RESUMEN
The Daam1 protein regulates Wnt-induced cytoskeletal changes during vertebrate gastrulation though its full mode of action and binding partners remain unresolved. Here we identify Reversion Induced LIM domain protein (RIL) as a new interacting protein of Daam1. Interaction studies uncover binding of RIL to the C-terminal actin-nucleating portion of Daam1 in a Wnt-responsive manner. Immunofluorescence studies showed subcellular localization of RIL to actin fibers and co-localization with Daam1 at the plasma membrane. RIL gain- and loss-of-function approaches in Xenopus produced severe gastrulation defects in injected embryos. Additionally, a simultaneous loss of Daam1 and RIL synergized to produce severe gastrulation defects indicating RIL and Daam1 may function in the same signaling pathway. RIL further synergizes with another novel Daam1-interacting protein, Formin Binding Protein 1 (FNBP1), to regulate gastrulation. Our studies altogether show RIL mediates Daam1-regulated non-canonical Wnt signaling that is required for vertebrate gastrulation.
Asunto(s)
Citoesqueleto de Actina , Gastrulación , Proteínas de Microfilamentos , Vía de Señalización Wnt , Proteínas de Xenopus , Xenopus laevis , Animales , Femenino , Humanos , Ratas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Unión Proteica , Vía de Señalización Wnt/fisiología , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMEN
The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the characterization of Formin Binding Protein 1 (FNBP1) as a binding partner of Daam1. The interaction of Daam1 with FNBP1 and its domains required for this interaction were delineated. Immunofluorescence studies showed FNBP1 co-localizes with Daam1, and is an integral component of the actin cytoskeletal complex that is responsive to Wnt stimulation. Specifically, FNBP1 can induce intracellular tubule-like structures and localize to focal adhesions suggesting a role for FNBP1 in cell migration. Functional FNBP1 studies in Xenopus embryos uncover a critical role for FNBP1 in regulating vertebrate gastrulation. Additionally, suboptimal doses of Daam1 and FNBP1 synergize to produce severe gastrulation defects, indicating FNBP1 and Daam1 may function within the same signaling pathway. These results together show FNBP1 is an integral component of Daam1-regulated non-canonical Wnt signaling required for vertebrate gastrulation.
Asunto(s)
Gastrulación , Vía de Señalización Wnt , Proteínas de Xenopus , Xenopus laevis , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Movimiento Celular , Embrión no Mamífero/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Unión Proteica , Proteínas de Unión al GTP rho , Vía de Señalización Wnt/fisiología , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , FemeninoRESUMEN
Even after idiopathic macular hole (MH) surgery and with successful closure of MH, aniseikonia is a common postoperative symptom. We investigated the correlation of MH diameter, retinal displacement and retinal layer thicknesses with aniseikonia in 41 eyes of 41 patients undergoing MH surgery with internal limiting membrane peeling. Aniseikonia was measured with the New Aniseikonia Test. Retinal displacement (RD%) was defined as change of retinal distance between the temporal margin of the optic papilla and the intersection of the retinal vessels. Changes of thicknesses of the inner nuclear layer (INL%) and the outer retinal layer (OR%) were calculated. Aniseikonia improved postoperatively. Preoperative aniseikonia and their improvement at 6 months correlated with MH diameters (P = 0.004-0.046). Improvement of aniseikonia correlated with temporal RD% (P = 0.002-0.012). Improvement of vertical aniseikonia correlated with INL% at 2 weeks and with the nasal OR% at 1, 3, and 6 months (P = < 0.001-0.028). MH diameter and age were significant predictors for improvement of aniseikonia. The greater the temporal retina displacement, and the thinner the postoperative INL and OR, the greater the improvement of aniseikonia. MH diameter and age are strong predictors for improvement of aniseikonia after MH surgery.
