RESUMO
Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.
Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas Desgrenhadas/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quinases Relacionadas a NIMA/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilação , Via de Sinalização WntRESUMO
Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Peixe-Zebra , Camundongos , Animais , Progranulinas , Peixe-Zebra/metabolismo , Neurônios Motores/metabolismo , Granulinas , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
The family of VPS10p-Domain (D) receptors comprises five members named SorLA, Sortilin, SorCS1, SorCS2 and SorCS3. While their physiological roles remain incompletely resolved, they have been recognized for their signaling engagements and trafficking abilities, navigating a number of molecules between endosome, Golgi compartments, and the cell surface. Strikingly, recent studies connected all the VPS10p-D receptors to Alzheimer's disease (AD) development. In addition, they have been also associated with diseases comorbid with AD such as diabetes mellitus and major depressive disorder. This systematic review elaborates on genetic, functional, and mechanistic insights into how dysfunction in VPS10p-D receptors may contribute to AD etiology, AD onset diversity, and AD comorbidities. Starting with their functions in controlling cellular trafficking of amyloid precursor protein and the metabolism of the amyloid beta peptide, we present and exemplify how these receptors, despite being structurally similar, regulate various and distinct cellular events involved in AD. This includes a plethora of signaling crosstalks that impact on neuronal survival, neuronal wiring, neuronal polarity, and synaptic plasticity. Signaling activities of the VPS10p-D receptors are especially linked, but not limited to, the regulation of neuronal fitness and apoptosis via their physical interaction with pro- and mature neurotrophins and their receptors. By compiling the functional versatility of VPS10p-D receptors and their interactions with AD-related pathways, we aim to further propel the AD research towards VPS10p-D receptor family, knowledge that may lead to new diagnostic markers and therapeutic strategies for AD patients.
Assuntos
Doença de Alzheimer , Transtorno Depressivo Maior , Humanos , Peptídeos beta-Amiloides , Transporte Proteico/fisiologia , Fatores de Crescimento NeuralRESUMO
WNTs are lipid-modified proteins that control multiple functions in development and disease via short- and long-range signaling. However, it is unclear how these hydrophobic molecules spread over long distances in the mammalian brain. Here we show that WNT5A is produced by the choroid plexus (ChP) of the developing hindbrain, but not the telencephalon, in both mouse and human. Since the ChP produces and secretes the cerebrospinal fluid (CSF), we examine the presence of WNT5A in the CSF and find that it is associated with lipoprotein particles rather than exosomes. Moreover, since the CSF flows along the apical surface of hindbrain progenitors not expressing Wnt5a, we examined whether deletion of Wnt5a in the ChP controls their function and find that cerebellar morphogenesis is impaired. Our study thus identifies the CSF as a route and lipoprotein particles as a vehicle for long-range transport of biologically active WNT in the central nervous system.
Assuntos
Lipoproteínas/líquido cefalorraquidiano , Rombencéfalo/embriologia , Proteína Wnt-5a/metabolismo , Animais , Transporte Biológico , Plexo Corióideo/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos ICR , Morfogênese , Rombencéfalo/metabolismo , Transdução de Sinais , Proteína Wnt-5a/genéticaRESUMO
BACKGROUND: Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/ß-catenin signaling via its interaction with the ß-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. METHODS: Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/ß-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. RESULTS: Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/ß-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and ß-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/ß-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. CONCLUSIONS: Our study shows for the first time that LRRK2 activates the WNT/PCP signaling pathway through its interaction to multiple WNT/PCP components. We suggest that LRRK2 regulates the balance between WNT/ß-catenin and WNT/PCP signaling, depending on the binding partners. Since this balance is crucial for homeostasis of midbrain dopaminergic neurons, we hypothesize that its alteration may contribute to the pathophysiology of Parkinson's disease.
Assuntos
Polaridade Celular/fisiologia , Neurônios Dopaminérgicos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Via de Sinalização Wnt/fisiologia , Caderinas/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , beta Catenina/metabolismoRESUMO
The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.
Assuntos
Ciclo Celular/genética , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Algoritmos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/patologiaRESUMO
The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/ß-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.
Assuntos
Proliferação de Células/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , Sistema Cardiovascular/citologia , Diferenciação Celular/genética , Células Cultivadas , Coração Fetal/citologia , Perfilação da Expressão Gênica/métodos , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Humanos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Laminina/metabolismo , Células-Tronco Mesenquimais/citologia , Microscopia de Fluorescência , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismoRESUMO
Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage.