ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA–adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III–dependent division system in Sulfolobus acidocaldarius , one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III–dependent membrane remodeling.

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems sequential changes in the composition of ESCRT-III polymers induced by the AAA ATPase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organised and remodelled in space and time in cells. Here, taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relative of eukaryotes, we use super-resolution microscopy and computational modelling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring which contracts and is disassembled by Vps4 to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodelling.

STimulated Emission Depletion (STED) nanoscopy uniquely combines a high spatial resolution (20-50nm in cells) with relatively fast imaging (frame rate of ~1-30Hz), straightforward sample preparation and direct image output (no postprocessing required). Although these characteristics in principle make STED very suitable for high-throughput imaging, only few steps towards automation have been made. Here, we have developed fully automated STED imaging, eliminating all manual steps including the selection and characterisation of the relevant (cellular) regions, sample focusing and positioning, and microscope adjustments. This automatic STED image acquisition increases the data output by roughly two orders of magnitude, resulting in a more efficient use of the high-end microscope, and the ability to detect and characterise objects that are only present in a small subset of the sample.