RESUMEN
Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We report that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the self-renewal of hPSCs. Further, we show that TTBK1, a related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation. Importantly, we demonstrate that while TTBK1 fails to localize to the mother centriole, it regulates primary cilia formation in the differentiated, but not the undifferentiated hPSCs. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes.
Asunto(s)
Proteínas Hedgehog , Células Madre Pluripotentes , Humanos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre Pluripotentes/metabolismo , Centriolos/metabolismo , Cilios/metabolismoRESUMEN
Primary cilia are hair-like sensory organelles protruding from the surface of most human cells. As cilia are dynamic, several aspects of their biology can only be revealed by real-time analysis in living cells. Here we describe the generation of primary cilia reporter cell lines. Furthermore, we provide a detailed protocol of how to use the reporter cell lines for live-cell imaging microscopy analysis of primary cilia to study their growth as well as intraciliary transport. For complete details on the use and execution of this protocol, please refer to Bernatik et al. (2020) and Pejskova et al. (2020).
Asunto(s)
Cilios , Procesamiento de Imagen Asistido por Computador , Línea Celular , Cilios/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodosRESUMEN
Cilia formation is essential for human life. One of the earliest events in the ciliogenesis program is the recruitment of tau-tubulin kinase 2 (TTBK2) by the centriole distal appendage component CEP164. Due to the lack of high-resolution structural information on this complex, it is unclear how it is affected in human ciliopathies such as nephronophthisis. Furthermore, it is poorly understood if binding to CEP164 influences TTBK2 activities. Here, we present a detailed biochemical, structural, and functional analysis of the CEP164-TTBK2 complex and demonstrate how it is compromised by two ciliopathic mutations in CEP164. Moreover, we also provide insights into how binding to CEP164 is coordinated with TTBK2 activities. Together, our data deepen our understanding of a crucial step in cilia formation and will inform future studies aimed at restoring CEP164 functionality in a debilitating human ciliopathy.
Asunto(s)
Ciliopatías/genética , Proteínas de Microtúbulos/química , Proteínas de Microtúbulos/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Sitios de Unión , Dicroismo Circular , Células HEK293 , Humanos , Proteínas de Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Estabilidad ProteicaRESUMEN
Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-ß. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/ß-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/ß-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/ß-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/ß-catenin pathway, ß-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/ß-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/ß-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/ß-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/ß-catenin pathway in some systems seems to moderately suppress ciliogenesis.
RESUMEN
Motile cilia, hairlike structures present on the cell surface, have a well-appreciated role in human physiology, including sweeping mucus, dirt and debris out of the respiratory tract. However, we are only beginning to understand the mechanisms governing cilia growth, maintenance and function. In this issue, Arora et al. reveal new details about the control of cilia growth. They identify a previously unrecognized connection between adenylate cyclase 6 (AC6), a cilia signaling mediator, and the autophagy-mediated regulation of motile cilia length via kinesin Kif19a, a regulator of cilia length. These findings provide new insights into motile cilia biology and may lead to novel ciliopathy treatments.
Asunto(s)
Cilios , Cinesinas , Adenilil Ciclasas , Animales , Humanos , Cinesinas/genética , Mamíferos , MicrotúbulosRESUMEN
Primary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling.
Asunto(s)
Aurora Quinasa A/metabolismo , Ciclo Celular/genética , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Cinesinas/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Cuerpos Basales/metabolismo , Cromatografía Liquida , Cilios/genética , Cilios/patología , Células HEK293 , Humanos , Interfase/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinesinas/genética , Mitosis/genética , Proteínas Oncogénicas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Transducción de Señal/genética , Canales de Sodio/metabolismo , Espectrometría de Masas en TándemRESUMEN
Primary cilia are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling the final step of cilia initiation. The function of TTBK2 in ciliogenesis is critically dependent on its kinase activity; however, the precise mechanism of TTBK2 action has so far not been fully understood due to the very limited information about its relevant substrates. In this study, we demonstrate that CEP83, CEP89, CCDC92, Rabin8, and DVL3 are substrates of TTBK2 kinase activity. Further, we characterize a set of phosphosites of those substrates and CEP164 induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we show that TTBK2 is also required for efficient phosphorylation of many S/T sites in CEP164 and provide evidence that TTBK2-induced phosphorylations of CEP164 modulate its function, which in turn seems relevant for the process of cilia formation. In summary, our work provides important insight into the substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.
