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1.
Elife ; 112022 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-35762204

RESUMEN

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.


Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.


Asunto(s)
Espectrina , Tubulina (Proteína) , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Espectrina/metabolismo , Tubulina (Proteína)/metabolismo
2.
Arch Histol Cytol ; 73(2): 65-72, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-21566332

RESUMEN

The fluorescent dye PKH26, which binds mainly to the cell membrane, has long stability that enables the tracing of PKH26-labeled transplanted cells in host tissue. In the present study, we examined whether this fluorescent dye works as a retrograde or anterograde tracer to label neural networks within the central nervous system of adult and postnatal day 3 (P3) mice. A small injection of the dye into the medullospinal junction resulted in the retrograde labeling of corticospinal tract (CST) neurons in layer V of the sensory-motor cortex both in the adult mice and pups. Injection of the dye into the motor cortex of the P3 pups resulted in the anterograde labeling of CST fibers at a single fiber resolution level, although a similar injection of the dye into the motor cortex of the adult mice failed to stain CST fibers anterogradely. These results suggest that, while PKH26 works as a retrograde or anterograde tracer, anterograde labeling of the adult tracts can not be expected.


Asunto(s)
Colorantes Fluorescentes/metabolismo , Compuestos Orgánicos/metabolismo , Animales , Axones/metabolismo , Fluorescencia , Inyecciones , Ratones , Ratones Endogámicos C57BL , Corteza Motora/citología , Corteza Motora/metabolismo , Tractos Piramidales/citología , Tractos Piramidales/metabolismo , Coloración y Etiquetado
3.
Mol Cell Biol ; 25(6): 2191-9, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15743817

RESUMEN

Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.


Asunto(s)
Válvula Aórtica/anomalías , Válvula Aórtica/embriología , Válvula Pulmonar/anomalías , Válvula Pulmonar/embriología , Fosfolipasas de Tipo C/fisiología , Alelos , Animales , Válvula Aórtica/inmunología , Defecto del Tabique Aortopulmonar/genética , Proteínas Morfogenéticas Óseas/fisiología , Cardiomiopatía Dilatada/etiología , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Receptores ErbB/deficiencia , Receptores ErbB/genética , Receptores ErbB/fisiología , Marcación de Gen , Enfermedades de las Válvulas Cardíacas/complicaciones , Enfermedades de las Válvulas Cardíacas/genética , Ventrículos Cardíacos/patología , Ratones , Ratones Mutantes , Mutación/genética , Fosfoinositido Fosfolipasa C , Fosfoproteínas/análisis , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Válvula Pulmonar/inmunología , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas Smad , Proteína Smad1 , Proteína Smad5 , Proteína Smad8 , Transactivadores/análisis , Transactivadores/genética , Transactivadores/metabolismo , Fosfolipasas de Tipo C/análisis , Fosfolipasas de Tipo C/genética
4.
Brain Res ; 1140: 205-15, 2007 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-17173877

RESUMEN

The causative gene for the reeler mouse is reelin which encodes Reelin protein, an extracellular molecule. In the present study, we have examined the cytoarchitecture, myeloarchitecture, and afferent/efferent systems of the superior colliculus (SC) of the reeler mouse. In the reeler, the laminar structures of the superficial three layers of the SC were disorganized and intermingled into a single layer, i.e., the superficial fused layer (SuF), as previously reported in the reelin-deficient SRK rat (Sakakibara et al., Develop. Brain Res. 141:1-13). Next, we have investigated the course and terminals of visual corticotectal and retinotectal projections with an injection of biocytin into the visual cortex or an injection of cholera toxin subunit B into the retina, respectively. In the reeler, anterogradely labeled visual corticotectal and retinotectal fibers took an aberrant course within the SuF, resulting in abnormal myeloarchitecture of the superficial SC of the reeler. Retrograde labeling of tectospinal tract neurons could not show any differences between the normal and reeler mice, suggesting that the deep layers of the reeler SC are cytoarchitectually normal. In situ hybridization and immunohistochemical studies have shown that reelin mRNA and Reelin protein were both recognized in the normal SC. These results suggest that Reelin protein plays some roles in histogenesis of the superficial layers of the SC.


