Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

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

Mapping fluctuations in biomembranes adhered to mictopatterns

We studied biomembrane fluctuations by calculating the instantaneous shape of model membranes adhered to micro-patterned substrates, using micro-interferometry. The model consisted of partially adherent giant unilamellar vesicles (GUVs) which were osmotically deflated. Adhesion was effected via the specific ligand-receptor interaction of biotin-neutravidin. Special micro-structured adhesive substrates were developed where the receptors were distributed in the form of grids or lines. Dual-wavelength reflection interference contrast microscopy (DW-RICM) measurements revealed that on the structured adhesive substrates GUVs exhibit regions of bound and fluctuating membrane, in accordance with the underlying pattern. In the fluctuating zone, the membrane presented itself as a flat-topped hill for an initial osmotic difference of 70 mOsm l(-1). The membrane-substrate distance saturated at a plateau of 79 +/- 9 nm. In this plateau, the fluctuation amplitude was found to be 10 +/- 3 nm. Variation of the shape (grid versus lines) or size (grids of 3.5 or 7 mu m lattice constant) influenced neither the height nor the fluctuation amplitude in the plateau. Fourier analysis revealed that the mode corresponding to a wavelength of twice the pattern size always contributed, and, depending on the substrate, additional modes were sometimes present. The plateau height could be tuned from 0 to 538 nm by changing the initial osmotic gradient between the inside and outside of the GUV, which effectively tuned the membrane tension. The corresponding fluctuation amplitude ranged from non-detectable to a maximum of 17 nm. Our results can be interpreted in the light of a tension dependent effective interaction potential