Asunto(s)
Aniseiconia , Membrana Epirretinal , Perforaciones de la Retina , Humanos , Perforaciones de la Retina/cirugía , Perforaciones de la Retina/diagnóstico , Aniseiconia/cirugía , Membrana Epirretinal/cirugía , Agudeza Visual , Vitrectomía , Tomografía de Coherencia Óptica , Retina/cirugía , Estudios RetrospectivosRESUMEN
Metamorphopsia is an important visual symptom of macular disease. We determined predictors for metamorphopsia investigating the relationships of macular hole (MH) diameter and retinal layer thicknesses with metamorphopsia after MH surgery. Forty-two eyes of 42 consecutive patients undergoing MH surgery were retrospectively studied. Metamorphopsia was measured with M-CHARTS. Inner nuclear layer (INL) and outer retinal layer (OR) thicknesses were measured 1000 µm away from central fovea at using Spectralis. Preoperative M-CHARTS scores correlated with MH diameters (P = 0.007-0.031) and changes of temporal OR thickness (P = 0.008-0.010). Postoperative M-CHARTS score at 3 months correlated with preoperative nasal and inferior OR thicknesses (P = 0.003 and 0.016) and with changes of superior INL at 3 and 6 months (P = 0.011 and 0.025), and score at 1 month with change of temporal OR at 6 months (P = 0.033). Postoperative improvement of M-CHARTS scores correlated with changes of temporal INL and superior OR (P = 0.026 and 0.002). Multiple regression analysis revealed that MH diameter was a significant predictor for metamorphopsia. Photoreceptor displacement and inner retinal change may generate metamorphopsia in MH undergoing surgery, however MH diameter is the most powerful predictor.
Asunto(s)
Perforaciones de la Retina , Humanos , Perforaciones de la Retina/cirugía , Estudios Retrospectivos , Agudeza Visual , Vitrectomía/efectos adversos , Tomografía de Coherencia Óptica , Trastornos de la Visión/diagnósticoRESUMEN
PURPOSE: To evaluate the outer retinal microstructure and visual function after idiopathic macular hole (MH) surgery using internal limiting membrane (ILM) peeling with and without Brilliant Blue G (BBG) staining. STUDY DESIGN: Retrospective, consecutive case series. METHODS: A total of 49 eyes of 47 patients were enrolled: 23 eyes of 23 patients with MH who underwent ILM peeling without dyes (control group) and 26 eyes of 26 patients who underwent BBG staining (BBG group). The lengths of defects of the photoreceptor ellipsoid zone (EZ), external limiting membrane (ELM), and interdigitation zone (IZ) were measured. RESULTS: The rate of MH closure after initial surgery was 95.6% (22/23 eyes) for the control group versus 100% (26/26 eyes) for the BBG group. In the 48 eyes with MH closure, the recovery rate of ELM deficiency and change in IZ deficiency showed no difference between the groups. The changes in EZ deficiency at 1 and 12 months were greater in the BBG group than in the control group. (P = 0.047 and 0.031). Visual acuity was better in the BBG group than in the control group during 12 months postoperatively (P < 0.001-0.038). CONCLUSION: Eyes undergoing BBG-assisted MH surgery achieved faster recovery of the outer retinal structures and greater visual improvement than those of eyes without BBG.
Asunto(s)
Perforaciones de la Retina , Humanos , Perforaciones de la Retina/diagnóstico , Perforaciones de la Retina/cirugía , Vitrectomía , Estudios Retrospectivos , Colorantes de Rosanilina , Membrana Basal/cirugía , Tomografía de Coherencia ÓpticaRESUMEN
PURPOSE: To investigate the correlations of thickness of three retinal layers with retinal displacement after idiopathic macular hole surgery. STUDY DESIGN: Retrospective, consecutive, case series. METHODS: 42 eyes of 42 patients undergoing macular hole surgery with internal limiting membrane peeling were studied. Retinal distance was measured with near-infrared images between the optic nerve and the intersection of retinal vessels at four quadrants. Retinal thicknesses of inner retinal layer, inner nuclear layer and outer retinal layer were measured 1000 µm away from the central fovea using Spectralis. RESULTS: Retinal distances other than the nasal quadrant decreased postoperatively (p < 0.001). Retinal displacement (%) correlated significantly with the change in inner nuclear layer thickness in the temporal sector at 1, 3, and 6 months, in the superior sector at 2 weeks, 1, and 6 months, and in the inferior sector at 3 and 6 months postoperatively (r = 0.319-0.570, p < 0.001-0.040), but not in the inner or outer retinal layers. CONCLUSION: Internal limiting membrane peeling for macular hole enhances retinal displacement toward the optic disc, whose distances correlate with the changes in inner nuclear layer thickness.
