具有可变角度荧光显微镜植物细胞表面动力学的实时成像
Summary
该协议的目的是演示如何监测荧光标记蛋白动力学上的植物细胞表面与可变角荧光显微镜，表示闪烁GFP标记PATROL1，一个膜运输蛋白质的点，在所述气孔络合物在细胞皮质拟南芥。
Abstract
A plant’s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed ‘residence time’) of the dot-like structures. The use of VAEM is discussed in the context of this example.
Introduction
The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell’s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant’s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass–water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.
Protocol
Representative Results
Discussion
在这个视频文章，方案给出了监测和测量GFP-PATROL1点对拟南芥的气孔复杂的动态行为。如图所示，VAEM观察是一个强大的工具，用于植物细胞表面的实时成像。下这里使用的GFP-PATROL1监测实验条件下，有用于视频捕捉1分钟的样品中很少荧光光漂白，因为高度敏感的EM-CCD的允许使用在VAEM光学相对弱激发激光的。激光定心和聚焦，每次实验开始前，对于成功VAEM观察重要。用户应该由专业的工作?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.
Materials
| Inverted microscope | Olympus | IX-73 | |
| TIRF unit | Olympus | IX3-RFAEVAW | |
| TIRF objective lens | Olympus | UAPON 100 × OTIRF | NA = 1.49 |
| Laser angle control box | Chuo Seiki | QT-AK | |
| Optically pumped semiconductor laser | Coherent | SapphireTM LP USB 488-20 CDRH Laser | |
| 510–550 nm band-pass filter | Olympus | U-FBNA | |
| EM CCD camera | Hamamatsu Photonics | ImagEM C9100-13 | |
| C-mount camera magnification change unit | Olympus | U-TVCAC | |
| MetaMorph software | Molecular Devices | MetaMorph version 7.7.11.0 | |
| TIRF microscopy manual | Olympus | AX7385 | Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012) |
| Immersion oil | Olympus | Immersion Oil Typr-F | ne = 1.518 (23 degrees) |
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