The division of chloroplasts is a critical process in photosynthetic eukaryotes. These organelles arose by endosymbiosis from a free-living cyanobacterium and inherited several of their division components from the cell division machinery in their prokaryotic ancestor. Foremost among these is FtsZ, a member of the FtsZ/tubulin superfamily of cytoskeletal proteins. FtsZ self-assembles into a central "Z ring" to initiate division of the cell or organelle and generate force for membrane constriction. A crucial difference between bacterial cell and chloroplast division systems is that the bacterial Z ring is composed of a single form of FtsZ that assembles as a homopolymer whereas the chloroplast Z ring is composed of two distinct forms of FtsZ called FtsZ1 and FtsZ2 that coassemble as a heteropolymer and function together in division of the organelle. The funded research will combine a series of complementary approaches to probe the mechanisms, dynamic properties and regulation of FtsZ1 and FtsZ2 assembly with the overall goal of advancing understanding of the functional and evolutionary significance of the involvement of two FtsZ types in chloroplast division. The experimental objectives are as follows: 1) Analyze the kinetics of FtsZ1 and FtsZ2 assembly separately and together to establish how assembly is nucleated and whether the assembly subunit is an FtsZ1/FtsZ2 heterodimer. These and other analyses will also be extended to related chloroplast FtsZ pairs from one red algal and one green algal species to expand the evolutionary scope of these studies. 2) Analyze force generation at different FtsZ1:FtsZ2 ratios using a novel liposome deformation assay recently developed for bacterial FtsZ, and use a similar system to reconstitute chloroplast Z rings inside liposomes. These experiments will reveal how heteropolymer formation affects chloroplast Z-ring assembly and constrictive force. 3) Analyze FtsZ1 and FtsZ2 assembly behavior and dynamics in a heterologous fission yeast system using fluorescence recovery after photobleaching as a powerful complement to in vitro and in vivo studies. 4) Use quantitative fluorescence microscopy with tagged, functional fusion proteins in Arabidopsis to investigate Z-ring composition and dynamics in vivo, including whether the FtsZ1:FtsZ2 ratio changes dynamically during Z-ring constriction or is regulated in concert with developmental changes in chloroplast division activity. 5) Initiate functional analysis of plant-specific regulators of chloroplast Z-ring assembly towards understanding how FtsZ self-assembly is controlled in chloroplasts.
Broader impacts The research brings together the complementary expertise of the project leaders and their lab members. One group, at Michigan State University, studies FtsZ from chloroplasts using biochemical, genetic, and cell biological approaches in the model plant Arabidopsis thaliana. The other group, at Duke University, studies FtsZ from E. coli and other bacteria using in vitro biochemical and biophysical approaches. Interaction among project personnel by email, video calling, and exchange visits will provide an interdisciplinary perspective that has the potential to catalyze new ideas and research directions. The new studies on algal FtsZs will lay the groundwork for understanding how chloroplast FtsZs have evolved in all photosynthetic eukaryotes. Since mitochondria, like chloroplasts, originated from a bacterial endosymbiont and some organisms retain FtsZ for mitochondrial division, the research will be relevant to understanding FtsZ evolution in mitochondria as well as chloroplasts. In addition, because the microtubule subunits alpha- and beta-tubulin evolved from FtsZ, the research on chloroplast FtsZ heteropolymers may be relevant to understanding evolution of the microtubule cytoskeleton. Finally, Arabidopsis ftsZ and other chloroplast division mutants will be exploited to develop a teaching module for undergraduate ecology students to explore natural selection on chloroplast division and morphology. This will encourage critical thinking on the connection between molecular events, cellular structure and population processes. The research will also provide training for undergraduates in biological research.