Asunto(s)
Membrana Epirretinal , Perforaciones de la Retina , Membrana Epirretinal/cirugía , Humanos , Perforaciones de la Retina/diagnóstico , Perforaciones de la Retina/cirugía , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodos , Vitrectomía/métodosRESUMEN
Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel's pore. Knockout (KO) of TRPM7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg2+ efflux activities that contribute to CNNMs' control of cellular Mg2+ homeostasis.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPM/metabolismo , Proteínas de Transporte de Catión/fisiología , Cationes Bivalentes/metabolismo , Línea Celular Tumoral , Ciclinas/fisiología , Células HEK293 , Humanos , Magnesio/metabolismo , Técnicas de Placa-Clamp , Proteínas Serina-Treonina Quinasas/fisiología , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/fisiologíaRESUMEN
PURPOSE: Vital dyes are frequently used to visualize the internal limiting membrane (ILM) of the neuroretina. This study evaluated and compared the microstructure of the inner retina and visual function with and without brilliant blue G (BBG) staining for ILM peeling during idiopathic epiretinal membrane (ERM) surgery. STUDY DESIGN: Retrospective, consecutive, interventional case series. METHODS: Fifty-five patients (55 eyes) with ERM underwent ILM peeling without dyes (non-dye group) and 55 patients (55 eyes) underwent ILM peeling with BBG staining (BBG group). The logMAR visual acuity (VA) and ganglion cell complex (GCC) thickness were measured using optical coherence tomography at baseline and 12 months after surgery. RESULTS: LogMAR VA improved significantly in both groups at 12 months and the BBG group tended to be better than the non-dye group but with no significant difference between the groups (unpaired t-test, P = 0.490). The average GCC thickness significantly decreased in both groups; however, there was no difference in the rates of change in GCC thickness between the groups. The ratio of GCC thickness to total retinal thickness (%) was significantly higher in the BBG group in the superior quadrant at 12 months postoperatively (P = 0.010). CONCLUSION: BBG-assisted ERM surgery resulted in better visual improvement and fewer structural changes in the inner retinal layers. BBG-assisted ILM peeling is safe both functionally and anatomically.
Asunto(s)
Membrana Epirretinal , Membrana Basal/cirugía , Membrana Epirretinal/diagnóstico , Membrana Epirretinal/cirugía , Humanos , Retina/diagnóstico por imagen , Estudios Retrospectivos , Colorantes de Rosanilina , Tomografía de Coherencia Óptica , VitrectomíaRESUMEN
The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.
Asunto(s)
Proteínas de Microfilamentos/genética , Placentación/genética , Proteínas de Unión al GTP rho/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Portadoras/metabolismo , Polaridad Celular , Citoesqueleto/metabolismo , Desarrollo Embrionario , Femenino , Forminas/genética , Forminas/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones/embriología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Placenta/embriología , Embarazo , Vía de Señalización Wnt , Proteínas de Unión al GTP rho/metabolismoRESUMEN
A common theme in organogenesis is how the final structure of organs emerge from epithelial tube structures, with the formation of the neural tube being one of the best examples. Two types of cell movements co-occur during neural tube closure involving the migration of cells toward the midline of the embryo (mediolateral intercalation or convergent extension) as well as the deep movement of cells from inside the embryo to the outside of the lateral side of the neural plate (radial intercalation). Failure of either type of cell movement will prevent neural tube closure, which can produce a range of neural tube defects (NTDs), a common congenital disease in humans. Numerous studies have identified signaling pathways that regulate mediolateral intercalation during neural tube closure. Less understood are the pathways that govern radial intercalation. Using the Xenopus laevis system, our group reported the identification of transient receptor potential (TRP) channels, TRPM6 and TRPM7, and the Mg2+ ion they conduct, as novel and key factors regulating both mediolateral and radial intercalation during neural tube closure. Here we broadly discuss tubulogenesis and cell intercalation from the perspective of neural tube closure and the respective roles of TRPM7 and TRPM6 in this critical embryonic process.