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

[Abstract unavailable

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Here we report that such dual regulation arises from surprising Ca2+-transduction capabilities of CaM. First, although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. Second, expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. Finally, the bifunctional capability of CaM arises from bifurcation of Ca2+ signalling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex.

Oscillatory firing patterns are an intrinsic property of some neurons and have an important function in information processing. In some cells, low voltage-activated calcium channels have been proposed to underlie a depolarizing potential that regulates bursting. The sequence of a rat brain calcium channel alpha 1 subunit (rbE-II) was deduced. Although it is structurally related to high voltage-activated calcium channels, the rbE-II channel transiently activated at negative membrane potentials, required a strong hyperpolarization to deinactivate, and was highly sensitive to block by nickel. In situ hybridization showed that rbE-II messenger RNA is expressed in regions throughout the central nervous system. The electrophysiological properties of the rbE-II current are consistent with a type of low voltage-activated calcium channel that requires membrane hyperpolarization for maximal activity, which suggests that rbE-II may be involved in the modulation of firing patterns.