Asunto(s)
Ratones Mutantes Neurológicos/anatomía & histología , Proteína Básica de Mielina/metabolismo , Colículos Superiores/metabolismo , Colículos Superiores/ultraestructura , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Toxina del Cólera/metabolismo , Vías Eferentes/metabolismo , Vías Eferentes/ultraestructura , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Hibridación in Situ/métodos , Lisina/análogos & derivados , Lisina/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteína Reelina , Retina/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Corteza Visual/metabolismo , Vías Visuales/metabolismo , Vías Visuales/ultraestructura
5.
Acta Otolaryngol ; 127(2): 213-20, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17364355

RESUMEN

CONCLUSIONS: The present study confirmed that ambiguus motoneurons innervating intrinsic laryngeal and esophageal muscles are radially malpositioned in the brainstem of the Shaking Rat Kawasaki (SRK), a reelin-deficient mutant rat. OBJECTIVES: Ambiguus motoneurons innervating the striated muscles of the larynx and esophagus take a long migration from their original birth plate in the floor of the fourth ventricle to their final settlement in the ventral margin of the medulla oblongata. To examine whether the migration of ambiguus nucleus neurons is affected in SRK, we studied localization of ambiguus motoneurons of postnatal day 21 (P21) normal and SRK rats. MATERIALS AND METHODS: To label ambiguus motoneurons retrogradely, horseradish peroxidase (HRP) was injected into some laryngeal muscles including cricothyroid, thyroarytenoid and posterior cricoarytenoid muscles, and the cervical and abdominal esophageal muscles of the SRK and normal controls 2 days before sacrifice. RESULTS: In the P21 normal rat, HRP-positive laryngeal and esophageal motoneurons were found in the nucleus ambiguus, whereas in the P21 SRK, they were scattered from the base of the fourth ventricle to the ventro-lateral margin of the medulla, suggesting that radial migration of ambiguus motoneurons from their birthplace to their final settlement is guided by Reelin protein.


Asunto(s)
Esófago/inervación , Músculos Laríngeos/inervación , Neuronas Motoras/patología , Músculo Esquelético/inervación , Animales , Movimiento Celular , Peroxidasa de Rábano Silvestre , Fenotipo , Ratas , Ratas Mutantes , Proteína Reelina
6.
Brain Res Dev Brain Res ; 159(2): 127-34, 2005 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-16139369

RESUMEN

The cytoarchitecture of dorsal cochlear nucleus (DCN), characterized by a distinct laminar structure similar to the cerebellar cortex of the normal mouse, is known to be disrupted in the Reelin-deficient mouse, reeler. Here, we have reexamined both the cytoarchitecture and myeloarchitecture of this nucleus and described expression pattern of Reelin protein during perinatal periods. Reelin-immunopositive granule cells were firstly recognized in the external granular layer of the DCN at embryological day 16 (E16). Next, we examined the cytoarchitecture of the DCN of the normal and reeler mice with Ca2+/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) immunostaining. CaMKIIalpha-immunoreactive cartwheel cells were laminarly distributed in the layer II of the normal DCN, but scattered throughout the reeler DCN. Injection of retrograde tracer, Fluoro-Gold (FG) into the inferior colliculus of the reeler mouse resulted in that retrogradely labeled neurons in the DCN were radially scattered instead of being confined to a single layer as seen in the normal mouse. To examine whether CaMKIIalpha-immunopositive cartwheel cells are neurons projecting to the inferior colliculus or not, double labeling with CaMKIIalpha immunohistochemistry and retrograde labeling with an injection of FG into the inferior colliculus were made, which revealed that CaMKIIalpha-immunoreactive cartwheel cells do not send axons to the inferior colliculus. The present findings imply that Reelin may have some roles in the formation of laminar structures of the DCN.


