The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.      

Cellular functions of NSF: not just SNAPs and SNAREs

N-ethylmaleimide sensitive factor (NSF) is an ATPases associated with various cellular activities protein (AAA), broadly required for intracellular membrane fusion. NSF functions as a SNAP receptor (SNARE) chaperone which binds, through soluble NSF attachment proteins (SNAPs), to SNARE complexes and utilizes the energy of ATP hydrolysis to disassemble them thus facilitating SNARE recycling. While this is a major function of NSF, it does seem to interact with other proteins, such as the AMPA receptor subunit, GluR2, and beta2-AR and is thought to affect their trafficking patterns. New data suggest that NSF may be regulated by transient post-translational modifications such as phosphorylation and nitrosylation. These new aspects of NSF function as well as its role in SNARE complex dynamics will be discussed.     

甘薯根腐病菌侵染对甘薯内源激素水平的影响

甘薯根腐病病原菌[Fusarium solani(Mart.)Sacc.f.sp.batatas McClure,简称FSB]侵染及其培养液滤液处理高敏感性甘薯品种‘胜利百号’后,引起甘薯叶片、茎尖和根部组织内源ABA含量大幅升高。其中在根部出现最早,但茎尖中积累浓度最高。侵染后甘薯叶片、茎尖和根部组织内源GA1/3含量显著低于对照。甘薯组培苗经FSB培养滤液处理9h后,ABA含量显著上升,处理15h,ABA含量呈下降趋势,而GA1/3含量在101和102稀释液处理15h(103稀释液处理12h)时出现显著上升。这些结果有助于解释甘薯根腐病株矮小不产生藤蔓,并在秋季大量现蕾开花的生理现象。     

盐敏感性高血压大鼠纹蛋白水平变化对醛固酮及p38MAPK通路的影响

摘要 目的：探讨盐敏感性高血压大鼠纹蛋白水平变化对醛固酮及p38MAPK通路的影响。方法：通过向新生大鼠皮下注射辣椒素和高盐饮食构建盐敏感性高血压大鼠模型,分别使用醛固酮（ALDO）和醛固酮、依普利酮联合给药,实验分组为：1）正常对照组;2）模型组;3）假手术组;4）ALDO组;5）ALDO+依普利酮组。通过ELISA检测大鼠血浆中ALDO含量和外周血肾素含量,通过qPCR和WB检测大鼠心脏、肾脏、血管平滑肌中striatin和p38 MAPK的mRNA和蛋白的表达变化。结果：造模后在给药前大鼠血压有不同程度的升高,给药后ALDO组和ALDO+ Eplerenone组血压均有一定程度的下降;给药后ALDO+Eplerenone组大鼠血浆中ALDO和外周血中肾素含量升高;在肾脏与Control组和Model组相比,ALDO+Eplerenone组的striatin和p38 MAPK蛋白表达水平显著升高,p38 MAPK的 mRNA表达水平显著升高;在心脏中与Model组相比,ALDO+Eplerenone组的p38 MAPK蛋白表达水平显著升高;在主动脉中与Control组和Model组相比,ALDO+Eplerenone组的striatin和p38 MAPK蛋白表达水平显著降低。结论：纹蛋白水平的改变与盐敏感高血压具有相关性,其可通过调节ALDO/MR→p38MAPK相关通路影响盐敏感性高血压。     

Proteoglycan 4 deficiency protects against glucose intolerance and fatty liver disease in diet-induced obese mice

Objective

Proteoglycan 4 (Prg4) has emerged from human association studies as a possible factor contributing to weight gain, dyslipidemia and insulin resistance. In the current study, we investigated the causal role of Prg4 in controlling lipid and glucose metabolism in mice.

LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress

We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12–30 dynes/cm²). Under the fluid shear stress, an increase of intracellular Ca²⁺ concentration of neutrophils was observed. This increase of intracellular Ca²⁺ concentration was caused by Ca²⁺ influx, and the blockage of the flux by NiCl₂ suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl₂-sensitive Ca²⁺ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress. © 1996 Wiley-Liss, Inc.     

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection

Abstract Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE ) gene of Escherichia coli , a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB -like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium .     

受脱落酸调节的拟南芥F-box基因的差异表达分析

脱落酸(Abscisic acid,ABA)是一种重要的植物激素,在种子休眠的建立、种子萌发、根发育和非生物胁迫反应过程中发挥作用。F-box蛋白是E3泛素连接酶SCF复合体的组成部分,通过特异识别和调节底物蛋白水平而调控植物生长发育过程。通过分析GEO基因芯片,筛选到38个受ABA调节的拟南芥候选F-box基因。选择其中6个F-box基因,进行实时荧光定量PCR分析。研究结果与基因芯片结果基本一致。分析启动子,发现候选基因含有大量ABA、干旱和胁迫相关的顺式作用元件。分析基因表达谱,发现部分基因在保卫细胞、种皮、花粉和衰老叶片中呈现高表达;大部分基因在ABA处理、胁迫和种子吸胀过程中表达量改变显著。这些分析结果为深入研究ABA调节植物生长发育和抗逆的分子机制提供了线索。