RESUMEN

De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart defects. Here, we use Xenopus laevis to investigate whether Fbrsl1 plays a role in heart development. Xenopus laevis fbrsl1 is expressed in tissues relevant for heart development, and morpholino-mediated knockdown of Fbrsl1 results in severely hypoplastic hearts. Our data suggest that Fbrsl1 is required for the development of the first heart field, which contributes to the ventricle and the atria, but not for the second heart field, which gives rise to the outflow tract. The morphant heart phenotype could be rescued using a human N-terminal FBRSL1 isoform that contains an alternative exon, but lacks the AUTS2 domain. N-terminal isoforms carrying patient variants failed to rescue. Interestingly, a long human FBRSL1 isoform, harboring the AUTS2 domain, also did not rescue the morphant heart defects. Thus, our data suggest that different FBRSL1 isoforms may have distinct functions and that only the short N-terminal isoform, appears to be critical for heart development.

Asunto(s)

Cardiopatías Congénitas , Corazón , Proteínas de Xenopus , Xenopus laevis , Animales , Humanos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Corazón/embriología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Fenotipo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Xenopus laevis/embriología , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética

RESUMEN

Truncating variants in specific exons of Fibrosin-like protein 1 (FBRSL1) were recently reported to cause a novel malformation and intellectual disability syndrome. The clinical spectrum includes microcephaly, facial dysmorphism, cleft palate, skin creases, skeletal anomalies and contractures, postnatal growth retardation, global developmental delay as well as respiratory problems, hearing impairment and heart defects. The function of FBRSL1 is largely unknown, but pathogenic variants in the FBRSL1 paralog Autism Susceptibility Candidate 2 (AUTS2) are causative for an intellectual disability syndrome with microcephaly (AUTS2 syndrome). Some patients with AUTS2 syndrome also show additional symptoms like heart defects and contractures overlapping with the phenotype presented by patients with FBRSL1 mutations. For AUTS2, a dual function, depending on different isoforms, was described and suggested for FBRSL1. Both, nuclear FBRSL1 and AUTS2 are components of the Polycomb subcomplexes PRC1.3 and PRC1.5. These complexes have essential roles in developmental processes, cellular differentiation and proliferation by regulating gene expression via histone modification. In addition, cytoplasmic AUTS2 controls neural development, neuronal migration and neurite extension by regulating the cytoskeleton. Here, we review recent data on FBRSL1 in respect to previously published data on AUTS2 to gain further insights into its molecular function, its role in development as well as its impact on human genetics.

RESUMEN

In green microalgae, prolonged exposure to inorganic carbon depletion requires long-term acclimation responses, involving modulated gene expression and the adjustment of photosynthetic activity to the prevailing supply of carbon dioxide. Here, we describe a microalgal regulatory cycle that adjusts the light-harvesting capacity at photosystem II (PSII) to the prevailing supply of carbon dioxide in Chlamydomonas (Chlamydomonas reinhardtii). It engages low carbon dioxide response factor (LCRF), a member of the squamosa promoter-binding protein (SBP) family of transcription factors, and the previously characterized cytosolic translation repressor nucleic acid-binding protein 1 (NAB1). LCRF combines a DNA-binding SBP domain with a conserved domain for protein-protein interaction. LCRF transcription is rapidly induced by carbon dioxide depletion. LCRF activates NAB1 transcription by specifically binding to tetranucleotide motifs present in its promoter. Accumulation of the NAB1 protein enhances translational repression of its prime target mRNA, encoding the PSII-associated major light-harvesting protein LHCBM6. The resulting truncation of the PSII antenna size helps maintaining a low excitation during carbon dioxide limitation. Analyses of low carbon dioxide acclimation in nuclear insertion mutants devoid of a functional LCRF gene confirm the essentiality of this novel transcription factor for the regulatory circuit.

Asunto(s)

Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/fisiología , Redes Reguladoras de Genes , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/genética , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Sitio de Iniciación de la Transcripción

RESUMEN

Caveolins are essential structural proteins driving the formation of caveolae, specialized invaginations of the plasma membrane. Loss of Caveolin-1 (Cav1) function in mice causes distinct neurological phenotypes leading to impaired motor control, however, the underlying developmental mechanisms are largely unknown. In this study we find that loss-of-function of Xenopus Cav1 results in a striking swimming defect characterized by paralysis of the morphants. High-resolution imaging of muscle cells revealed aberrant sarcomeric structures with disorganized actin fibers. As cav1 is expressed in motor neurons, but not in muscle cells, the muscular abnormalities are likely a consequence of neuronal defects. Indeed, targeting cav1 Morpholino oligonucleotides to neural tissue, but not muscle tissue, disrupts axonal outgrowth of motor neurons and causes swimming defects. Furthermore, inhibition of voltage-gated sodium channels mimicked the Cav1 loss-of-function phenotype. In addition, analyzing axonal morphology we detect that Cav1 loss-of-function causes excessive filopodia and lamellipodia formation. Using rescue experiments, we show that the Cav1 Y14 phosphorylation site is essential and identify a role of RhoA, Rac1, and Cdc42 signaling in this process. Taken together, these results suggest a previously unrecognized function of Cav1 in muscle development by supporting axonal outgrowth of motor neurons.

