Arabidopsis ANGUSTIFOLIA3 (AN3) is associated with the promoter of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) to regulate light‐mediated stomatal development

Light signals are perceived by multiple photoreceptors that converge to suppress the RING E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) for the regulation of stomatal development. Thus, COP1 is a point of integration between light signaling and stomatal patterning. However, how light signaling is collected into COP1 for the production and spacing of stomata is still unknown. Here, we report that the loss‐of‐function mutant of ANGUSTIFOLIA3 (AN3) delays asymmetric cell division, which leads to decreased stomatal index. Furthermore, overexpression of AN3 accelerates asymmetric cell division, which results in clusters of stomata. In addition, the stomatal development through AN3 regulation is mediated by light signaling. Finally, we find that an3 is a light‐signaling mutant, and that AN3 protein is light regulated. Self‐activation by AN3 contributes to the control of AN3 expression. Thus, AN3 is a point of collection between light signaling and stomatal patterning. Target‐gene analysis indicates that AN3 is associated with COP1 promoter for the regulation of light‐controlling stomatal development. Together, these components for regulating stomatal development form an AN3–COP1–E3 ubiquitin ligase complex, allowing the integration of light signaling into the production and spacing of stomata.     

Calcium influx signals normal flagellar RNA induction following acid shock of Chlamydomonas reinhardtii

Acid shock of Chlamydomonas results in flagellar excision and induction of flagellar protein RNAs. The magnitude of flagellar RNA accumulations after flagellar excision by mechanical shear depends on the extracel]ular Ca2+ concentration. In this report, we demonstrate that the magnitude and duration of flagellar RNA accumulations are signaled by an acid shock-induced Ca2+ influx. RNA accumulations were greater in cells acid shocked in 500 µM CaCl2 than in 200 µM CaCl2, although the accumulation durations were similar. RNA accumulations of lower magnitude and shorter duration were observed in cells in Ca2+-containing buffer treated with CdCl2. RNA accumulations were of still lower magnitude and shorter duration in cells shocked in buffer without added CaCl2 than in cells shocked in 200 or 500 µM CaCl2 or in the presence of CdCl2. RNA accumulations similar to those in cells shocked in buffer without added CaCl2 were measured in cells following acid shock in buffer containing 200 µM CaCl2 and supplemented with neomycin, ruthenium red, or LaCl3. Acid shock of the adf-1 mutant resulted in RNA accumulations of shorter duration and lower magnitude than those measured in adf-1 cells stimulated by mechanical shear. These results are consistent with an hypothesis that acid shock generates two genetically and pharmacologically distinct signals governing flagellar RNA induction; the first signal is independent of a Ca2+ influx and flagellar excision and results in low magnitude accumulations of short duration, and the second is a consequence of a Ca2+ influx and results in accumulations of high magnitude and long duration.     

Receptor-Mediated Calcium Influx in Chlamydomonas reinhardtii

ABSTRACT. Alcian blue acts as a secretagogue and chemorepellent in a variety of unicellular eukaryotes. We report that alcian blue stimulates flagellar excision and induction of RNA encoding flagellar proteins in Chlamydomonas reinhardtii . Flagellar excision by alcian blue is dependent on extracellular Ca²⁺ and is blocked by La³⁺, ruthenium red, and neomycin, and so is similar to flagellar excision by acid shock. However, the adf-l mutant excises its flagella following alcian blue treatment, but not following acid shock, thus genetically distinguishing alcian-blue-induced excision from acid-shock-induced excision. Wild-type, but not adf-1, cells regrow their flagella in the continued presence of alcian blue. Wild-type cells that regrow flagella in the presence of alcian blue fail to excise their flagella in response to either increased concentrations of alcian blue or to acid shock. Alcian blue treatment of cells also induces RNA encoding flagellar components, but in a manner distinct from other means of stimulation. These results suggest that treating Chlamydomonas with the secretagogue alcian blue initiates a Ca²⁺ influx pathway and that prolonged treatment with alcian blue desensitizes the acid-shock-activated Ca²⁺ influx pathway to acid treatment. Alcian blue will thus be a useful excitatory ligand in future studies of receptor-mediated Ca²⁺ signaling in the Chlamydomonas flagellar regeneration system.     

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.     

Endothelial microparticles-mediated transfer of microRNA-19b promotes atherosclerosis via activating perivascular adipose tissue inflammation in apoE−/− mice

Microparticles(MPs) are the major carriers of circulating microRNAs. Our previous study has shown that microRNA (miR)-19b in endothelial cell-derived microparticles (EMPs) is significantly increased in patients with unstable angina. However, little is known about the relationship between miR-19b in EMPs and the progression of atherosclerosis. The aim of the present study was to define the role and potential mechanism of miR-19b incorporated in EMPs in the development of atherosclerosis.Western-diet-fed apoE^?/? mice were injected with phosphate buffered solution(PBS), EMP carrying microRNA control(EMP^control) or miR-19b mimic (EMP^miR19b) intravenously. Systemic treatment with EMP^miR19b significantly accelerated carotid artery atherosclerosis progression by increasing lipid, macrophages and smooth muscle cells and decreasing collagen content in atherosclerotic plaque. Fluorescence-labelled EMP^miR19b injection proved that miR-19b could be transported into perivascular adipose tissue(PVAT) by EMPs. EMP^miR19b treatment also promoted inflammatory cytokines secretion and macrophages infiltration in PVAT. In further experiment, apoE^?/? mice were divided into 3 groups: EMP^controlPVAT(+), EMP^miR19bPVAT(+) and EMP^miR19bPVAT(-), based on removing or keeping pericarotid adipose tissue and injected with EMP^control or EMP^miR19b. Loss of PVAT attenuated EMP^miR19b-mediated effects on increasing carotid atherosclerosis formation and inflammatory cytokines level in plaque. EMP^miR19b inhibited suppressor of cytokine signaling 3 (SOCS3) expression in PVAT. Our findings demonstrate that miR-19b in EMPs exaggerates atherosclerosis progression by augmenting PVAT-specific inflammation proceeded by downregulating SOCS3 expression.