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1.
Nat Methods ; 21(5): 868-881, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38374263

RESUMO

The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.


Assuntos
Diferenciação Celular , Hematopoese , Células-Tronco Pluripotentes Induzidas , Organoides , Humanos , Organoides/citologia , Organoides/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Camundongos , Células-Tronco Hematopoéticas/citologia , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo
2.
Blood ; 144(3): 308-322, 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-38657197

RESUMO

ABSTRACT: Thrombotic microangiopathy (TMA) is characterized by immunothrombosis and life-threatening organ failure but the precise underlying mechanism driving its pathogenesis remains elusive. In this study, we hypothesized that gasdermin D (GSDMD), a pore-forming protein that serves as the final downstream effector of the pyroptosis/interleukin-1ß (IL-1ß) pathway, contributes to TMA and its consequences by amplifying neutrophil maturation and subsequent necrosis. Using a murine model of focal crystalline TMA, we found that Gsdmd deficiency ameliorated immunothrombosis, acute tissue injury, and failure. Gsdmd-/- mice exhibited a decrease in mature IL-1ß, as well as in neutrophil maturation, ß2-integrin activation, and recruitment to TMA lesions, in which they formed reduced neutrophil extracellular traps in both arteries and interstitial tissue. The GSDMD inhibitor disulfiram dose-dependently suppressed human neutrophil pyroptosis in response to cholesterol crystals. Experiments with GSDMD-deficient, human-induced, pluripotent stem cell-derived neutrophils confirmed the involvement of GSDMD in neutrophil ß2-integrin activation, maturation, and pyroptosis. Both prophylactic and therapeutic administration of disulfiram protected the mice from focal TMA, acute tissue injury, and failure. Our data identified GSDMD as a key mediator of focal crystalline TMA and its consequences, including ischemic tissue infarction and organ failure. GSDMD could potentially serve as a therapeutic target for the systemic forms of TMA.


Assuntos
Gasderminas , Neutrófilos , Proteínas de Ligação a Fosfato , Microangiopatias Trombóticas , Animais , Humanos , Camundongos , Antígenos CD18/metabolismo , Antígenos CD18/genética , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Inflamação/patologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Piroptose , Microangiopatias Trombóticas/patologia , Microangiopatias Trombóticas/metabolismo , Microangiopatias Trombóticas/imunologia , Microangiopatias Trombóticas/etiologia
3.
EMBO J ; 40(23): e108788, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34725842

RESUMO

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α/ß, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N-terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild-type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mitose , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Xenopus laevis
4.
Blood ; 141(6): 645-658, 2023 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-36223592

RESUMO

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.


Assuntos
Células-Tronco Pluripotentes Induzidas , Proteoma , Animais , Humanos , Peixe-Zebra , Genética Humana , Mamíferos , Proteínas Adaptadoras de Transdução de Sinal
5.
Blood ; 137(14): 1932-1944, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33512427

RESUMO

Vacuolar protein sorting 45 homolog (VPS45), a member of the Sec1/Munc18 (SM) family, has been implicated in the regulation of endosomal trafficking. VPS45 deficiency in human patients results in congenital neutropenia, bone marrow fibrosis, and extramedullary renal hematopoiesis. Detailed mechanisms of the VPS45 function are unknown. Here, we show an essential role of mammalian VPS45 in maintaining the intracellular organization of endolysosomal vesicles and promoting recycling of cell-surface receptors. Loss of VPS45 causes defective Rab5-to-Rab7 conversion resulting in trapping of cargos in early endosomes and impaired delivery to lysosomes. In this context, we demonstrate aberrant trafficking of the granulocyte colony-stimulating factor receptor in the absence of VPS45. Furthermore, we find that lack of VPS45 in mice is not compatible with embryonic development. Thus, we identify mammalian VPS45 as a critical regulator of trafficking through the endosomal system and early embryogenesis of mice.


Assuntos
Endossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Endossomos/genética , Deleção de Genes , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Camundongos Knockout , Transporte Proteico , Proteínas de Transporte Vesicular/genética
6.
STAR Protoc ; 4(1): 102073, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36853722

RESUMO

Mitochondrial metabolism is critical in hematopoietic stem cell maintenance and differentiation. Here, we present a step-by-step protocol to efficiently differentiate human induced pluripotent stem cells into myeloid progenitors by a robust feeder- and serum-free system. Furthermore, we provide a protocol to subsequently assess mitochondrial function in iPSC-derived myeloid progenitors. We comprehensively describe a protocol to analyze and to quantify key parameters of mitochondrial respiration of iPSC-derived myeloid progenitors by the Seahorse XFe96 Analyzer. Additionally, our protocol includes extensive troubleshooting suggestions. For complete details on the use and execution of this protocol, please refer to Fan et al. (2022).1.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Hematopoéticas , Células Progenitoras Mieloides/metabolismo , Respiração , Mitocôndrias/metabolismo
7.
J Clin Invest ; 132(9)2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35499078

RESUMO

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1-/- cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.


Assuntos
Neutrófilos , Proteostase , Proteínas Adaptadoras de Transdução de Sinal/genética , Granulócitos/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Neutrófilos/metabolismo
8.
Cell Syst ; 10(2): 133-155.e6, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32027860

RESUMO

Living systems integrate biochemical reactions that determine the functional state of each cell. Reactions are primarily mediated by proteins. In proteomic studies, these have been treated as independent entities, disregarding their higher-level organization into complexes that affects their activity and/or function and is thus of great interest for biological research. Here, we describe the implementation of an integrated technique to quantify cell-state-specific changes in the physical arrangement of protein complexes concurrently for thousands of proteins and hundreds of complexes. Applying this technique to a comparison of human cells in interphase and mitosis, we provide a systematic overview of mitotic proteome reorganization. The results recall key hallmarks of mitotic complex remodeling and suggest a model of nuclear pore complex disassembly, which we validate by orthogonal methods. To support the interpretation of quantitative SEC-SWATH-MS datasets, we extend the software CCprofiler and provide an interactive exploration tool, SECexplorer-cc.


Assuntos
Mitose/genética , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Humanos
9.
Trends Cell Biol ; 27(1): 26-41, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27528558

RESUMO

The preparation of eukaryotic cells for division requires an extensive cellular reorganization, affecting cytoskeletal elements, chromatin, and organelles. These drastic changes in cellular architecture ensure the proper segregation of chromosomes and inheritance of organelles. The morphological alterations occurring during mitotic entry are tightly coordinated with the cell cycle, mainly through the action of mitotic kinases. Conversely, the fidelity of these processes impacts mitotic progression and is important for organismal homeostasis and cell fate. Here, we provide an overview of major architectural changes observed during early mitosis and review recent progress in understanding their regulatory mechanisms, focusing on processes accompanying mitotic cell rounding and restructuring of organelles in mammalian cells.


Assuntos
Células/metabolismo , Actomiosina/metabolismo , Animais , Adesões Focais/metabolismo , Humanos , Mitose , Modelos Biológicos , Organelas/metabolismo
10.
Dev Cell ; 43(2): 141-156.e7, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-29065306

RESUMO

During interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Mitose/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Quinase CDC2 , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Células HeLa , Humanos , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-Like
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