RESUMEN
Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This "packaging" is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane expands and eventually closes upon itself is an area of intense study. One factor implicated in both membrane expansion and membrane fusion is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the pre-autophagosomal membrane and remains bound through the maturation process of the autophagosome. In this chapter, we discuss two approaches to the in vitro reconstitution of this lipidation reaction. We then describe methods to study Atg8-PE mediated membrane tethering and fusion, two functions implicated in Atg8's role in autophagosome maturation.
Asunto(s)
Membrana Celular/química , Proteínas Asociadas a Microtúbulos/química , Proteínas de Saccharomyces cerevisiae/química , Animales , Autofagia , Proteína 7 Relacionada con la Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Biocatálisis , Reactivos de Enlaces Cruzados/química , Microscopía por Crioelectrón , Humanos , Cinética , Luz , Liposomas/química , Maleimidas/química , Fusión de Membrana , Lípidos de la Membrana/química , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Modelos Biológicos , Nefelometría y Turbidimetría , Tamaño de la Partícula , Fagosomas/química , Fosfatidiletanolaminas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Dispersión de Radiación , Enzimas Ubiquitina-Conjugadoras/químicaRESUMEN
Serotonergic neurons project widely throughout the CNS and modulate many different brain functions. Particularly important, but controversial, are the contributions of serotonin (5-HT) neurons to respiratory and thermoregulatory control. To better define the roles of 5-HT neurons in breathing and thermoregulation, we took advantage of a unique conditional knock-out mouse in which Lmx1b is genetically deleted in Pet1-expressing cells (Lmx1b(f/f/p)), resulting in near-complete absence of central 5-HT neurons. Here, we show that the hypercapnic ventilatory response in adult Lmx1b(f/f/p) mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1b(f/f/p) mice rapidly became hypothermic when exposed to an ambient temperature of 4 degrees C, decreasing core temperature to 30 degrees C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO(2) chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO(2). We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.