Dynamics of the Synechococcus elongatus cytoskeletal GTPase FtsZ yields mechanistic and evolutionary insight into cyanobacterial and chloroplast FtsZs.

The division of cyanobacteria and their chloroplast descendants is orchestrated by filamenting temperature-sensitive Z (FtsZ), a cytoskeletal GTPase that polymerizes into protofilaments that form a "Z ring" at the division site. The Z ring has both a scaffolding function for division-complex assembly and a GTPase-dependent contractile function that drives cell or organelle constriction. A single FtsZ performs these functions in bacteria, whereas in chloroplasts, they are performed by two copolymerizing FtsZs, called AtFtsZ2 and AtFtsZ1 in Arabidopsis thaliana, which promote protofilament stability and dynamics, respectively. To probe the differences between cyanobacterial and chloroplast FtsZs, we used light scattering to characterize the in vitro protofilament dynamics of FtsZ from the cyanobacterium Synechococcus elongatus PCC 7942 (SeFtsZ) and investigate how coassembly of AtFtsZ2 or AtFtsZ1 with SeFtsZ influences overall dynamics. SeFtsZ protofilaments assembled rapidly and began disassembling before GTP depletion, whereas AtFtsZ2 protofilaments were far more stable, persisting beyond GTP depletion. Coassembled SeFtsZ-AtFtsZ2 protofilaments began disassembling before GTP depletion, similar to SeFtsZ. In contrast, AtFtsZ1 did not alter disassembly onset when coassembled with SeFtsZ, but fluorescence recovery after photobleaching analysis showed it increased the turnover of SeFtsZ subunits from SeFtsZ-AtFtsZ1 protofilaments, mirroring its effect upon coassembly with AtFtsZ2. Comparisons of our findings with previous work revealed consistent differences between cyanobacterial and chloroplast FtsZ dynamics and suggest that the scaffolding and dynamics-promoting functions were partially separated during evolution of two chloroplast FtsZs from their cyanobacterial predecessor. They also suggest that chloroplasts may have evolved a mechanism distinct from that in cyanobacteria for promoting FtsZ protofilament dynamics.

The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro.

Bacterial cell and chloroplast division are driven by a contractile "Z ring" composed of the tubulin-like cytoskeletal GTPase FtsZ. Unlike bacterial Z rings, which consist of a single FtsZ, the chloroplast Z ring in plants is composed of two FtsZ proteins, FtsZ1 and FtsZ2. Both are required for chloroplast division in vivo, but their biochemical relationship is poorly understood. We used GTPase assays, light scattering, transmission electron microscopy, and sedimentation assays to investigate the assembly behavior of purified Arabidopsis thaliana (At) FtsZ1 and AtFtsZ2 both individually and together. Both proteins exhibited GTPase activity. AtFtsZ2 assembled relatively quickly, forming protofilament bundles that were exceptionally stable, as indicated by their sustained assembly and slow disassembly. AtFtsZ1 did not form detectable protofilaments on its own. When mixed with AtFtsZ2, AtFtsZ1 reduced the extent and rate of AtFtsZ2 assembly, consistent with its previously demonstrated ability to promote protofilament subunit turnover in living cells. Mixing the two FtsZ proteins did not increase the overall GTPase activity, indicating that the effect of AtFtsZ1 on AtFtsZ2 assembly was not due to a stimulation of GTPase activity. However, the GTPase activity of AtFtsZ1 was required to reduce AtFtsZ2 assembly. Truncated forms of AtFtsZ1 and AtFtsZ2 consisting of only their conserved core regions largely recapitulated the behaviors of the full-length proteins. Our in vitro findings provide evidence that FtsZ1 counterbalances the stability of FtsZ2 filaments in the regulation of chloroplast Z-ring dynamics and suggest that restraining FtsZ2 self-assembly is a critical function of FtsZ1 in chloroplasts.

Allelic Variation in the Chloroplast Division Gene FtsZ2-2 Leads to Natural Variation in Chloroplast Size.

Chloroplast size varies considerably in nature, but the underlying mechanisms are unknown. By exploiting a near-isogenic line population derived from a cross between the Arabidopsis (Arabidopsis thaliana) accessions Cape Verde Islands (Cvi-1), which has larger chloroplasts, and Landsberg erecta (Ler-0), with smaller chloroplasts, we determined that the large-chloroplast phenotype in Cvi-1 is associated with allelic variation in the gene encoding the chloroplast-division protein FtsZ2-2, a tubulin-related cytoskeletal component of the contractile FtsZ ring inside chloroplasts. Sequencing revealed that the Cvi-1 FtsZ2-2 allele encodes a C-terminally truncated protein lacking a region required for FtsZ2-2 interaction with inner-envelope proteins, and functional complementation experiments in a Columbia-0 ftsZ2-2 null mutant confirmed this allele as causal for the increased chloroplast size in Cvi-1. Comparison of FtsZ2-2 coding sequences in the 1001 Genomes database showed that the Cvi-1 allele is rare and identified additional rare loss-of-function alleles, including a natural null allele, in three other accessions, all of which had enlarged-chloroplast phenotypes. The ratio of nonsynonymous to synonymous substitutions was higher among the FtsZ2-2 genes than among the two other FtsZ family members in Arabidopsis, FtsZ2-1, a close paralog of FtsZ2-2, and the functionally distinct FtsZ1-1, indicating more relaxed constraint on the FtsZ2-2 coding sequence than on those of FtsZ2-1 or FtsZ1-1 Our results establish that allelic variation in FtsZ2-2 contributes to natural variation in chloroplast size in Arabidopsis, and they also demonstrate that natural variation in Arabidopsis can be used to decipher the genetic basis of differences in fundamental cell biological traits, such as organelle size.

