Summary
With current light microscopy and laboratory-level computational capability, many questions in organelle assembly and membrane trafficking that were once treated in a qualitative manner can now be treated quantitatively. We present here an overview of the principles involved in doing quantitative fluorescence microscopy. We illustrate these with examples drawn from our work with the Golgi apparatus and endosomes in cultured mammalian cells. The principles themselves can be applied to any system.
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References
Abbe, E. (1873) Beiträge zur Theorie des Mikroskops und der mikroskopishen Wahrmehmung. Arch. Microskop. Anat. 9, 413–420.
Pawley, J. B. (2006) Handbook of biological confocal microscopy. Springer, New York, NY.
Murphy, D. B. (2001) Fundamentals of light microscopy and electronic imaging. Wiley-Liss, New York, NY.
Jiang, S., Rhee, S. W., Gleeson, P. A., and Storrie, B. (2006) Capacity of the Golgi apparatus for cargo transport prior to complete assembly. Mol. Biol. Cell 17, 4105–4117.
Rhee, S. W., Starr, T., Forsten-Williams, K., and Storrie, B. (2005) The steady-state distribution of glycosyltransferaes between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10. Traffic 6, 978–990.
Röttger, S., White, J., Wandall, H. H., et al. (1998) Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. J. Cell Sci. 111, 45–60.
Starr, T., Forsten-Williams, K., and Storrie, B. (2007) Both post-Golgi and intra-Golgi cycling affect the distribution of the Golgi phosphoprotein GPP130. Traffic 8, 1265–1279.
Gniadek, T. J., and Warren, G. (2007) WatershedCounting3D: a new method for segmenting and counting punctate structures from confocal image data. Traffic 8, 339–346.
Andag, U., and Schmitt, H. D. (2003) Ds1p, an essential component of the Golgi-endoplasmic reticulum retrieval system in yeast, uses the same sequence motlf to interact with different subunits of the COPI vesicle coat protein. J. Biol. Chem. 278, 51722–51734.
Hirose, H., Arasaki, K., Dohmae, N., et al. (2004) Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi. EMBO J. 23, 1267–1278.
Sun, Y., Shestakova, A., Lupashin, V., and Storrie, B. (2007). Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis. Mol. Biol. Cell 18, 4129–4142.
Puthenveedu, M. A., Bachert, C., Puri, S., Lanni, F., and Linstedt, A. D. (2006) GM130 and GRASP65-dependent lateral cisternal fusion allows uniform Golgi-enzyme distribution. Nat. Cell Biol. 8, 238–248.
Antony, C., Cibert, C., Geraud, G., et al. (1992) The GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscope approach. J. Cell Sci. 103, 785–796.
Shima, D. T., Haldar, K., Pepperkok, R., Watson, R., and Warren, G. (1997) Partitioning of the Golgi apparatus during mitosis in living HeLa cell. J. Cell Biol. 137, 1211–1228.
Dejgaard, S.Y., Murshid, A., Dee, K. M., and Presley, J. F. (in press) Confocal microscopy based linescan methodologies for intra-Golgi localization of proteins. J. Histochem. Cytochem. 55, 709–719.
Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., and Hell, S. W. (2006) STED microscopy revels that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939.
Acknowledgments
This chapter builds on past efforts by laboratory members and was supported in part by a grant from the National Science Foundation (MCB-0549001).
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Storrie, B., Starr, T., Forsten-Williams, K. (2008). Using Quantitative Fluorescence Microscopy to Probe Organelle Assembly and Membrane Trafficking. In: Vancura, A. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 457. Humana Press. https://doi.org/10.1007/978-1-59745-261-8_13
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DOI: https://doi.org/10.1007/978-1-59745-261-8_13
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