RESUMEN
Chk1 (encoded by CHEK1 in mammals) is an evolutionarily conserved protein kinase that transduces checkpoint signals from ATR to Cdc25A during the DNA damage response (DDR). In mammals, Chk1 also controls cellular proliferation even in the absence of exogenous DNA damage. However, little is known about how Chk1 regulates unperturbed cell cycle progression, and how this effect under physiological conditions differs from its regulatory role in DDR. Here, we have established near-diploid HCT116 cell lines containing endogenous Chk1 protein tagged with a minimum auxin-inducible degron (mAID) through CRISPR/Cas9-based gene editing. Establishment of these cells enabled us to induce specific and rapid depletion of the endogenous Chk1 protein, which resulted in aberrant accumulation of DNA damage factors that induced cell cycle arrest at S or G2 phase. Cdc25A was stabilized upon Chk1 depletion before the accumulation of DNA damage factors. Simultaneous depletion of Chk1 and Cdc25A partially suppressed the defects caused by Chk1 single depletion. These results indicate that, similar to its function in DDR, Chk1 controls normal cell cycle progression mainly by inducing Cdc25A degradation.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN , Puntos de Control de la Fase G2 del Ciclo Celular , Proteolisis , Puntos de Control de la Fase S del Ciclo Celular , Fosfatasas cdc25/metabolismo , Sistemas CRISPR-Cas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Edición Génica , Células HCT116 , Humanos , Fosfatasas cdc25/genéticaRESUMEN
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure. We have previously shown using afadin-deficient mice that afadin plays multiple roles in the structural and functional differentiations of this synapse. We investigated here using a co-culture system with cultured hippocampal neurons and non-neuronal COS-7 cells expressing synaptogenic cell adhesion molecules (CAMs) whether afadin is involved in the presynaptic differentiation of hippocampal synapses. Postsynaptic CAMs NGL-3 (alias, a Lrrc4b gene product) and neuroligin induced presynaptic differentiation by trans-interacting with their respective presynaptic binding CAMs LAR (alias, a Ptprf gene product) and neurexin. This activity of NGL-3, but not neuroligin, was dependent on afadin, but not the afadin-binding presynaptic CAM nectin-1. The afadin-binding postsynaptic CAM nectin-3 did not induce presynaptic differentiation. Immunofluorescence and immunoelectron microscopy analyses showed that afadin was localized mainly at puncta adherentia junctions, but partly at synaptic junctions, of the mossy fiber synapse. ß-Catenin and γ-catenin known to bind to LAR were co-immunoprecipitated with afadin from the lysate of mouse brain. These results suggest that afadin is involved in the NGL-3-LAR system-induced presynaptic differentiation of hippocampal neurons cooperatively with ß-catenin and γ-catenin in a nectin-1-independent manner.
Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Hipocampo/metabolismo , Proteínas de Microfilamentos/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Neuronas/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas Ligadas a GPI/genética , Hipocampo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas de Microfilamentos/genética , Fibras Musgosas del Hipocampo/ultraestructura , Nectinas/genética , Nectinas/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Unión Proteica , beta Catenina/metabolismo , gamma Catenina/metabolismoRESUMEN
The hippocampal formation with tightly packed neurons, mainly at the dentate gyrus, CA3, CA2, and CA1 regions, constitutes a one-way neural circuit, which is associated with learning and memory. We previously showed that the cell adhesion molecules nectins and its binding protein afadin play roles in the formation of the mossy fiber synapses which are formed between the mossy fibers of the dentate gyrus granule cells and the dendrites of the CA3 pyramidal cells. We showed here that in the afadin-deficient hippocampal formation, the dentate gyrus granules cells and the CA3, CA2, and CA1 pyramidal cells were abnormally located; the mossy fiber trajectory was abnormally elongated; the CA3 pyramidal cells were abnormally differentiated; and the densities of the presynaptic boutons on the mossy fibers and the apical dendrites of the CA3 pyramidal cells were decreased. These results indicate that afadin plays roles not only in the formation of the mossy fiber synapses but also in the formation of the cellular architecture of the hippocampus and the dentate gyrus.