Asunto(s)
Cilios/metabolismo , Complejos Multiproteicos/metabolismo , Organogénesis , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencias de Aminoácidos , Quinasa de la Caseína I/metabolismo , Células HEK293 , Humanos , Fosforilación , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Serina-Treonina Quinasas/química , Especificidad por SustratoRESUMEN
BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced ß-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.
Asunto(s)
Proteínas Dishevelled/metabolismo , Células Cultivadas , Proteínas Dishevelled/química , Células HEK293 , Humanos , Espectrometría de Masas , Quinasas Relacionadas con NIMA/metabolismo , Fosforilación , Conformación Proteica , Transducción de SeñalRESUMEN
Development of neural tube has been extensively modeled in vitro using human pluripotent stem cells (hPSCs) that are able to form radially organized cellular structures called neural rosettes. While a great amount of research has been done using neural rosettes, studies have only inadequately addressed how rosettes are formed and what the molecular mechanisms and pathways involved in their formation are. Here we address this question by detailed analysis of the expression of pluripotency and differentiation-associated proteins during the early onset of differentiation of hPSCs towards neural rosettes. Additionally, we show that the BMP signaling is likely contributing to the formation of the complex cluster of neural rosettes and its inhibition leads to the altered expression of PAX6, SOX2 and SOX1 proteins and the rosette morphology. Finally, we provide evidence that the mechanism of neural rosettes formation in vitro is reminiscent of the process of secondary neurulation rather than that of primary neurulation in vivo. Since secondary neurulation is a largely unexplored process, its understanding will ultimately assist the development of methods to prevent caudal neural tube defects in humans.
Asunto(s)
Diferenciación Celular , Células-Madre Neurales/citología , Tubo Neural/embriología , Neurulación , Células Madre Pluripotentes/citología , Factor de Transcripción COUP II/genética , Factor de Transcripción COUP II/metabolismo , Células Cultivadas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células-Madre Neurales/metabolismo , Tubo Neural/citología , Tubo Neural/metabolismo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Células Madre Pluripotentes/metabolismoRESUMEN
Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs.
Asunto(s)
Diferenciación Celular , Autorrenovación de las Células , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proliferación Celular , Centrosoma/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Mitosis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.
Asunto(s)
Sistemas CRISPR-Cas , Células Madre Embrionarias/metabolismo , Técnicas de Inactivación de Genes/métodos , Animales , Línea Celular , Células Cultivadas , Regulación hacia Abajo , Células Madre Embrionarias/citología , Humanos , Ratones , MicroARNs/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Wnt signaling cascade has developed together with multicellularity to orchestrate the development and homeostasis of complex structures. Wnt pathway components - such as ß-catenin, Dishevelled (DVL), Lrp6, and Axin-- are often dedicated proteins that emerged in evolution together with the Wnt signaling cascade and are believed to function primarily in the Wnt cascade. It is interesting to see that in recent literature many of these proteins are connected with cellular functions that are more ancient and not limited to multicellular organisms - such as cell cycle regulation, centrosome biology, or cell division. In this review, we summarize the recent literature describing this crosstalk. Specifically, we attempt to find the answers to the following questions: Is the response to Wnt ligands regulated by the cell cycle? Is the centrosome and/or cilium required to activate the Wnt pathway? How do Wnt pathway components regulate the centrosomal cycle and cilia formation and function? We critically review the evidence that describes how these connections are regulated and how they help to integrate cell-to-cell communication with the cell and the centrosomal cycle in order to achieve a fine-tuned, physiological response.
