In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are 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 monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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We examine the role of synaptic activity in the development of identified Drosophila embryonic motorneurons. Synaptic activity was blocked by both pan-neuronal expression of tetanus toxin light chain (TeTxLC) and by reduction of acetylcholine (ACh) using a temperature-sensitive allele of choline acetyltransferase (Cha ts2 ). In the absence of synaptic activity, aCC and RP2 motorneurons develop with an apparently normal morphology and retain their capacity to form synapses. However, blockade of synaptic transmission results in significant changes in the electrical phenotype of these neurons. Specifically, increases are seen in both voltage-gated inward Na ϩ and voltage-gated outward K ϩ currents. Voltage-gated Ca 2ϩ currents do not change. The changes in conductances appear to promote neuron excitability. In the absence of synaptic activity, the number of action potentials fired by a depolarizing ramp (Ϫ60 to ϩ60 mV) is increased and, in addition, the amplitude of the initial action potential fired is also significantly larger. Silencing synaptic input to just aCC, without affecting inputs to other neurons, demonstrates that the capability to respond to changing levels of synaptic excitation is intrinsic to these neurons. The alteration to electrical properties are not permanent, being reversed by restoration of normal synaptic function. Whereas our data suggest that synaptic activity makes little or no contribution to the initial formation of embryonic neural circuits, the electrical development of neurons that constitute these circuits seems to depend on a process that requires synaptic activity.

In a genetic screen for genes that control synapse development, we have identified spinster (spin), which encodes a multipass transmembrane protein. spin mutant synapses reveal a 200% increase in bouton number and a deficit in presynaptic release. We demonstrate that spin is expressed in both nerve and muscle and is required both pre- and postsynaptically for normal synaptic growth. We have localized Spin to a late endosomal compartment and present evidence for altered endosomal/lysosomal function in spin. We also present evidence that synaptic overgrowth in spin is caused by enhanced/misregulated TGF-beta signaling. TGF-beta receptor mutants show dose-dependent suppression of synaptic overgrowth in spin. Furthermore, mutations in Dad, an inhibitory Smad, cause synapse overgrowth. We present a model for synaptic growth control with implications for the etiology of lysosomal storage and neurodegenerative disease.