ARC3 Activation by PARC6 Promotes FtsZ-Ring Remodeling at the Chloroplast Division Site.

Chloroplast division is initiated by assembly of the stromal Z ring, composed of cytoskeletal Filamenting temperature-sensitive Z (FtsZ) proteins. Midplastid Z-ring positioning is governed by the chloroplast Min (Minicell) system, which inhibits Z-ring assembly everywhere except the division site. The central Min-system player is the FtsZ-assembly inhibitor ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3). Here, we report Arabidopsis (Arabidopsis thaliana) chloroplasts contain two pools of ARC3: one distributed throughout the stroma, which presumably fully inhibits Z-ring assembly at nondivision sites, and the other localized to a midplastid ring-like structure. We show that ARC3 is recruited to the middle of the plastid by the inner envelope membrane protein PARALOG OF ARC6 (PARC6). ARC3 bears a C-terminal Membrane Occupation and Recognition Nexus (MORN) domain; previous yeast two-hybrid experiments with full-length and MORN-truncated ARC3 showed the MORN domain mediates ARC3-PARC6 interaction but prevents ARC3-FtsZ interaction. Using yeast three-hybrid experiments, we demonstrate that the MORN-dependent ARC3-PARC6 interaction enables full-length ARC3 to bind FtsZ. The resulting PARC6/ARC3/FtsZ complex enhances the dynamics of Z rings reconstituted in a heterologous system. Our findings lead to a model whereby activation of midplastid-localized ARC3 by PARC6 facilitates Z-ring remodeling during chloroplast division by promoting Z-ring dynamics and reveal a novel function for MORN domains in regulating protein-protein interactions.

Tissue- and isoform-specific phytochrome regulation of light-dependent anthocyanin accumulation in Arabidopsis thaliana.

Phytochromes regulate light- and sucrose-dependent anthocyanin synthesis and accumulation in many plants. Mesophyll-specific phyA alone has been linked to the regulation of anthocyanin accumulation in response to far-red light in Arabidopsis thaliana. However, multiple mesophyll-localized phytochromes were implicated in the photoregulation of anthocyanin accumulation in red-light conditions. Here, we report a role for mesophyll-specific phyA in blue-light-dependent regulation of anthocyanin levels and novel roles for individual phy isoforms in the regulation of anthocyanin accumulation under red illumination. These results provide new insight into spatial- and isoform-specific regulation of pigmentation by phytochromes in A. thaliana.

The pathogen-actin connection: a platform for defense signaling in plants.

The cytoskeleton, a dynamic network of cytoplasmic polymers, plays a central role in numerous fundamental processes, such as development, reproduction, and cellular responses to biotic and abiotic stimuli. As a platform for innate immune responses in mammalian cells, the actin cytoskeleton is a central component in the organization and activation of host defenses, including signaling and cellular repair. In plants, our understanding of the genetic and biochemical responses in both pathogen and host that are required for virulence and resistance has grown enormously. Additional advances in live-cell imaging of cytoskeletal dynamics have markedly altered our view of actin turnover in plants. In this review, we outline current knowledge of host resistance following pathogen perception, both in terms of the genetic interactions that mediate defense signaling, as well as the biochemical and cellular processes that are required for defense signaling.

Regulation of lipopolysaccharide-induced inflammatory response and endotoxemia by beta-arrestins.

Beta-arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that beta-arrestin-1 (beta-arr-1) and -2 knockout (KO) mice are protected from TLR4-mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild-type (WT) and beta-arr-1 and -2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS-induced inflammatory cytokine levels in the plasma were markedly decreased in both beta-arr-1 and -2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11(b+) and CD11(b-) populations) from WT, beta-arr-1, and -2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS-induced inflammatory cytokines were significantly blocked in both splenocyte populations from the beta-arr-2 KO compared to the WT mice. This effect in the beta-arr-1 KO mice, however, was restricted to the CD11(b-) splenocytes. Our studies further indicate that regulation of cytokine production by beta-arrestins is likely independent of MAPK and IkappaBalpha-NFkappaB pathways. Our results, however, suggest that LPS-induced chromatin modification is dependent on beta-arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by beta-arrestins in vivo. Taken together, these results indicate that beta-arr-1 and -2 mediate LPS-induced cytokine secretion in a cell-type specific manner and that both beta-arrestins have overlapping but non-redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice.

G-protein-coupled-receptor kinases mediate TNFα-induced NFκB signalling via direct interaction with and phosphorylation of IκBα.

Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in Raw264.7 macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibits TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene, macrophage inflammatory protein-1ß. Consistent with these results, over-expression of GRK2 or 5 enhances TNFα-induced NFκB activity. In addition,we show that GRK2 and 5 interact with IκBα via the N-terminal domain of IκBα and that IκBα isa substrate for GRK2 and 5 in vitro. Furthermore, we also find that GRK5 but not GRK2 phosphorylates IκBα at the same amino acid residues (Ser32/36) as that of IKKß. Interestingly,associated with these results, knockdown of IKKß in Raw264.7 macrophages did not affect TNFα-induced IκBα phosphorylation. Taken together, these results demonstrate that both GRK2 and 5 are important and novel mediators of a non-traditional IκBα-NFκB signaling pathway.