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Magnesio/química , Proteínas Serina-Treonina Quinasas/fisiología , Canales Catiónicos TRPM/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/metabolismo , Células 3T3 , Animales , Movimiento Celular , Desarrollo Embrionario , Humanos , Iones , Magnesio/metabolismo , Ratones , Placa Neural/metabolismo , Tubo Neural/metabolismo , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo , Neurulación , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Proteínas de Xenopus/genética , Pez CebraRESUMEN
PURPOSE: To determine the relationship between the subfoveal choroidal thickness (SCT) and intraocular pressure (IOP) following 25-gauge (25G) and 23-gauge (23G) vitrectomy for idiopathic epiretinal membrane (ERM). STUDY DESIGN: Retrospective, consecutive, interventional case series. METHODS: Sixty-two patients undergoing 25G vitrectomy and 56 patients undergoing 23G vitrectomy for ERM participated. SCT was measured using enhanced depth imaging optical coherence tomography and IOP were measured both at baseline and postoperatively. RESULTS: In both groups, the IOPs on day one and one week after surgery were significantly lower than at baseline (P < 0.001 for both). The rates of changes of IOP were significantly greater in 23G compared to 25G on day one (P = 0.026). In 23G the SCTs on day one and one week after surgery were significantly thicker (P < 0.001) than baseline. The rates of changes in SCT between baseline and day one negatively correlated with those of IOP in 23G (r = -0.559, P < 0.001) but no correlation was observed with 25G (r = -0.129, P = 0.316). CONCLUSION: Choroidal thickness increases soon after 23G vitrectomy for ERM which is probably due to the transient hypotony, however, early SCT change does not appear in 25G vitrectomy. Twenty-five-gauge vitrectomy may have an advantage in minimizing postoperative choroidal changes.
Asunto(s)
Coroides/patología , Membrana Epirretinal/cirugía , Presión Intraocular/fisiología , Vitrectomía , Anciano , Anciano de 80 o más Años , Coroides/diagnóstico por imagen , Membrana Epirretinal/fisiopatología , Femenino , Fóvea Central , Humanos , Masculino , Persona de Mediana Edad , Tamaño de los Órganos , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Tonometría Ocular , Agudeza Visual/fisiologíaRESUMEN
In humans, germline mutations in Trpm6 cause autosomal dominant hypomagnesemia with secondary hypocalcemia disorder. Loss of Trpm6 in mice also perturbs cellular magnesium homeostasis but additionally results in early embryonic lethality and neural tube closure defects. To define the mechanisms by which TRPM6 influences neural tube closure, we functionally characterized the role of TRPM6 during early embryogenesis in Xenopus laevis. The expression of Xenopus TRPM6 (XTRPM6) is elevated at the onset of gastrulation and is concentrated in the lateral mesoderm and ectoderm at the neurula stage. Loss of XTRPM6 produced gastrulation and neural tube closure defects. Unlike XTRPM6's close homologue XTRPM7, whose loss interferes with mediolateral intercalation, depletion of XTRPM6 but not XTRPM7 disrupted radial intercalation cell movements. A zinc-influx assay demonstrated that TRPM6 has the potential to constitute functional channels in the absence of TRPM7. The results of our study indicate that XTRPM6 regulates radial intercalation with little or no contribution from XTRPM7 in the region lateral to the neural plate, whereas XTRPM7 is mainly involved in regulating mediolateral intercalation in the medial region of the neural plate. We conclude that both TRPM6 and TRPM7 channels function cooperatively but have distinct and essential roles during neural tube closure.
