分类： 生物学 >> 植物学 >> 植物细胞学与植物遗传学、植物形态学 提交时间： 2016-05-04
摘要： Fe deficiency is a widespread nutritional disorder in plants. The basic helix-loop-helix (bHLH) transcription factors (TFs), especially Ib subgroup bHLH TFs which are involved in iron uptake, have been identified. In this study, an IVc subgroup bHLH TF MdbHLH104 was identified and characterized as a key component in the response to Fe deficiency in apple. The overexpression of the MdbHLH104 gene noticeably increased the H+-ATPase activity under iron limitation conditions and the tolerance to Fe deficiency in transgenic apple plants and calli. Further investigation showed that MdbHLH104 proteins bonded directly to the promoter of the MdAHA8 gene, thereby positively regulating its expression, the plasma membrane (PM) H+-ATPase activity and Fe uptake. Similarly, MdbHLH104 directly modulated the expression of three Fe-responsive bHLH genes, MdbHLH38, MdbHLH39 and MdPYE. In addition, MdbHLH104 interacted with 5 other IVc subgroup bHLH proteins to coregulate the expression of the MdAHA8 gene, the activity of PM H+-ATPase and the content of Fe in apple calli. Therefore, MdbHLH104 acts together with other apple bHLH TFs to regulate Fe uptake by modulating the expression of the MdAHA8 gene and the activity of PM H+-ATPase in apple.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
摘要： Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
Li, Dong
Shao, Lin
Chen, Bi-Chang
Betzig, Eric
Zhang, Xi
Zhang, Mingshu
Xu, Pingyong
Zhang, Xi
Zhang, Mingshu
Xu, Pingyong
Zhang, Xi
Moses, Brian
Milkie, Daniel E.
Beach, Jordan R.
Hammer, John A., III
Baird, Michelle A.
Pasham, Mithun
Kirchhausen, Tomas
Pasham, Mithun
Kirchhausen, Tomas
摘要： Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and a-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
Chen, Xiulan
Wei, Shasha
Ji, Yanlong
Guo, Xiaojing
Yang, Fuquan
Chen, Xiulan
Wei, Shasha
Ji, Yanlong
Guo, Xiaojing
Yang, Fuquan
Ji, Yanlong
摘要： SILAC is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods. The great advantages of SILAC lie in its straight-forward implementation, quantitative accuracy, and reproducibility over chemical labeling or label-free quantification strategies, favoring its adoption for proteomic research. SILAC has been widely applied to characterize the proteomic changes between different biological samples, to investigate dynamic changes of protein PTMs, to distinguish specific interacting proteins in interaction proteomic analysis, and to analyze protein turnover in the proteome-wide scale. The present review summarizes the principles of SILAC technology, its applications in biological research, and the present state of this technology.
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