Approaches to the study of Atg8-mediated membrane dynamics in vitro.

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.

SNARE proteins are required for macroautophagy.

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

Defects in breathing and thermoregulation in mice with near-complete absence of central serotonin neurons.

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.