Asunto(s)
Región CA3 Hipocampal/citología , Giro Dentado/citología , Proteínas de Microfilamentos/metabolismo , Células Piramidales/citología , Animales , Región CA3 Hipocampal/crecimiento & desarrollo , Región CA3 Hipocampal/metabolismo , Células Cultivadas , Giro Dentado/crecimiento & desarrollo , Giro Dentado/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Fibras Musgosas del Hipocampo/metabolismo , Neurogénesis , Células Piramidales/metabolismo , Sinapsis/metabolismoRESUMEN
Ectopic protein with proper steric structure was efficiently loaded onto the envelope of the F gene-defective BC-PIV vector derived from human parainfluenza virus type 2 (hPIV2) by a reverse genetics method of recombinant virus production. Further, ectopic antigenic peptide was successfully loaded either outside, inside, or at both sides of the envelope of the vector. The BC-PIV vector harboring the Ebola virus GP gene was able to elicit neutralizing antibodies in mice. In addition, BC-PIV with antigenic epitopes of both melanoma gp100 and WT1 tumor antigen induced a CD8+ T-cell-mediated response in tumor-transplanted syngeneic mice. Considering the low pathogenicity and recurrent infections of parental hPIV2, BC-PIV can be used as a versatile vector with high safety for recombinant vaccine development, addressing unmet medical needs.
Asunto(s)
Vectores Genéticos/genética , Virus de la Parainfluenza 2 Humana/genética , Vacunas Sintéticas/genética , Vacunología/métodos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Epítopos/genética , Epítopos/inmunología , Orden Génico , Ingeniería Genética , Humanos , Ratones , Pruebas de Neutralización , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células VeroRESUMEN
The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin-like cell adhesion molecules (CAMs) nectin-2α and nectin-2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin-2α, but not nectin-2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin-2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin-2α at this boundary was not colocalized with the nectin-2α-binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin-2α forms a cell adhesion apparatus different from the Kv4.2-associated MSs. Genetic ablation of nectin-2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin-2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.
Asunto(s)
Neuronas Colinérgicas/metabolismo , Habénula/citología , Nectinas/metabolismo , Terminales Presinápticos/metabolismo , Canales de Potasio Shal/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Animales Recién Nacidos , Neuronas Colinérgicas/citología , Regulación de la Expresión Génica/genética , Habénula/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nectinas/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfopiruvato Hidratasa/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.
Asunto(s)
Hipocampo/citología , Proteínas de Microfilamentos/metabolismo , Morfogénesis/fisiología , Fibras Musgosas del Hipocampo/ultraestructura , Neuronas/ultraestructura , Sinapsis/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Dendritas/metabolismo , Dendritas/ultraestructura , Regulación de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Modelos Neurológicos , Fibras Musgosas del Hipocampo/metabolismo , Nectinas/metabolismo , Neuronas/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de potasio activados por Sodio , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Sinapsis/ultraestructura , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules. In the nervous system, among four members (nectin-1, -2, -3, and -4), nectin-1 and -3 are asymmetrically localized at puncta adherentia junctions formed between the mossy fiber terminals and the dendrites of CA3 pyramidal neurons in the mouse hippocampus and heterophilic trans-interactions between nectin-1 and nectin-3 are involved in the selective interaction of axons and dendrites of cultured neurons. By contrast, nectin-2, which has two splicing variants, nectin-2α and -2δ, has not been well characterized in the brain. We showed here that nectin-2α was expressed in both cultured mouse neurons and astrocytes whereas nectin-2δ was selectively expressed in the astrocytes. Nectin-2δ was localized at the adhesion sites between adjacent cultured astrocytes, but in the brain it was localized on the plasma membranes of astrocytic perivascular endfoot processes facing the basement membrane of blood vessels. Genetic ablation of nectin-2 caused degeneration of astrocytic perivascular endfoot processes and neurons in the cerebral cortex. These results uncovered for the first time the localization and critical functions of nectin-2 in the brain.