Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.

We have developed two types of fluorescent probes, DALGreen and DAPGreen, for monitoring autophagy, that exhibit fluorescence upon being incorporated into autophagosomes. DALGreen enhances its fluorescence at acidic pH, which is favorable for monitoring late‐phase autophagy, whereas DAPGreen remains fluorescent with almost constant brightness during the autophagic process. With these probes that stain autophagosomes as they are being formed, the real‐time change of autophagic phenomena of live cells may be traced, which is an advantage over conventional approaches with small molecules that stain mature autophagosomes. The use of both dyes allows monitoring of the membrane dynamics of autophagy in any type of cell without the need for genetic engineering, and therefore, will be useful as a tool to study autophagic phenomena.

ORCID ID: 0000-0001-7441-0203 (H.T.S.)RAB5 GTPases act as molecular switches that regulate various endosomal functions in animal cells, including homotypic fusion of early endosomes, endosomal motility, endosomal signaling, and subcompartmentalization of the endosomal membrane. RAB5 proteins fulfill these diverse functions through interactions with downstream effector molecules. Two canonical RAB5 members, ARA7 and RAB HOMOLOG1 (RHA1), are encoded in the Arabidopsis thaliana genome. ARA7 and RHA1 play crucial roles in endocytic and vacuolar trafficking pathways. Plant RAB5 GTPases function via interactions with effector molecules, whose identities and functions are currently unclear. In this study, we searched for canonical RAB5 effector molecules of Arabidopsis and identified a candidate, which we called ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN (EREX). The intimate genetic interaction between EREX and RAB5 members, the results from subcellular colocalization experiments, and the direct interaction observed in an in vitro pull-down assay strongly suggest that EREX is a genuine effector of canonical RAB5s in Arabidopsis. We further found that close homologs of EREX play partially redundant functions with EREX in the transport of seed storage proteins. Our results indicate that canonical plant RAB5s acquired distinct effector molecules from those of non-plant systems to fulfill their functions.

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