लंबी अवधि के, उच्च संकल्प Confocal समय चूक की इमेजिंग<em> Arabidopsis अंकुर</emअंकुरण के दौरान> एपिडर्मिस
Summary
हम एक कक्ष स्लाइड्स और मीडिया का उपयोग करने के लिए विकास के कई दिनों में epidermis के confocal इमेजिंग के लिए संयंत्र बीजपत्र स्थिर, रंध्रीय भेदभाव का दस्तावेजीकरण प्रोटोकॉल का वर्णन करता है. Fluorophore टैग प्रोटीन अभिव्यक्ति और subcellular स्थानीयकरण गतिशील से लगाया जा सकता है, कोशिका विभाजन और भेदभाव सेल प्रकार के दौरान उनके संभव भूमिकाओं की समझ बढ़ रही है.
Abstract
Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.
Protocol
Representative Results
Discussion
यह समय चूक confocal तकनीक fluorescently टैग प्रोटीन अभिव्यक्ति और Arabidopsis बीजपत्र epidermis, जो ध्रुवीय और अन्य प्रोटीन गत्यात्मक रूप से बदल के मामले में उनके कार्य का एक उचित समझ के लिए महत्वपूर्ण है की व्यक्ति की कोशिका?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम Amanda Rychel POLAR :: POLAR-EGFP के निर्माण के लिए समय चूक प्रोटोकॉल और लिन Pillitteri के विकास में सहायता के लिए धन्यवाद. हम भी प्रदान करने बजे आरबी का निर्माण लिए ABRC के लिए आभारी हैं. इस प्रोटोकॉल जापान विज्ञान प्रौद्योगिकी और एजेंसी से PRESTO पुरस्कार से समर्थन के माध्यम से विकसित किया गया था. ध्रुवीय पर अनुसंधान भी वाशिंगटन रॉयल्टी रिसर्च फंड (RRF-4098) और राष्ट्रीय विज्ञान फाउंडेशन (MCB 0,855,659) के विश्वविद्यालय द्वारा समर्थित किया गया. KMP एक NSF ग्रेजुएट रिसर्च फैलो (डीजीई 0,718,124) है, और KUT एक अन्वेषक HHMI GBMF है.
Materials
| Name of reagent | Company | Catalog Number | Comments |
| Bacto Agar | BD | 214010 | |
| One-chamber slide | Nunc (Thermo Scientific) | 155360 | Or two-chamber (155379) |
| Laser scanning confocal microscope | Zeiss | LSM700 | Zen 2009 software |
| 20x objective lens | Zeiss | 420650-9901 | NA 0.8, Plan-APOCHROMAT |
| Dissecting microscope | Benz (National) | 431TBL | Illuminates from below |
| #5 forceps, biology tip | Roboz Surgical Instrument | RS-4978 | Very fine tips are critical |
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