Asunto(s)
Ciclo Celular , Centrosoma/metabolismo , Vía de Señalización Wnt , Animales , Comunicación Celular , Polaridad Celular , HumanosRESUMEN
Centrioles function as core components of centrosomes and as basal bodies for the formation of cilia and flagella. Thus, effective control of centriole numbers is essential for embryogenesis, tissue homeostasis and genome stability. In mammalian cells, the centriole duplication cycle is governed by Polo-like kinase 4 (Plk4). Here, we identify the E3 ubiquitin ligase Mind bomb (Mib1) as a new interaction partner of Plk4. We show that Mib1 localizes to centriolar satellites but redistributes to centrioles in response to conditions that induce centriole amplification. The E3 ligase activity of Mib1 triggers ubiquitylation of Plk4 on multiple sites, causing the formation of Lys11-, Lys29- and Lys48-ubiquitin linkages. These modifications control the abundance of Plk4 and its ability to interact with centrosomal proteins, thus counteracting centriole amplification induced by excess Plk4. Collectively, these results identify the interaction between Mib1 and Plk4 as a new and important element in the control of centriole homeostasis.
Asunto(s)
Centriolos/metabolismo , Biogénesis de Organelos , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Unión Proteica , UbiquitinaciónRESUMEN
Primary cilia play critical roles in development and disease. Their assembly is triggered by mature centrioles (basal bodies) and requires centrosomal protein 164kDa (Cep164), a component of distal appendages. Here we show that loss of Cep164 leads to early defects in ciliogenesis, reminiscent of the phenotypic consequences of mutations in TTBK2 (Tau tubulin kinase 2). We identify Cep164 as a likely physiological substrate of TTBK2 and demonstrate that Cep164 and TTBK2 form a complex. We map the interaction domains and demonstrate that complex formation is crucial for the recruitment of TTBK2 to basal bodies. Remarkably, ciliogenesis can be restored in Cep164-depleted cells by expression of chimeric proteins in which TTBK2 is fused to the C-terminal centriole-targeting domain of Cep164. These findings indicate that one of the major functions of Cep164 in ciliogenesis is to recruit active TTBK2 to centrioles. Once positioned, TTBK2 then triggers key events required for ciliogenesis, including removal of CP110 and recruitment of intraflagellar transport proteins. In addition, our data suggest that TTBK2 also acts upstream of Cep164, contributing to the assembly of distal appendages.
Asunto(s)
Centriolos/metabolismo , Proteínas de Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Cuerpos Basales/metabolismo , Centriolos/genética , Cilios/genética , Cilios/metabolismo , Eliminación de Gen , Células HEK293 , Humanos , Proteínas de Microtúbulos/genética , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Centrioles function in the assembly of centrosomes and cilia. Structural and numerical centrosome aberrations have long been implicated in cancer, and more recent genetic evidence directly links centrosomal proteins to the etiology of ciliopathies, dwarfism and microcephaly. To better understand these disease connections, it will be important to elucidate the biogenesis of centrioles as well as the controls that govern centriole duplication during the cell cycle. Moreover, it remains to be fully understood how these organelles organize a variety of dynamic microtubule-based structures in response to different physiological conditions. In proliferating cells, centrosomes are crucial for the assembly of microtubule arrays, including mitotic spindles, whereas in quiescent cells centrioles function as basal bodies in the formation of ciliary axonemes. In this short review, we briefly introduce the key gene products required for centriole duplication. Then we discuss recent findings on the centriole duplication factor STIL that point to centrosome amplification as a potential root cause for primary microcephaly in humans. We also present recent data on the role of a disease-related centriole-associated protein complex, Cep164-TTBK2, in ciliogenesis.
Asunto(s)
Centriolos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microcefalia/genética , Animales , Proteínas de Ciclo Celular , Centriolos/genética , Centriolos/patología , Proteínas del Citoesqueleto , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.
