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1.
Sci Adv ; 7(17)2021 04.
Article in English | MEDLINE | ID: mdl-33893097

ABSTRACT

Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.

2.
Front Cell Dev Biol ; 8: 588941, 2020.
Article in English | MEDLINE | ID: mdl-33178701

ABSTRACT

Neural rosettes (NPC rosettes) are radially arranged groups of cells surrounding a central lumen that arise stochastically in monolayer cultures of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPC). Since NPC rosette formation is thought to mimic cell behavior in the early neural tube, these rosettes represent important in vitro models for the study of neural tube morphogenesis. However, using current protocols, NPC rosette formation is not synchronized and results are inconsistent among different hPSC lines, hindering quantitative mechanistic analyses and challenging live cell imaging. Here, we report a rapid and robust protocol to induce rosette formation within 6 h after evenly-sized "colonies" of NPC are generated through physical cutting of uniformly polarized NESTIN+/PAX6+/PAX3+/DACH1+ NPC monolayers. These NPC rosettes show apically polarized lumens studded with primary cilia. Using this assay, we demonstrate reduced lumenal size in the absence of PODXL, an important apical determinant recently identified as a candidate gene for juvenile Parkinsonism. Interestingly, time lapse imaging reveals that, in addition to radial organization and apical lumen formation, cells within cut NPC colonies initiate rapid basally-driven spreading. Further, using chemical, genetic and biomechanical tools, we show that NPC rosette morphogenesis requires this basal spreading activity and that spreading is tightly regulated by Rho/ROCK signaling. This robust and quantitative NPC rosette platform provides a sensitive system for the further investigation of cellular and molecular mechanisms underlying NPC rosette morphogenesis.

3.
J Cell Biol ; 216(12): 3981-3990, 2017 12 04.
Article in English | MEDLINE | ID: mdl-29021220

ABSTRACT

Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.


Subject(s)
Cytokinesis , Germ Layers/ultrastructure , Morphogenesis/genetics , Pluripotent Stem Cells/ultrastructure , Actins/genetics , Actins/metabolism , Biomarkers/metabolism , Calcium/metabolism , Calnexin/genetics , Calnexin/metabolism , Cell Line , Cell Polarity , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Gene Expression , Germ Layers/cytology , Germ Layers/metabolism , Humans , Interphase , Lamin Type A/genetics , Lamin Type A/metabolism , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Mitosis , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Single-Cell Analysis , Time-Lapse Imaging
4.
Microbiology (Reading) ; 156(Pt 10): 3085-3095, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20634239

ABSTRACT

Expression of the extensive arsenal of virulence factors by Streptococcus pyogenes is controlled by many regulators, of which CovRS is one of the best characterized and can influence ∼15 % of the genome. Animal models have established that mutants of covRS arise spontaneously in vivo resulting in highly invasive organisms. We analysed a pharyngeal and a blood isolate of S. pyogenes recovered from the same individual 13 days apart. The two isolates varied in many phenotypic properties including SpeB production, which were reflected in transcriptomic analyses. PFGE, multilocus sequence typing and partial sequencing of some key genes failed to show any differences except for an 11 bp insert in the covS gene in the blood isolate which caused a premature termination of transcription. Complementation of a fully functional covS gene into the blood isolate resulted in high expression of CovS and expression of speB. These results, showing a pharyngeal and a blood isolate from a single individual differing by a simple insertion, provide evidence for the model that regulatory gene mutations allow S. pyogenes to invade different niches in the body.


Subject(s)
Bacterial Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Streptococcus pyogenes/genetics , Virulence Factors/genetics , Base Sequence , Blood/microbiology , Child , DNA, Bacterial/genetics , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Histidine Kinase , Humans , Male , Molecular Sequence Data , Multilocus Sequence Typing , Mutagenesis, Insertional , Oligonucleotide Array Sequence Analysis , Pharynx/microbiology , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Streptococcus pyogenes/classification , Streptococcus pyogenes/isolation & purification , Streptococcus pyogenes/pathogenicity
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