Asunto(s)

Caveolina 1/metabolismo , Neuronas Motoras/metabolismo , Músculo Esquelético/metabolismo , Xenopus laevis/metabolismo , Animales , Células Musculares/metabolismo

RESUMEN

We report truncating de novo variants in specific exons of FBRSL1 in three unrelated children with an overlapping syndromic phenotype with respiratory insufficiency, postnatal growth restriction, microcephaly, global developmental delay and other malformations. The function of FBRSL1 is largely unknown. Interestingly, mutations in the FBRSL1 paralogue AUTS2 lead to an intellectual disability syndrome (AUTS2 syndrome). We determined human FBRSL1 transcripts and describe protein-coding forms by Western blot analysis as well as the cellular localization by immunocytochemistry stainings. All detected mutations affect the two short N-terminal isoforms, which show a ubiquitous expression in fetal tissues. Next, we performed a Fbrsl1 knockdown in Xenopus laevis embryos to explore the role of Fbrsl1 during development and detected craniofacial abnormalities and a disturbance in neurite outgrowth. The aberrant phenotype in Xenopus laevis embryos could be rescued with a human N-terminal isoform, while the long isoform and the N-terminal isoform containing the mutation p.Gln163* isolated from a patient could not rescue the craniofacial defects caused by Fbrsl1 depletion. Based on these data, we propose that the disruption of the validated N-terminal isoforms of FBRSL1 at critical timepoints during embryogenesis leads to a hitherto undescribed complex neurodevelopmental syndrome.

Asunto(s)

Discapacidad Intelectual/genética , Linfocinas/genética , Mutación/genética , Anomalías Múltiples/genética , Adolescente , Animales , Niño , Exones/genética , Humanos , Masculino , Fenotipo , Isoformas de Proteínas/genética , Síndrome , Factores de Transcripción/genética

RESUMEN

PTK7 (protein tyrosine kinase 7) is an evolutionarily conserved transmembrane receptor regulating various processes in embryonic development and tissue homeostasis. On a cellular level PTK7 affects the establishment of cell polarity, the regulation of cell movement and migration as well as cell invasion. The PTK7 receptor has been shown to interact with ligands, co-receptors, and intracellular transducers of Wnt signaling pathways, pointing to a function in the fine-tuning of the Wnt signaling network. Here we will review recent findings implicating PTK7 at the crossroads of Wnt signaling pathways in development and disease.

RESUMEN

Protein tyrosine kinase 7 (PTK7) is an evolutionarily conserved transmembrane receptor with important roles in embryonic development and disease. Originally identified as a gene upregulated in colon cancer, it was later shown to regulate planar cell polarity (PCP) and directional cell movement. PTK7 is a Wnt co-receptor; however, its role in Wnt signaling remains controversial. Here, we find evidence that places PTK7 at the intersection of canonical and non-canonical Wnt signaling pathways. In presence of canonical Wnt ligands PTK7 is subject to caveolin-mediated endocytosis, while it is unaffected by non-canonical Wnt ligands. PTK7 endocytosis is dependent on the presence of the PTK7 co-receptor Fz7 (also known as Fzd7) and results in lysosomal degradation of PTK7. As we previously observed that PTK7 activates non-canonical PCP Wnt signaling but inhibits canonical Wnt signaling, our data suggest a mutual inhibition of canonical and PTK7 Wnt signaling. PTK7 likely suppresses canonical Wnt signaling by binding canonical Wnt ligands thereby preventing their interaction with Wnt receptors that would otherwise support canonical Wnt signaling. Conversely, if canonical Wnt proteins interact with the PTK7 receptor, they induce its internalization and degradation.

Asunto(s)

Caveolina 1/genética , Moléculas de Adhesión Celular/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Wnt/genética , Vía de Señalización Wnt , Proteína Wnt3A/genética , Animales , Caveolina 1/metabolismo , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Clatrina/genética , Clatrina/metabolismo , Embrión no Mamífero , Endocitosis , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ligandos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Células MCF-7 , Unión Proteica , Estabilidad Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3A/metabolismo , Xenopus laevis , beta Catenina/genética , beta Catenina/metabolismo

RESUMEN

Photosynthetic eukaryotes are challenged by a fluctuating light supply, demanding for a modulated expression of nucleus-encoded light-harvesting proteins associated with photosystem II (LHCII) to adjust light-harvesting capacity to the prevailing light conditions. Here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. In the green unicellular alga Chlamydomonas reinhardtii, the cytosolic RNA-binding protein NAB1 represses translation of certain LHCII isoform mRNAs. Specific nitrosylation of Cys-226 decreases NAB1 activity and could be demonstrated in vitro and in vivo. The less active, nitrosylated form of NAB1 is found in cells acclimated to limiting light supply, which permits accumulation of light-harvesting proteins and efficient light capture. In contrast, elevated light supply causes its denitrosylation, thereby activating the repression of light-harvesting protein synthesis, which is needed to control excitation pressure at photosystem II. Denitrosylation of recombinant NAB1 is efficiently performed by the cytosolic thioredoxin system in vitro. To our knowledge, NAB1 is the first example of stimulus-induced denitrosylation in the context of photosynthetic acclimation. By identifying this novel redox cross-talk pathway between chloroplast and cytosol, we add a new key element required for drawing a precise blue print of the regulatory network of light harvesting.

