Chloroplast Division Protein ARC3: Effects on FtsZ2 Assembly and GTPase Activity

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by assembly of cytoskeletal FtsZ proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts except that MinC was lost and replaced by the eukaryotic ARC3. Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, light microscopy, GTPase assay and transmission electron microscopy in conjunction with single particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division: FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. 3D reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6-binding MORN domain are in close proximity and suggests a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression

ObjectiveThe mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling.MethodsVascular remodeling was induced in CD44+/+ and CD44−/− mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs.ResultsTransluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44−/− mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor.ConclusionsLMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression.Clinical RelevanceAngioplasty is widely used in clinical practice to treat various stenostic vascular disorders, but the postangioplasty reocclusion has been a big limit and the mechanism underlying the vascular remodeling remains poorly understood. LMW heparin has been a promising medicine to inhibit VSMC proliferation. However, the mechanism of LMW heparin inhibition against smooth muscle cell (SMC) proliferation and its clinical usefulness is still not clear. Our present data, which were based on in vivo and in vitro studies, suggested that LMW heparin induced higher CD44 expression in VSMCs, and through the CD44 pathway, LMW heparin significantly reduced SMC proliferation and injury-induced femoral artery remodeling. Our study clarified the roles of LMW heparin in vascular occlusive diseases. This study helps to elucidate the underlying cellular and molecular mechanism by which LMW heparin inhibits injury-induced remodeling and could promote creating new therapeutics to control the exaggerated neointimal hyperplasia

Characterization of macroautophagic flux in vivo using a leupeptin-based assay

Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice