In situ structure of FtsZ mini-rings in Arabidopsis chloroplasts

Chloroplasts are essential plant organelles that divide by binary fission through a coordinated ring-shaped division machinery located both on the outside and inside of the chloroplast. The first step in chloroplast division is the assembly of an internal division ring (Z-ring) that is composed of the key filamentous chloroplast division proteins FtsZ1 and FtsZ2. How the individual FtsZ filaments assemble into higher-order structures to form the dividing Z-ring is not well understood and the most detailed insights have so far been gleaned from prokaryotic FtsZ. Here, we present in situ data of chloroplast FtsZ making use of a smaller ring-like FtsZ assembly termed mini-rings that form under well-defined conditions. Structured illumination microscopy (SIM) permitted their mean diameter to be determined as 208 nm and also showed that 68 % of these rings are terminally attached to linear FtsZ filaments. A correlative microscopy-compatible specimen preparation based on freeze substitution after high-pressure freezing is presented addressing the challenges such as autofluorescence and specific fluorescence attenuation. Transmission electron microscopy (TEM) and scanning TEM (STEM) imaging of thin sections exhibited ring-like densities that matched in size with the SIM data, and TEM tomography revealed insights into the molecular architecture of mini-rings demonstrating the following key features: (1) overall, a roughly bipartite split into a more ordered/curved and less ordered/curved half is readily discernible; (2) the density distribution in individual strands matches with the X-ray data, suggesting they constitute FtsZ protofilaments; (3) in the less ordered half of the ring, the protofilaments are able to assemble into higher-order structures such as double helices and supercoiled structures. Taken together, the data suggest that the state of existence of mini-rings could be described as metastable and their possible involvement in filament storage and Z-ring assembly is discussed.