Asunto(s)

Proteínas Algáceas/metabolismo , Chlamydomonas/fisiología , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/genética , Núcleo Celular/metabolismo , Chlamydomonas/efectos de la radiación , Cisteína/metabolismo , Citosol/metabolismo , Luz , Complejos de Proteína Captadores de Luz/efectos de la radiación , Modelos Moleculares , Oxidación-Reducción , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema II/efectos de la radiación , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Tiorredoxinas/metabolismo , Tilacoides/metabolismo

RESUMEN

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.

Asunto(s)

Movimiento Celular , Cresta Neural/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Cresta Neural/citología , Cresta Neural/embriología , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Vía de Señalización Wnt , Xenopus , Proteínas de Xenopus/genética

RESUMEN

The unicellular green alga Chlamydomonas reinhardtii is capable of using organic and inorganic carbon sources simultaneously, which requires the adjustment of photosynthetic activity to the prevailing mode of carbon assimilation. We obtained novel insights into the regulation of light-harvesting at photosystem II (PSII) following altered carbon source availability. In C. reinhardtii, synthesis of PSII-associated light-harvesting proteins (LHCBMs) is controlled by the cytosolic RNA-binding protein NAB1, which represses translation of particular LHCBM isoform transcripts. This mechanism is fine-tuned via regulation of the nuclear NAB1 promoter, which is activated when linear photosynthetic electron flow is restricted by CO(2)-limitation in a photoheterotrophic context. In the wild-type, accumulation of NAB1 reduces the functional PSII antenna size, thus preventing a harmful overexcited state of PSII, as observed in a NAB1-less mutant. We further demonstrate that translation control as a newly identified long-term response to prolonged CO(2)-limitation replaces LHCII state transitions as a fast response to PSII over-excitation. Intriguingly, activation of the long-term response is perturbed in state transition mutant stt7, suggesting a regulatory link between the long- and short-term response. We depict a regulatory circuit operating on distinct timescales and in different cellular compartments to fine-tune light-harvesting in photoheterotrophic eukaryotes.

Asunto(s)

Carbono/metabolismo , Chlamydomonas reinhardtii/fisiología , Chlamydomonas reinhardtii/efectos de la radiación , Luz , Fotosíntesis/efectos de la radiación , Aclimatación/efectos de los fármacos , Aclimatación/efectos de la radiación , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Dióxido de Carbono/metabolismo , Dióxido de Carbono/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/crecimiento & desarrollo , Complejos de Proteína Captadores de Luz/metabolismo , Modelos Biológicos , Fotosíntesis/efectos de los fármacos , Complejo de Proteína del Fotosistema II/metabolismo , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/efectos de la radiación , Transcripción Genética/efectos de los fármacos , Transcripción Genética/efectos de la radiación

RESUMEN

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor.

Asunto(s)

Proteínas Portadoras/metabolismo , Chlamydomonas reinhardtii/metabolismo , Hielo , Lolium/enzimología , Proteínas de Plantas/metabolismo , Dióxido de Carbono/metabolismo , Proteínas Portadoras/genética , Chlamydomonas reinhardtii/genética , Medios de Cultivo/química , Medios de Cultivo/economía , Luz , Lolium/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Microalgae are diverse photosynthetic microbes which offer the potential for production of a number of high value products (HVP) such as pigments, oils, and bio-active compounds. Fast growth rates, ease of photo-autotrophic cultivation, unique metabolic properties and continuing progress in algal transgenics have raised interest in the use of microalgae systems for recombinant protein (RP) production. This work demonstrates the development of an advanced RP production and secretion system for the green unicellular model alga Chlamydomonas reinhardtii. We generated a versatile expression vector that employs the secretion signal of the native extracellular C. reinhardtii carbonic anhydrase for efficient RP secretion into the culture medium. Unique restriction sites were placed between the regulatory elements to allow fast and easy sub-cloning of sequences of interest. Positive transformants can rapidly be identified by high-throughput plate-level screens via a coupled Gaussia luciferase marker. The vector was tested in Chlamydomonas wild type CC-1883 (WT) and in the transgene expression transformant UVM4. Compared to the native secretion signal of the Gaussia luciferase, up to 84% higher RP production could be achieved. With this new expression system we could generate transformants that express up to 10 mg RP per liter culture without further optimization. The target RP is found exclusively in culture medium and can therefore easily be isolated and purified. We conclude that this new expression system will be a valuable tool for many heterologous protein expression applications from C. reinhardtii in the future.

Asunto(s)

Núcleo Celular/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transgenes , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Núcleo Celular/metabolismo , Clonación Molecular/métodos , Expresión Génica , Vectores Genéticos , Luciferasas/biosíntesis , Luciferasas/genética , Luciferasas/metabolismo