RESUMEN
Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Bioensayo , Microtúbulos/ultraestructura , Imagen Molecular/métodos , Neuronas/ultraestructura , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Polaridad Celular , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Expresión Génica , Hipocampo/citología , Hipocampo/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Cultivo Primario de Células , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Grabación en VideoRESUMEN
In central mammalian neurons, activation of metabotropic glutamate receptor type1 (mGluR1) evokes a complex synaptic response consisting of IP3 receptor-dependent Ca(2+) release from internal Ca(2+) stores and a slow depolarizing potential involving TRPC3 channels. It is largely unclear how mGluR1 is linked to its downstream effectors. Here, we explored the role of stromal interaction molecule 1 (STIM1) in regulating neuronal Ca(2+) signaling and mGluR1-dependent synaptic transmission. By analyzing mouse cerebellar Purkinje neurons, we demonstrate that STIM1 is an essential regulator of the Ca(2+) level in neuronal endoplasmic reticulum Ca(2+) stores. Both mGluR1-dependent synaptic potentials and IP3 receptor-dependent Ca(2+) signals are strongly attenuated in the absence of STIM1. Furthermore, the Purkinje neuron-specific deletion of Stim1 causes impairments in cerebellar motor behavior. Together, our results demonstrate that in the mammalian nervous system STIM1 is a key regulator of intracellular Ca(2+) signaling, metabotropic glutamate receptor-dependent synaptic transmission, and motor coordination.