Asunto(s)
Cilios/fisiología , Proteínas del Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Tubulina (Proteína)/metabolismo , Línea Celular Tumoral , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cilios/genética , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Interferente Pequeño/genéticaRESUMEN
Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1(-/-) mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1(-/-);Wnt5a(-/-) mice revealed a greater loss of Nurr1(+) cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Mesencéfalo/crecimiento & desarrollo , Neurogénesis/fisiología , Células Madre/citología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Proteína Wnt1/metabolismo , Análisis de Varianza , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Recuento de Células , Diferenciación Celular/fisiología , Neuronas Dopaminérgicas/metabolismo , Inmunohistoquímica , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/terapia , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Proteína Wnt-5a , Proteína Wnt1/deficienciaRESUMEN
Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson's disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Quinasa de la Caseína I/metabolismo , Diferenciación Celular , Proteínas Dishevelled , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Humanos , Inmunoprecipitación , Mesencéfalo/citología , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Ratones , Neuropéptidos/genética , Fosfoproteínas/genética , Transducción de Señal , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteínas Wnt/genética , Vía de Señalización Wnt , Proteína Wnt-5a , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1RESUMEN
Secreted Frizzled related proteins (sFRPs) are a family of proteins that modulate Wnt signaling, which in turn regulates multiple aspects of ventral midbrain (VM) and dopamine (DA) neuron development. However, it is not known which Wnt signaling branch and what aspects of midbrain DA neuron development are regulated by sFRPs. Here, we show that sFRP1 and sFRP2 activate the Wnt/planar-cell-polarity/Rac1 pathway in DA cells. In the developing VM, sFRP1 and sFRP2 are expressed at low levels, and sFRP1-/- or sFRP2-/- mice had no detectable phenotype. However, compound sFRP1-/-;sFRP2-/- mutants revealed a Wnt/PCP phenotype similar to that previously described for Wnt5a-/- mice. This included an anteroposterior shortening of the VM, a lateral expansion of the Shh domain and DA lineage markers (Lmx1a and Th), as well as an accumulation of Nurr1+ precursors in the VM. In vitro experiments showed that, while very high concentrations of SFRP1 had a negative effect on cell survival, low/medium concentrations of sFRP1 or sFRP2 promoted the DA differentiation of progenitors derived from primary VM cultures or mouse embryonic stem cells (ESCs), mimicking the effects of Wnt5a. We thus conclude that the main function of sFRP1 and sFRP2 is to enhance Wnt/PCP signaling in DA cells and to regulate Wnt/PCP-dependent functions in midbrain development. Moreover, we suggest that low-medium concentrations of sFRPs may be used to enhance the DA differentiation of ESCs and improve their therapeutic application.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Células Madre Embrionarias/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Mesencéfalo/embriología , Proteínas del Tejido Nervioso/metabolismo , Animales , Neuronas Dopaminérgicas/citología , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/citología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Mesencéfalo/citología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
WNT signaling pathways play an important role in both development and disease. By analyzing the signaling capabilities of commercially available WNT3a preparations towards the PI3K/AKT/GSK3 signaling pathway, we discovered unexpected inconsistencies from lot to lot of recombinant WNT3a. We provide evidence that: (1) The ability to trigger AKT/GSK3 signaling varies dramatically between different lots of WNT3a, without any variation in their ability to activate the canonical WNT/ß-catenin signaling. (2) sFRP1, a WNT signaling inhibitor, is unable to interfere with the activation of AKT/GSK3 signaling induced by some of the WNT3a lots. (3) Pharmacological inhibition of AKT/GSK3 phosphorylation by PI3K inhibitors fails to affect the stabilization of ß-catenin, the central effector of the canonical WNT/ß-catenin signaling pathway. In summary, while all tested lots of recombinant WNT3a activated WNT/ß-catenin pathway, our results suggest that individual lots of recombinant WNT3a activate the PI3K/AKT/GSK3 pathway in a WNT-independent manner, hampering thus the analysis of regulation of PI3K/AKT/GSK3 by WNT ligand.