Asunto(s)
Desarrollo Embrionario/genética , Placa Neural/crecimiento & desarrollo , Tubo Neural/crecimiento & desarrollo , Canales Catiónicos TRPM/genética , Proteínas de Xenopus/genética , Animales , Calcio/metabolismo , Movimiento Celular/genética , Regulación del Desarrollo de la Expresión Génica , Mutación de Línea Germinal/genética , Humanos , Hipercalciuria/genética , Hipercalciuria/metabolismo , Hipercalciuria/patología , Hipocalcemia/genética , Hipocalcemia/metabolismo , Hipocalcemia/patología , Magnesio/metabolismo , Nefrocalcinosis/genética , Nefrocalcinosis/metabolismo , Nefrocalcinosis/patología , Placa Neural/metabolismo , Defectos Congénitos del Transporte Tubular Renal/genética , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Defectos Congénitos del Transporte Tubular Renal/patología , Xenopus laevisRESUMEN
Mutations in protein kinase C substrate 80K-H (PRKCSH), which encodes for an 80 KDa protein named hepatocystin (80K-H, PRKCSH), gives rise to polycystic liver disease (PCLD). Hepatocystin functions as the noncatalytic beta subunit of Glucosidase II, an endoplasmic reticulum (ER)-resident enzyme involved in processing and quality control of newly synthesized glycoproteins. Patients harboring heterozygous germline mutations in PRKCSH are thought to develop renal cysts as a result of somatic loss of the second allele, which subsequently interferes with expression of the TRP channel polycystin-2 (PKD2). Deletion of both alleles of PRKCSH in mice results in embryonic lethality before embryonic day E11.5. Here, we investigated the function of hepatocystin during Xenopus laevis embryogenesis and identified hepatocystin as a binding partner of the TRPM7 ion channel, whose function is required for vertebrate gastrulation. We find that TRPM7 functions synergistically with hepatocystin. Although other N-glycosylated proteins are critical to early development, overexpression of TRPM7 in Xenopus laevis embryos was sufficient to fully rescue the gastrulation defect caused by loss of hepatocystin. We observed that depletion of hepatocystin in Xenopus laevis embryos decreased TRPM7 expression, indicating that the early embryonic lethality caused by loss of hepatocystin is mainly due to impairment of TRPM7 protein expression.
Asunto(s)
Embrión no Mamífero/embriología , Desarrollo Embrionario/fisiología , Gástrula/embriología , Glucosidasas/metabolismo , Canales Catiónicos TRPM/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Línea Celular , Glucosidasas/genética , Humanos , Ratones , Canales Catiónicos TRPM/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Magnesium (Mg(2+)) is the second most abundant cellular cation and is essential for all stages of life, from the early embryo to adult. Mg(2+) deficiency causes or contributes to many human diseases, including migraine headaches, Parkinson's disease, Alzheimer's disease, hypotension, type 2 diabetes mellitus and cardiac arrhythmias. Although the concentration of Mg(2+) in the extracellular environment can vary significantly, the total intracellular Mg(2+) concentration is actively maintained within a relatively narrow range (14 - 20 mM) via tight, yet poorly understood, regulation of intracellular Mg(2+)by Mg(2+) transporters and Mg(2+)-permeant ion channels. Recent studies have continued to add to the growing number of Mg(2+) transporters and ion channels involved in Mg(2+) homeostasis, including TRPM6 and TRPM7, members of the transient receptor potential (TRP) ion channel family. Mutations in TRPM6, including amino acid substitutions that prevent its heterooligomerization with TRPM7, occur in the rare autosomal-recessive disease hypomagnesemia with secondary hypocalcemia (HSH). Genetic ablation of either gene in mice results in early embryonic lethality, raising the question of whether these channels' capacity to mediate Mg(2+) influx plays an important role in embryonic development. Here we review what is known of the function of Mg(2+) in early development and summarize recent findings regarding the function of the TRPM6 and TRPM7 ion channels during embryogenesis.
Asunto(s)
Desarrollo Embrionario/fisiología , Magnesio/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Ratones , Ratones NoqueadosRESUMEN
Biopolymer-ceramic composites are thought to be particularly promising materials for bone tissue engineering as they more closely mimic natural bone. Here, we demonstrate the fabrication by electrospinning of fibrous chitosan-hydroxyapatite composite scaffolds with low (1 wt %) and high (10 wt %) mineral contents. Scanning electron microscopy (FESEM), energy dispersive spectroscopy (EDS) and unidirectional tensile testing were performed to determine fiber surface morphology, elemental composition, and tensile Young's modulus (E) and ultimate tensile strength (σUTS ), respectively. EDS scans of the scaffolds indicated that the fibers, crosslinked with either hexamethylene-1,6-diaminocarboxysulfonate (HDACS) or genipin, have a crystalline hydroxyapatite mineral content at 10 wt % additive. Moreover, FESEM micrographs showed that all electrospun fibers have diameters (122-249 nm), which fall within the range of those of fibrous collagen found in the extracellular matrix of bone. Young's modulus and ultimate tensile strength of the various crosslinked composite compositions were in the range of 116-329 MPa and 2-15 MPa, respectively. Osteocytes seeded onto the mineralized fibers were able to demonstrate good biocompatibility enhancing the potential use for this material in future bone tissue engineering applications.