Asunto(s)
Vías Auditivas/citología , Moléculas de Adhesión Celular Neuronal/biosíntesis , Núcleo Coclear/citología , Núcleo Coclear/metabolismo , Núcleo Coclear/patología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Serina Endopeptidasas/biosíntesis , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Mutantes , Mutación , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , ARN Mensajero/análisis , Proteína Reelina , Serina Endopeptidasas/genética
7.
Kaibogaku Zasshi ; 79(1): 15-23, 2004 Mar.
Artículo en Japonés | MEDLINE | ID: mdl-15101173

RESUMEN

Dissection of the brachial plexus is an important part in the anatomical course, but it is difficult for medical students to identify individual nerves of the brachial plexus due to its complexity and numerous variations. We have recently adopted the Grant method (1991) to guide students in the successful identification of this plexus. However, according to the Grant method the part of the upper limb including the brachial plexus is dissected before the neck part, which makes it impossible to identify the roots, trunks, and cords of the brachial plexus, and to identify the nerve branches extending from the brachial plexus. Here, we propose of anatomical dissection protocol of the brachial plexus a modified Grant method for medical students and instructors. The points of the modified protocols are: (1) to dissect the brachial plexus after the dissection of the neck part, (2) to identify the nerve trunks at the scalenus gap after dissecting the lateral, medial and posterior cords. The modified Grant method can be adapted to any other dissecting protocol of the brachial plexus, and will allow students to cope with many variations of the brachial plexus when they occur.


Asunto(s)
Anatomía/educación , Anatomía/métodos , Plexo Braquial/anatomía & histología , Disección/educación , Disección/métodos , Extremidad Superior/inervación , Educación de Pregrado en Medicina , Humanos
8.
PLoS One ; 8(1): e53490, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23308235

RESUMEN

We describe a novel spontaneous mouse mutant, laggard (lag), characterized by a flat head, motor impairment and growth retardation. The mutation is inherited as an autosomal recessive trait, and lag/lag mice suffer from cerebellar ataxia and die before weaning. lag/lag mice exhibit a dramatic reduction in brain size and slender optic nerves. By positional cloning, we identify a splice site mutation in Kif14. Transgenic complementation with wild-type Kif14-cDNA alleviates ataxic phenotype in lag/lag mice. To further confirm that the causative gene is Kif14, we generate Kif14 knockout mice and find that all of the phenotypes of Kif14 knockout mice are similar to those of lag/lag mice. The main morphological abnormality of lag/lag mouse is severe hypomyelination in central nervous system. The lag/lag mice express an array of myelin-related genes at significantly reduced levels. The disrupted cytoarchitecture of the cerebellar and cerebral cortices appears to result from apoptotic cell death. Thus, we conclude that Kif14 is essential for the generation and maturation of late-developing structures such as the myelin sheath, cerebellar and cerebral cortices. So far, no Kif14-deficient mice or mutation in Kif14 has ever been reported and we firstly define the biological function of Kif14 in vivo. The discovery of mammalian models, laggard, has opened up horizons for researchers to add more knowledge regarding the etiology and pathology of brain malformation.


Asunto(s)
Ataxia Cerebelosa/genética , Cerebelo/patología , Corteza Cerebral/patología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Cinesinas/genética , Enfermedades Mitocondriales/genética , Mutación , Vaina de Mielina/genética , Trastornos Psicomotores/genética , Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Antiportadores/deficiencia , Antiportadores/genética , Antiportadores/metabolismo , Apoptosis , Secuencia de Bases , Ataxia Cerebelosa/metabolismo , Ataxia Cerebelosa/patología , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Femenino , Prueba de Complementación Genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Masculino , Ratones , Ratones Transgénicos , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Datos de Secuencia Molecular , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Fenotipo , Isoformas de Proteínas/genética , Trastornos Psicomotores/metabolismo , Trastornos Psicomotores/patología
9.
Kobe J Med Sci ; 56(2): E50-9, 2010 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-21063146

RESUMEN

Both in the Reelin-deficient reeler and Dab1-deficient yotari mice, layer V corticospinal tract neurons in the sensory-motor cortex are radially spread instead of being confined to a single cortical layer. In the present study, we examined distribution pattern of cortical layer V neurons in the visual and auditory cortices of reeler and yotari mice with the injection of HRP into the superior and inferior colliculi of the adult animals, respectively. After the injection of HRP into the superior colliculus of the normal mouse, retrogradely labeled cells were distributed in layer V of the visual cortex, while the similar injection of HRP in the reeler and yotari mice produced radial dispersion of retrograde labeling through all of the depths of the visual cortex of these mutant mice. Next, we injected HRP into the inferior colliculus of the normal, reeler and yotari mice. Retrogradely labeled neurons were distributed in layer V of the normal auditory cortex, whereas they were again radially scattered in the auditory cortex of the reeler and yotari mice. Taken together with the previous and present findings, layer V cortical efferent neurons are radially scattered in the sensory-motor, visual and auditory cortices of the reeler and yotari mice.


