Autophagy is an evolutionarily conserved bulk-protein degradation pathway in which isolation membranes engulf the cytoplasmic constituents, and the resulting autophagosomes transport them to lysosomes. Two ubiquitin-like conjugation systems, termed Atg12 and Atg8 systems, are essential for autophagosomal formation. In addition to the pathophysiological roles of autophagy in mammals, recent mouse genetic studies have shown that the Atg8 system is predominantly under the control of the Atg12 system. To clarify the roles of the Atg8 system in mammalian autophagosome formation, we generated mice deficient in Atg3 gene encoding specific E2 enzyme for Atg8. Atg3-deficient mice were born but died within 1 d after birth. Conjugate formation of mammalian Atg8 homologues was completely defective in the mutant mice. Intriguingly, Atg12-Atg5 conjugation was markedly decreased in Atg3-deficient mice, and its dissociation from isolation membranes was significantly delayed. Furthermore, loss of Atg3 was associated with defective process of autophagosome formation, including the elongation and complete closure of the isolation membranes, resulting in malformation of the autophagosomes. The results indicate the essential role of the Atg8 system in the proper development of autophagic isolation membranes in mice.

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.

ABSTRACT-The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), a voltagesensitive fluorescent dye, for the measurement of membrane potentials (MPs) was evaluated in HEK293 cells, where a or a plus b 1 subunits of large conductance Ca 2+-activated K + (BK) channels were expressed (HEKBKa and HEKBKab). The fluorescent intensity of DiBAC4(3) was measured at various potentials under voltage-clamp for calibration to estimate the absolute MP semi-quantitatively. The resting MPs measured with DiBAC4(3) were roughly comparable to those recorded with a microelectrode; the MP in HEKBKab was 10 -20 mV more negative than that in native HEK. In HEKBKa, the membrane hyperpolarization induced by 10 m M Evans blue, a BK channel opener, was detected with DiBAC4(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/FK even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBKab by application of 10 m M acetylcholine via increase in intracellular Ca 2+ concentration. The oscillatory hyperpolarization was, however, detected only as a slow hyperpolarization with DiBAC4(3). It can be concluded that relatively slow effects of BK channel modulators can be semi-quantitatively measured by use of DiBAC4(3) in HEKBK, while the limited temporal resolution and possible artifacts should be taken into account.

Effects of pimaric acid (PiMA) and eight closely related compounds on large-conductance K ϩ (BK) channels were examined using human embryonic kidney (HEK) 293 cells, in which either the ␣ subunit of BK channel (HEKBK␣) or both ␣ and ␤1 (HEKBK␣␤1) subunits were heterologously expressed. Effects of these compounds (10 M) on the membrane potential of HEKBK␣␤1 were monitored by use of DiBAC 4 (3), a voltagesensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid, and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BK␣␤1 opening under whole-cell voltage clamp showed that these six compounds activated BK␣␤1 in a very similar concentration range (1-10 M); in contrast, abietic acid, sclareol, and methyl pimarate had no effect. PiMA did not affect the charybdotoxin-induced block of macroscopic BK␣␤1 current. Single channel recordings of BK␣␤1 in insideout patches showed that 10 M PiMA did not change channel conductance but significantly increased its open probability as a result of increase in sensitivity to Ca 2ϩ and voltage. Because coexpression of the ␤1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BK␣ subunit. PiMA was selective to BK over cloned small and intermediate Ca 2ϩ activated K ϩ channels. In conclusion, PiMA (Ͼ1 M) increases Ca 2ϩ and voltage-sensitivity of BK␣ when applied from either side of the cell membrane. The marked difference in potency as BK channel openers between PiMA and abietic acid, despite only very small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BK␣ activation.