RESUMO
Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Filaminas/metabolismo , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Actinas/metabolismo , Animais , Caenorhabditis elegans/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Ratos Wistar , Semaforina-3A/metabolismoRESUMO
Alzheimer's disease (AD) is characterized by amyloid-ß (Aß) protein and tau deposition in the brain. Numerous studies have reported a central role of Aß in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of Aß neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of Aß25â35 peptide, a neurotoxic fragment of Aß protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of Aß25â35 peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of Aß25â35 peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for Aß-induced impairment of cognitive memory and synaptic plasticity.
Assuntos
Encéfalo/metabolismo , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Transtornos Cognitivos/induzido quimicamente , Hipocampo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/toxicidade , Fosforilação , Reconhecimento Psicológico/fisiologiaRESUMO
Biosensors selectively detecting a very small amount of biomarker protein in human blood are desired for early and reliable diagnoses of severe diseases. This paper reports the detection of protein (streptavidin: SA) in ultra-low concentration, with an ultra-high selectivity against contaminants, using photonic crystal nanolasers. For biotin-modified nanolasers in pure water with SA, an extremely-low detection limit of 16 zM is evaluated. Even in a mixture with 1 µM bovine serum albumin as the contaminant, 100 zM SA is detected, meaning a selectivity of 10(13). These are remarkable capabilities that are promising for practical biosensing in the medical applications mentioned above.
Assuntos
Técnicas Biossensoriais/instrumentação , Lasers , Nanotecnologia/instrumentação , Fotometria/instrumentação , Refratometria/instrumentação , Estreptavidina/sangue , Cristalização , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Sensibilidade e EspecificidadeRESUMO
Site-selective adsorption of protein molecules was found on sapphire surfaces that exhibit a phase separation into two domains: weakly charged hydrophobic domain and negatively charged hydrophilic one. Ferritin and bovine serum albumin molecules, which are negatively charged in a buffer solution, are adsorbed to the hydrophobic domains. Avidin molecules, which are positively charged, are adsorbed to the other domain. Fibrinogen molecules, which consist of both negative and positive modules, are adsorbed to the whole sapphire surface. Hemoglobin molecules, whose net charge is almost zero, are also adsorbed to the whole surfaces. These results indicate that electrostatic double layer interaction is the primary origin of the observed selectivity. Dependence of protein adsorption or desorption behaviors on the pH value can also be interpreted by the proposed model.
Assuntos
Óxido de Alumínio/química , Proteínas/isolamento & purificação , Adsorção , Animais , Avidina/isolamento & purificação , Bovinos , Ferritinas/isolamento & purificação , Fibrinogênio/isolamento & purificação , Cavalos , Concentração de Íons de Hidrogênio , Soroalbumina Bovina/isolamento & purificação , Propriedades de SuperfícieRESUMO
Self-organized step/terrace structures on a sapphire surface were used to investigate interface properties between a solid surface and a supported planar lipid bilayer (SPB). We prepared random-stepped, single-stepped and multistepped sapphire surfaces. Some multistepped surfaces covered with crossing steps exhibit phase-separation into hydrophilic and hydrophobic domains. We studied evolution of self-spreading lipid bilayers that are subject to the atomic structures and chemical states on the surfaces. The growth direction of SPBs in the self-spreading method is regulated by the atomic steps. While the SPBs were apparently uniform after a 1 h self-spreading, a density gradient of the lipid molecules was observed even after 24 h spreading. We found that various patterns of the SPBs that depend on the density of the lipid molecules are self-assembled on the phase-separated surfaces. Although the SPB is supported on the sapphire surface via an about 1 nm water layer, the self-spreading direction and the morphology of the SPBs are affected by the atomic steps, whose height is much smaller than that of the water layer.