Carol B. Johnson

Plant FtsZ1 and FtsZ2 expressed in a eukaryotic host: GTPase activity and self-assembly.

Plants and algae contain the FtsZ1 and FtsZ2 protein families that perform specific, non‐redundant functions in plastid division. In vitro studies of chloroplast division have been hampered by the lack of a suitable expression system. Here we report the expression and purification of FtsZ1‐1 and FtsZ2‐1 from Arabidopsis thaliana using a eukaryotic host. Specific GTPase activities were determined and found to be different for FtsZ1‐1 vs. FtsZ2‐1. The purified proteins readily assembled into previously unreported assembly products named type‐I and ‐II filaments. In contrast to bacterial FtsZ, the Arabidopsis proteins do not form bundled sheets in the presence of Ca2+.

FtsZ1/FtsZ2 Turnover in Chloroplasts and the Role of ARC3

Chloroplast division requires filamentation temperature-sensitive Z (FtsZ), a tubulin-like GTPase of cyanobacterial endosymbiotic origin. Plants and algae possess two distinct FtsZ protein families, FtsZ1 and FtsZ2 that co-assemble into a ring (Z-ring) at the division site. Z-ring assembly and disassembly and division site positioning is controlled by both positive and negative factors via their specific interactions with FtsZ1 and FtsZ2. Here we present the in planta analysis of Arabidopsis FtsZ1 and FtsZ2 turnover in the context of a native chloroplast division machinery. Fluorescence recovery after photobleaching analysis was conducted using fluorescently tagged FtsZ at wild-type (WT)-like levels. Rapid photobleaching, low signal-to-noise ratio, and phototropic movements of chloroplasts were overcome by (i) using progressive intervals in time-lapse imaging, (ii) analyzing epidermal rather than stromal chloroplasts, and (iii) employing image stack alignment during postprocessing. In plants of WT background, fluorescence recovery half-times averaged 117 and 325 s for FtsZ1 and FtsZ2, respectively. In plants lacking ARC3, the key negative regulator of FtsZ assembly, the turnover was threefold slower. The findings are discussed in the context of previous results conducted in a heterologous system.

Single particle tracking analysis of the chloroplast division protein FtsZ anchoring to the inner envelope membrane.

Replication of chloroplast in plant cells is an essential process that requires co-assembly of the tubulin-like plastid division proteins FtsZ1 and FtsZ2 at mid-chloroplast to form a ring structure called the Z-ring. The Z-ring is stabilized via its interaction with the transmembrane protein ARC6 on the inner envelope membrane of chloroplasts. Plants lacking ARC6 are defective in plastid division and contain only one or two enlarged chloroplasts per cell with abnormal localization of FtsZ: instead of a single Z-ring, many short FtsZ filaments are distributed throughout the chloroplast. ARC6 is thought to be the anchoring point for FtsZ assemblies. To investigate the role of ARC6 in FtsZ anchoring, the mobility of green fluorescent protein-tagged FtsZ assemblies was assessed by single particle tracking in mutant plants lacking the ARC6 protein. Mean square displacement analysis showed that the mobility of FtsZ assemblies is to a large extent characterized by anomalous diffusion behavior (indicative of intermittent binding) and restricted diffusion suggesting that besides ARC6-mediated anchoring, an additional FtsZ-anchoring mechanism is present in chloroplasts.

Oligomerization of plant FtsZ1 and FtsZ2 plastid division proteins.

FtsZ was identified in bacteria as the first protein to localize mid-cell prior to division and homologs have been found in many plant species. Bacterial studies demonstrated that FtsZ forms a ring structure that is dynamically exchanged with a soluble pool of FtsZ. Our previous work established that Arabidopsis FtsZ1 and FtsZ2-1 are capable of in vitro self-assembly into two distinct filament types, termed type-I and type-II and noted the presence of filament precursor molecules which prompted this investigation. Using a combination of electron microscopy, gel chromatography and native PAGE revealed that (i) prior to FtsZ assembly initiation the pool consists solely of dimers and (ii) during assembly of the Arabidopsis FtsZ type-II filaments the most common intermediate between the dimer and filament state is a tetramer. Three-dimensional reconstructions of the observed dimer and tetramer suggest these oligomeric forms may represent consecutive steps in type-II filament assembly and a mechanism is proposed, which is expanded to include FtsZ assembly into type-I filaments. Finally, the results permit a discussion of the oligomeric nature of the soluble pool in plants.

Protein screening using cold microwave technology

Protein detection is a common yet time-intensive task in many laboratories. Here we report a protocol that makes use of cold microwave technology to reduce the total processing time to less than 1 h with dot and Western blot applications while yielding lower background noise at similar signal strength when compared with conventional protocols. With dot blots, the time savings was accompanied by a decrease in reagent use. With Western blots, the visibility of prestained markers was maintained, in stark contrast to conventional procedures. Experiments kept at a constant temperature of 21 degrees C support the existence of a microwave radiation effect, whereas an additional thermal effect is noted when the temperature is increased to 37 degrees C from ambient. Microwave-assisted dot blotting is suggested as an effective way of facilitating large-scale screening of expressed proteins.

Improved Protein Detection Using Cold Microwave Technology

Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio would be highly beneficial to many researchers. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 45 min.

Cold Microwave-Enabled Protein Detection and Quantification

Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio is highly beneficial in many research areas. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, Western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 24-45 min.

In Situ FtsZ Mini-Ring Structure revealed by TEM Tomography and STEM

Both bacteria and chloroplasts divide by the process of binary fission that is initiated by assembly of a cytoskeletal protein FtsZ into a ring structure (Z-ring) at the division site. FtsZ assembly and Z-ring structure depend on the balance between stabilizing and destabilizing agents [1]. In chloroplasts, the absence of stabilizing agents causes the formation of additional types of assemblies, termed mini-rings [2,3]. While in vitro studies of these mini-rings revealed a diameter of ~200 nm and proposed that they could represent an energy-minimized state of FtsZ [4], their in situ structure remained elusive. Here we describe the structure of FtsZ mini-rings in Arabidopsis thaliana leaf chloroplasts using TEM tomography and STEM after high pressure freezing and freeze substitution (HPF-FS).

Arabidopsis Plastid Division Proteins FtsZ1 and FtsZ2: Macromolecular Assembly and Subunit Exchange Dynamics

The bacterial cell division protein FtsZ (Filamentous temperature sensitive Z) plays an essential role in prokaryotic division l[VS1]ocating mid-cell and forming a contractile ring. Unlike bacteria, chloroplasts harbor[VS2] two distinct families of FtsZ (1 and 2) that are[VS3] essential, non-redundant and localize to a mid-plastid ring [1,2]. The[VS4] division components in chloroplasts are a mixture of proteins displaying homology to bacterial counterparts and novel proteins that consequently demand a strikingly different division mechanism as compared to bacteria. Understanding[VS5] the mechanism of chloroplast division may have commercial applications that could improve wetmilling efficiency and produce a domestic savings of

In situ structure of FtsZ mini-rings in Arabidopsis chloroplasts

280 million annually [3].