Abstract
The success of meiosis depends on intricate coordination of a series of unique cellular processes to ensure proper chromosome segregation. Many proteins involved in these cellular events are directly or indirectly associated with chromosomes, especially those required for homologous recombination. These meiotic processes have been explored extensively by conventional light microscopy. However, many features of interest, such as chromatin organization, recombination nodules, or the synaptonemal complex are beyond the resolution of conventional wide-field microscopy. Moreover, in most sample preparation techniques for light microscopy, meiotic cells are squashed, which destroys the spatial organization of the nucleus. Here, I describe a protocol to analyze maize meiotic chromosomes by three-dimensional structured illumination microscopy (3D-SIM), a recently developed high-resolution microscopy technique. This protocol can be used to examine protein localizations at a high resolution level by immunofluorescence.
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References
Harper L, Golubovskaya I, Cande WZ (2004) A bouquet of chromosomes. J Cell Sci 117:4025–4032
Hunter N (2006) Meiotic recombination. In: Aguilera A, Rothstein R (eds) Topics in current genetics: molecular genetics of recombination. Springer, Heidelberg, pp 381–442
Zickler D, Kleckner N (1999) Meiotic chromosomes: integrating structure and function. Annu Rev Genet 33:603–754
Hauf S, Watanabe Y (2004) Kinetochore orientation in mitosis and meiosis. Cell 119:317–327
Rieder CL (1999) Methods in cell biology: mitosis and meiosis. Academic, San Diego, CA
Hamant O, Ma H, Cande WZ (2006) Genetics of meiotic prophase I in plants. Annu Rev Plant Biol 57:267–302
Mercier R, Grelon M (2008) Meiosis in plants: ten years of gene discovery. Cytogenet Genome Res 120:281–290
Toomre D, Bewersdorf J (2010) A new wave of cellular imaging. Annu Rev Cell Dev Biol 26:285–314
Abbe E (1873) Beitrage zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv Mikroskop Anatomie 9:413–468
Gu M (1996) Principles of three-dimesional imaging in confocal microscopes. World Scientific, Singapore
Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A 97:8206–8210
Willig KI, Kellner RR, Medda R, Hein B, Jakobs S, Hell SW (2006) Nanoscale resolution in GFP-based microscopy. Nat Methods 3:721–723
Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645
Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272
Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795
Egner A, Geisler C, von Middendorff C, Bock H, Wenzel D, Medda R et al (2007) Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J 93:3285–3290
Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813
Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82–87
Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P et al (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332–1336
Gustafsson MG, Shao L, Carlton PM, Wang CJ, Golubovskaya IN, Cande WZ et al (2008) Three-dimensional resolution doubling in widefield fluorescence microscopy by structured illumination. Biophys J 94:4957–4970
Gustafsson MG (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A 102:13081–13086
Shao L, Kner P, Rego EH, Gustafsson MG (2011) Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods 8:1044–1046
Kafri O, Glatt I (1990) The physics of moiré metrology. Wiley, New York, NY
Wang CR, Carlton PM, Golubovskaya IN, Cande WZ (2009) Interlock formation and coiling of meiotic chromosome axes during synapsis. Genetics 183:905–915
Urata Y, Parmelee SJ, Agard DA, Sedat JW (1995) A three-dimensional structural dissection of Drosophila polytene chromosomes. J Cell Biol 131:279–295
Bass HW, Marshall WF, Sedat JW, Agard DA, Cande WZ (1997) Telomeres cluster de novo before the initiation of synapsis: a three-dimensional spatial analysis of telomere positions before and during meiotic prophase. J Cell Biol 137:5–18
Wignall SM, Deehan R, Maresca TJ, Heald R (2003) The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts. J Cell Biol 161:1041–1051
Chang MT, Neuffer MG (1994) Chromosomal behavior during microsporogenesis. In: Freeling M, Walbot V (eds) The maize handbook. Springer, New York, NY, pp 460–475
Acknowledgments
I thank Zac Cande (UC Berkeley) for supporting this protocol development, as well as John Sedat and Jennifer Feng (UCSF) for access to the SIM microscope. I thank Peter Carlton for help in image processing.
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Wang, CJ.R. (2013). Analyzing Maize Meiotic Chromosomes with Super-Resolution Structured Illumination Microscopy. In: Pawlowski, W., Grelon, M., Armstrong, S. (eds) Plant Meiosis. Methods in Molecular Biology, vol 990. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-333-6_7
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DOI: https://doi.org/10.1007/978-1-62703-333-6_7
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