Asunto(s)
Quitosano/química , Reactivos de Enlaces Cruzados/química , Ensayo de Materiales , Osteocitos/metabolismo , Animales , Línea Celular , Módulo de Elasticidad , Ratones , Osteocitos/citologíaRESUMEN
Chitosan is a naturally occurring polysaccharide, which has proven to be an attractive candidate for bone tissue engineering, due to its ability to promote osteoblast mineralization. Electrospinning has become a well-established cell scaffold processing technique, as it produces a high surface area to volume fibrous material that can mimic the three dimensionality of the extracellular matrix of a cell. In this study, we have investigated the osteoblast response to two different chemically crosslinked (hexamethylene-1,6-diaminocarboxysulfonate (HDACS) and genipin) electrospun chitosan scaffolds and their film counterparts in order to determine how material chemistry influences cellular behavior in conjunction with material topology. In addition, material properties of each fiber scaffold such as porosity and tensile strength were considered. MLO-A5 osteoblast cells grown on chitosan-HDACS scaffolds were found to display a more organized cellular network, along with significantly more filopodia extensions, compared to those grown on chitosan-genipin scaffolds. After 2 days of growth on chitosan-HDACS fibers, a higher level of alkaline phosphatase expression in MLO-A5 cells was reported compared to that of either chitosan-genipin fibers or films. These results indicate that not only chemistry, but also surface topology is an important effecter of cellular behavior. Ultimately, chitosan-HDACS fiber scaffolds provided an adequate substrate for osteoblast attachment and proliferation.
Asunto(s)
Materiales Biocompatibles/química , Quitosano/química , Osteoblastos/citología , Andamios del Tejido/química , Fosfatasa Alcalina/metabolismo , Animales , Sustitutos de Huesos/química , Adhesión Celular , Línea Celular , Proliferación Celular , Supervivencia Celular , Reactivos de Enlaces Cruzados/química , Diaminas/química , Ensayo de Materiales , Ratones , Microscopía Electrónica de Rastreo , Osteoblastos/metabolismo , Ingeniería de TejidosRESUMEN
ß-Catenin is a central effector of the Wnt pathway and one of the players in Ca(+)-dependent cell-cell adhesion. While many wnts are present and expressed in vertebrates, only one ß-catenin exists in the majority of the organisms. One intriguing exception is zebrafish that carries two genes for ß-catenin. The maternal recessive mutation ichabod presents very low levels of ß-catenin2 that in turn affects dorsal axis formation, suggesting that ß-catenin1 is incapable to compensate for ß-catenin2 loss and raising the question of whether these two ß-catenins may have differential roles during early axis specification. Here we identify a specific antibody that can discriminate selectively for ß-catenin1. By confocal co-immunofluorescent analysis and low concentration gain-of-function experiments, we show that ß-catenin1 and 2 behave in similar modes in dorsal axis induction and cellular localization. Surprisingly, we also found that in the ich embryo the mRNAs of the components of ß-catenin regulatory pathway, including ß-catenin1, are more abundant than in the Wt embryo. Increased levels of ß-catenin1 are found at the membrane level but not in the nuclei till high stage. Finally, we present evidence that ß-catenin1 cannot revert the ich phenotype because it may be under the control of a GSK3ß-independent mechanism that required Axin's RGS domain function.
Asunto(s)
Proteína Axina/metabolismo , Mutación/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Animales , Especificidad de Anticuerpos , Proteína Axina/genética , Blástula/efectos de los fármacos , Blástula/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Dominantes , Inmunohistoquímica , Cloruro de Litio/farmacología , Fenotipo , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , beta Catenina/metabolismoRESUMEN
Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of ß-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited ß-catenin/TCF-dependent transcription. Custos binds to ß-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of ß-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-ß-catenin signal transduction cascade.