The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division.

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.

Physical mechanisms of ESCRT-III-driven cell division.

Living systems propagate by undergoing rounds of cell growth and division. Cell division is at heart a physical process that requires mechanical forces, usually exerted by assemblies of cytoskeletal polymers. Here we developed a physical model for the ESCRT-III-mediated division of archaeal cells, which despite their structural simplicity share machinery and evolutionary origins with eukaryotes. By comparing the dynamics of simulations with data collected from live cell imaging experiments, we propose that this branch of life uses a previously unidentified division mechanism. Active changes in the curvature of elastic cytoskeletal filaments can lead to filament perversions and supercoiling, to drive ring constriction and deform the overlying membrane. Abscission is then completed following filament disassembly. The model was also used to explore how different adenosine triphosphate (ATP)-driven processes that govern the way the structure of the filament is changed likely impact the robustness and symmetry of the resulting division. Comparisons between midcell constriction dynamics in simulations and experiments reveal a good agreement with the process when changes in curvature are implemented at random positions along the filament, supporting this as a possible mechanism of ESCRT-III-dependent division in this system. Beyond archaea, this study pinpoints a general mechanism of cytokinesis based on dynamic coupling between a coiling filament and the membrane.

Heimdallarchaea encodes profilin with eukaryotic-like actin regulation and polyproline binding.

It is now widely accepted that the first eukaryotic cell emerged from a merger of an archaeal host cell and an alphaproteobacterium. However, the exact sequence of events and the nature of the cellular biology of both partner cells is still contentious. Recently the structures of profilins from some members of the newly discovered Asgard superphylum were determined. In addition, it was found that these profilins inhibit eukaryotic rabbit actin polymerization and that this reaction is regulated by phospholipids. However, the interaction with polyproline repeats which are known to be crucial for the regulation of profilin:actin polymerization was found to be absent for these profilins and was thus suggested to have evolved later in the eukaryotic lineage. Here, we show that Heimdallarchaeota LC3, a candidate phylum within the Asgard superphylum, encodes a putative profilin (heimProfilin) that interacts with PIP2 and its binding is regulated by polyproline motifs, suggesting an origin predating the rise of the eukaryotes. More precisely, we determined the 3D-structure of Heimdallarchaeota LC3 profilin and show that this profilin is able to: i) inhibit eukaryotic actin polymerization in vitro; ii) bind to phospholipids; iii) bind to polyproline repeats from enabled/vasodilator-stimulated phosphoprotein; iv) inhibit actin from Heimdallarchaeota from polymerizing into filaments. Our results therefore provide hints of the existence of a complex cytoskeleton already in last eukaryotic common ancestor.

NMR resonance assignment and dynamics of profilin from Heimdallarchaeota.

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

The proteasome controls ESCRT-III-mediated cell division in an archaeon.

Sulfolobus acidocaldarius is the closest experimentally tractable archaeal relative of eukaryotes and, despite lacking obvious cyclin-dependent kinase and cyclin homologs, has an ordered eukaryote-like cell cycle with distinct phases of DNA replication and division. Here, in exploring the mechanism of cell division in S. acidocaldarius, we identify a role for the archaeal proteasome in regulating the transition from the end of one cell cycle to the beginning of the next. Further, we identify the archaeal ESCRT-III homolog, CdvB, as a key target of the proteasome and show that its degradation triggers division by allowing constriction of the CdvB1:CdvB2 ESCRT-III division ring. These findings offer a minimal mechanism for ESCRT-III-mediated membrane remodeling and point to a conserved role for the proteasome in eukaryotic and archaeal cell cycle control.

NanoJ: a high-performance open-source super-resolution microscopy toolbox.

Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM designed to combine high performance and ease of use. We named it NanoJ-a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.