vitro protocol of intermediate mesoderm kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. ResultsWhile hiPSCs with homozygous CEP83 inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of CEP83-deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes (PAX8, EYA1, HOXB7) and an aberrant induction of LPM markers (e. g. FOXF1, FOXF2, FENDRR, HAND1, HAND2). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas CEP83-deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. ConclusionOur data suggest that CEP83 regulates the balance of intermediate mesoderm and lateral plate mesoderm formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.

developmental biology↗

bioRxiv2022

The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites.

The precise control of phagosome maturation is critical for innate and adaptive immunity, determining whether phagocytosed material is destroyed or used to present antigens. We observed previously that non-fusogenic contacts between the endoplasmic reticulum (ER) and phagosomes, called membrane contact sites (MCS), are tethered by the calcium regulator STIM1 and fine-tune phagosomal maturation. The secretory pathway SNARE protein Sec22b has been implicated in controlling phagocytosis, phagosome maturation and antigen presentation, though its effects are controversial, and its mechanism of action poorly understood. Recently, Sec22b was shown to tether MCS at the plasma membrane without mediating membrane fusion. Here, we show that Sec22b localizes to and regulates the frequency of ER-phagosome contacts independently of STIM proteins. Sec22b knockdown and overexpression of a an MCS-disrupting mutant Sec22b-P33 induced only mild or no effect on global and local calcium signalling. However, Sec22b knockdown altered phagosomal phospholipids including PI(3)P, PI(4)P and PS, but not PI(4,5)P2. Increased PI(4)P in shSec22b cells was rescued by re-expression of Sec22b or the artificial MCS tether MAPPER but not the P33 mutant. Moreover, Sec22b co-precipitated and was co-recruited to phagosomes with the PS/PI(4)P lipid exchange protein ORP8. Expression of wild-type, but not mutant ORP8, also rescued phagosomal PI(4)P. Concordantly, Sec22b, MAPPER and ORP8 but not P33 or the ORP8 mutant decreased phagolysosome fusion in shSec22b cells. These results clarify a novel mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

cell biology↗