In situ structure of FtsZ mini-rings in Arabidopsis chloroplasts

Abstract

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.

Conserved Dynamics of Chloroplast Cytoskeletal FtsZ Proteins Across Photosynthetic Lineages

“…2, A and B). Small Z rings have also been noted in transgenic Arabidopsis plants overexpressing FtsZ2-mYFP or ARC3-Myc and in minE and arc6 null mutants (Zhang et al, 2013;Johnson et al, 2015a). The combined results suggest that FtsZ2 and glaucophyte FtsZ proteins may have at least two distinct states of polymer geometry: a less curved and a more highly curved geometry.…”

The Arabidopsis arc5 and arc6 mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis

“…This result is in accordance with the plastid morphology observed in leaf mesophyll cells, cotyledons, petals, stamen filaments, stem endodermis, and roots [ 7 , 18 , 19 , 22 , 23 , 27 , 42 – 44 ]. In addition, FtsZ proteins (FtsZ1 and FtsZ2) exhibit similar localization patterns in the giant chloroplasts of arc6 and atminE1 (and arc12 , another mutant allele of AtMinE1 [ 45 , 46 ]) [ 9 , 27 , 34 , 46 – 49 ]. Thus, ARC6 and AtMinE1 may play equally important roles in plastid morphogenesis in leaf PCs as they do in mesophyll cells.…”

FtsZ Constriction Force – Curved Protofilaments Bending Membranes

“…Toroids ~200 nm in diameter were imaged by light microscopy in Arabidopsis , under conditions where FtsZ2-GFP was overexpressed, or when Z-ring modulating factors ARC6 or MinE were deleted (Johnson et al 2015). These toroids required super-resolution light microscopy for optimal resolution.…”