Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles

TRPML3 is a member of the TRPML subfamily of the transient receptor potential cation channel superfamily. The TRPML3(A419P) mutation causes a severe form, whereas the TRPML3(I362T/A419P) mutation results in a mild form of the varitint-waddler phenotype. The channel properties of TRPML3 and how the mutations cause each phenotype are not known. In this study, we report the first channel properties of TRPML3 as a strongly inward rectifying cation channel with a novel regulation by extracytosolic Na ؉ . Preincubating the extracytosolic face of TRPML3 in Na ؉ -free medium is required for channel activation, but then the channel slowly inactivates. The A419P mutation locks the channel in an open unregulated state. Similar gain of function was observed with the A419G mutation, which, like A419P, is expected to destabilize the ␣-helical fifth transmembrane domain of TRPML3. The I362T mutation results in an inactive channel, but the channel properties of TRPML3(I362T/A419P) are similar to those of TRPML3(A419P). However, the surface expression and current density of TRPML3(I362T/A419P) are lower than those of TRPML3(A419P). The A419P mutation also affects channel glycosylation and causes massive cell death. These findings show that the varitint-waddler phenotype is due to a gain of function of TRPML3(A419P) that is reduced by the TRPML3(I362T/A419P) mutant, resulting in a milder phenotype.TRPML3 is a putative channel belonging to the TRPML subfamily of the transient receptor potential channels (1). The TRPML subfamily consists of three members. Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (2, 3). The A419P mutations in the ␣-helical fifth transmembrane domain of TRPML3 cause the varitint-waddler phenotype, which is characterized by pigmentation defect, hearing loss, circling behavior, and embryonic lethality (4, 5). Homozygotes for the A419P mutation have a severe phenotype that results in embryonic lethality. A milder phenotype is observed with the ϩ/A419P heterozygote, when the I362T mutation occurs in the same allele as A419P (4, 5).The channel function of TRPML3 and the effect of the A419P and I362T mutations on channel function are unknown. We report here that TRPML3 functions as an inward rectifying cation channel that is inactivated by extracytosolic cations. The A419P mutation locks the channel in an open state and eliminates regulation of the channel by extracytosolic cations. The I362T mutation inhibits channel activity and reduces the surface expression and current density of the A419P mutant. We conclude that the varitint-waddler phenotype is the result of a gain-of-function mutation in TRPML3. EXPERIMENTAL PROCEDURESPlasmid Construction, Mutagenesis, and Reagents-Human TRPML3 was amplified from human placenta and cloned into the pEGFPC1 and p3XFLAG-CMV-7.1 vectors. Mutations were introduced with the QuikChange kit.Cell Culture, Transfection, and Western Blotting-HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells ...

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