लाइव सेल प्रवास के लिए जाते एक तीन आयामी मैट्रिक्स में प्रोटीन fluorescently टैग व्यक्त कोशिकाओं की इमेजिंग
Summary
सेल प्रवास जैसे सेलुलर प्रक्रियाओं को पारंपरिक रूप से दो आयामी, कठोर प्लास्टिक सतहों पर अध्ययन किया गया है. इस रिपोर्ट सीधे प्रोटीन स्थानीयकरण visualizing और अधिक physiologically प्रासंगिक, तीन आयामी मैट्रिक्स में पलायन कोशिकाओं में प्रोटीन गतिशीलता का विश्लेषण करने के लिए एक तकनीक का वर्णन करता है.
Abstract
Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix.
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.
Protocol
Discussion
हम यहाँ रहने कक्ष इमेजिंग का उपयोग करने के लिए एक तीन आयामी मैट्रिक्स में सेल प्रवास के तंत्र का अध्ययन करने के लिए एक विधि का वर्णन. इस तकनीक की सफलता "अच्छा" stably GFP टैग प्रोटीन व्यक्त क्लोन प्राप्त कर?…
Declarações
The authors have nothing to disclose.
Acknowledgements
हम पांडुलिपि के महत्वपूर्ण पढ़ने के लिए डॉ. अनुदान Sumida धन्यवाद. यह काम एक Beckman युवा अन्वेषक पुरस्कार (SY), एक हेलमैन परिवार नई संकाय पुरस्कार (SY), एक NIH यूरेका, कैलिफोर्निया कैंसर रिसर्च समन्वय समिति के विश्वविद्यालय द्वारा समर्थित किया गया.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Collagen, bovine, Type I | BD Biosciences | 354231 | Stock is about 3 mg/ml |
| 3-aminopropyltrimethoxysilane | Sigma Aldrich | 281778 | Dilute in water |
| glutaraldehyde | Sigma Aldrich | 340855 | Dilute in PBS |
| 1M Hepes | Invitrogen | 15630-080 | |
| Fluospheres polystyrene microspheres 1 µm, red fluorescence (580/605) | Invitrogen | F13083 | |
| Geneticin (G418) | Invitrogen | 11811-031 | |
| Culture media components: | |||
| DMEM | Invitrogen | 31600-034 | |
| Fetal Bovine Serum | Atlanta Biologicals | S115500 | |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| Kanamycin | Invitrogen | 15160-054 | |
Referências
- Shih, W., Yamada, S., Myosin, . A dependent retrograde flow drives 3D cell migration. Biophys. J. 98 (8), L29-L31 (2010).
- O’Brien, L. E., Yu, W., Tang, K., Jou, T. S., Zegers, M. M., Mostov, K. E. Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures. Methods Enzymol. 406, 676-691 (2006).
- Legant, W. R., Miller, J. S., Blakely, B. L., Cohen, D. M., Genin, G. M., Chen, C. S. Measurement of mechanical tractions exerted by cells in three-dimensional matrices. Nat. Methods. 7 (12), 969-971 (2010).
- Del Alamo, J. C., Meili, R., Alonso-Latorre, B., Rodriguez-Rodriguez, J., Aliseda, A., Firtel, R. A., Lasheras, J. C. Spatio-temporal analysis of eurkaryotic cell motility by improved force cytometry. Proc. Natl. Acad. Sci. U.S.A. 104 (33), 13343-13348 (2007).
- Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2 (6), 444-456 (2001).
- Phair, R. D., Misteli, T. Kinetic modeling approaches to in vivo imaging. Nat. Rev. Mol. Cell Biol. 2 (12), 898-907 (2001).
- Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. Fluorescent toolbox for assessing protein location and function. Science. 312 (5771), 217-224 (2006).
- Shaner, N. C., Steinbach, P. A., Tsien, R. Y. A guide to choosing fluorescent proteins. Nat. Methods. 2, 905-909 (2005).
- Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science. 294 (5547), 1708-1712 (2001).
