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1.
Cell ; 187(2): 276-293.e23, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38171360

RESUMO

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.


Assuntos
Estruturas da Membrana Celular , Miosinas , Tubo Neural , Transdução de Sinais , Animais , Camundongos , Transporte Biológico , Estruturas da Membrana Celular/metabolismo , Proteínas Hedgehog/metabolismo , Miosinas/metabolismo , Pseudópodes/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismo
2.
Nature ; 595(7869): 724-729, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234346

RESUMO

T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.


Assuntos
Imunidade Humoral , Fosfatidiletanolaminas/metabolismo , Receptores CXCR5/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linfócitos B/imunologia , Sistemas CRISPR-Cas , Diferenciação Celular , Cistina Difosfato , Feminino , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfotransferases (Aceptor do Grupo Álcool) , RNA Nucleotidiltransferases , Transdução de Sinais
3.
Mol Cell ; 61(4): 589-601, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26853145

RESUMO

Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis. The MLKL brace, proximal to the N-terminal helix bundle (NB), is involved in oligomerization to facilitate plasma membrane targeting through the low-affinity binding of NB to phosphorylated inositol polar head groups of phosphatidylinositol phosphate (PIP) phospholipids. At the membrane, the NB undergoes a "rolling over" mechanism to expose additional higher-affinity PIP-binding sites responsible for robust association to the membrane and displacement of the brace from the NB. PI(4,5)P2 is the preferred PIP-binding partner. We investigate the specific association of MLKL with PIPs and subsequent structural changes during necroptosis.


Assuntos
Fibroblastos/citologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Modelos Moleculares , Fosforilação , Proteínas Quinases/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
5.
Nat Commun ; 11(1): 5312, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082319

RESUMO

Evidence is lacking as to how developing neurons integrate mitogenic signals with microenvironment cues to control proliferation and differentiation. We determine that the Siah2 E3 ubiquitin ligase functions in a coincidence detection circuit linking responses to the Shh mitogen and the extracellular matrix to control cerebellar granule neurons (CGN) GZ occupancy. We show that Shh signaling maintains Siah2 expression in CGN progenitors (GNPs) in a Ras/Mapk-dependent manner. Siah2 supports ciliogenesis in a feed-forward fashion by restraining cilium disassembly. Efforts to identify sources of the Ras/Mapk signaling led us to discover that GNPs respond to laminin, but not vitronectin, in the GZ microenvironment via integrin ß1 receptors, which engages the Ras/Mapk cascade with Shh, and that this niche interaction is essential for promoting GNP ciliogenesis. As GNPs leave the GZ, differentiation is driven by changing extracellular cues that diminish Siah2-activity leading to primary cilia shortening and attenuation of the mitogenic response.


Assuntos
Cílios/metabolismo , Matriz Extracelular/metabolismo , Neurônios/citologia , Proteínas Nucleares/metabolismo , Células-Tronco/citologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Cerebelo/citologia , Cerebelo/metabolismo , Cílios/genética , Matriz Extracelular/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Proteínas Nucleares/genética , Transdução de Sinais , Células-Tronco/metabolismo , Ubiquitina-Proteína Ligases/genética
6.
Nat Commun ; 7: 11876, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27336173

RESUMO

Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin-tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical-basal polarity and in the maintenance of the epithelial barrier.


Assuntos
Actomiosina/metabolismo , Barreira Hematoencefálica , Proteínas de Ligação ao Cálcio/fisiologia , Plexo Corióideo/metabolismo , Junções Íntimas/metabolismo , Actinas/metabolismo , Animais , Polaridade Celular , Plexo Corióideo/ultraestrutura , Epêndima/ultraestrutura , Células Epiteliais/ultraestrutura , Hidrocefalia/etiologia , Camundongos , Camundongos Knockout , Proteína da Zônula de Oclusão-1/metabolismo
7.
Cell Rep ; 5(4): 878-85, 2013 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-24268776

RESUMO

Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death.


Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Células 3T3 , Animais , Hidroxianisol Butilado/farmacologia , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Camundongos , Mitofagia/efeitos dos fármacos , Necrose/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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