Asunto(s)
Corteza Auditiva/patología , Corteza Visual/patología , Animales , Corteza Auditiva/citología , Corteza Auditiva/enzimología , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Peroxidasa de Rábano Silvestre/administración & dosificación , Peroxidasa de Rábano Silvestre/farmacocinética , Ratones , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/enzimología , Neuronas/patología , Proteína Reelina , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Distribución Tisular , Corteza Visual/citología , Corteza Visual/enzimología
10.
Anat Sci Int ; 84(3): 200-9, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19221860

RESUMEN

The Reelin (Reln)-deficient mouse (reeler) and the Dab1-deficient mouse (yotari) are autosomal recessive mutant mice characterized by cerebellar ataxia. Previously, we reported that Reelin and Dab1 proteins have slightly different functions during the development of the cerebral cortex. To analyze the functional roles of Reelin and Dab1 proteins in detail, we attempted to generate a reelin/Dab1 compound-mutant mouse by breeding heterozygote reeler and yotari mice. We examined the cytoarchitecture of the cerebral and cerebellar cortices and the hippocampus of wild-type (Reln ( +/+ ); Dab1 ( +/+ )), double-heterozygote (Reln ( rl/+ ); Dab1 ( yot/+ )), reeler (Reln ( rl/rl ); Dab1 ( +/+ ), Reln ( rl/rl ); Dab1 ( yot/+ )), yotari (Reln ( +/+ ); Dab1 ( yot/yot ), Reln ( rl/+ ); Dab1 ( yot/yot )), and double-compound-deficient (Reln ( rl/rl ); Dab1 ( yot/yot )) mice. Nissl staining demonstrated that no abnormality was recognized in the mice of reelin/Dab1 double-heterozygote (Reln ( rl/+ ); Dab1 ( yot/+ )). The reelin/Dab1-compound mutant mouse (Reln ( rl/rl ); Dab1 ( yot/yot )) showed histological abnormalities in the cerebral and cerebellar cortices and the hippocampus, in addition to those of reeler and yotari mice. We injected HRP into the lumbar cord of these animals with various gene compositions to examine the distribution pattern of corticospinal tract (CST) neurons. CST neurons of the reelin/Dab1-compound mutant mice were not confined to layer V, but scattered throughout the motor cortex. This quantitative and statistical analysis shows that the distribution pattern of CST neurons of the reelin/Dab1-compound mutant mouse differs from those of either of the reeler or yotari counterparts. Taken together, although Reelin/Dab1 signal transduction is a primary cascade in neurons during developmental periods, other signaling cascades (e.g., the Cdk-5/Dab1 pathway) may lie in a parallel fashion to Reelin/Dab1 signal transduction.


Asunto(s)
Encéfalo/anomalías , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Proteínas del Tejido Nervioso/genética , Tractos Piramidales/anomalías , Serina Endopeptidasas/genética , Animales , Heterocigoto , Peroxidasa de Rábano Silvestre , Ratones , Ratones Endogámicos C57BL , Mutación , Técnicas de Trazados de Vías Neuroanatómicas , Proteína Reelina , Transducción de Señal
11.
Biochem Biophys Res Commun ; 363(1): 106-12, 2007 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-17826737

RESUMEN

A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1(-/-) mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1(-/-) yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1(-/-) embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.


Asunto(s)
Vasos Sanguíneos/anomalías , Vasos Sanguíneos/metabolismo , Pérdida del Embrión/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Morfogénesis , Neovascularización Patológica/embriología , Neovascularización Patológica/metabolismo , Animales , Femenino , Silenciador del Gen , Edad Gestacional , Masculino , Ratones , Ratones Endogámicos C57BL
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