Erratum: Publisher correction: In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters (Nature communications (2017) 8 (13660))

This corrects the article DOI: 10.1038/ncomms13660

In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters

Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release

OPA1 Processing Reconstituted in Yeast Depends on the Subunit Composition of the m-AAA Protease in Mitochondria

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field