Methods for Live Microscopy of Drosophila Spermatocytes

Abstract

The fruitfly Drosophila melanogaster offers a rich and varied source of differentiated cell types. This and its exceptional experimental tractability make it well suited for cell cycle or other developmental biology investigations. In particular, the sperm‐producing cells of the testes provide a powerful system for studying the mechanics of cell division. These meiotically dividing primary spermatocytes can be easily isolated from the testes of mutant or transgenic animals and maintained in short‐term primary cultures. The cells' large and flat geometry makes them amenable to a variety of live cell light‐microscopy‐based observation methods. Single‐plane transmitted light time‐lapse imaging and more advanced multi‐dimensional widefield or confocal fluorescence microscopy have been used to define the morphological and kinetic changes that accompany processes ranging from meiotic chromosome segregation to microtubule dynamics. The ability to document such dynamic changes underscores the power of live cell imaging in understanding cell physiology and function.

Key Concepts

  • Drosophila is an experimentally tractable source of different cell types.
  • Male meiotic primary spermatocytes provide a powerful model system for studying cell division.
  • These large flat cells are easily cultured for live cell light microscopy.
  • Transmitted light and fluorescence imaging methods can be used separately or in tandem to record different dynamic processes during cell division.

Keywords: live cell imaging; mitosis; meiosis; light microscopy; fluorescent protein

Figure 1. The primary spermatocyte culture system. (a) Testes (arrowheads) isolated from a pharate adult in phosphate buffered saline. (b) The aluminium slide and attached coverslip used as a culturing chamber. (c) Low magnification view of a culture. Primary spermatocyte cells entering meiosis (*) can be recognised by their large size and nuclear morphology. Also present in this culture are post‐meiotic cells before differentiation (arrowhead) as well as those that have matured into sperm as revealed by groups of flagella (arrow). Bar is 20 µm.
Figure 2. Selected frames from a multi‐dimensional recording of a spermatocyte with GFP‐tagged microtubules undergoing anaphase. At each time point, DIC transmitted light and widefield fluorescence images were acquired at six successive focal planes. The single centre‐most DIC optical section for each time point is shown. The corresponding fluorescence panels are the inverted superimposed projections of all six sections and reveal the distribution of microtubules throughout the cellular volume. The combination of transmitted light and fluorescence microscopy allows for the tracking of chromosomes (white arrowheads) as they segregate on individual bundles of shortening microtubules (black arrowheads). Bar is 20 µm.
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References

Church K and Lin HP (1985) Kinetochore microtubules and chromosome movement during prometaphase in Drosophila melanogaster spermatocytes studied in life and with the electron microscope. Chromosoma 92: 273–282.

Endow SA and Komma DJ (1996) Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd‐GFP fusion proteins. Journal of Cell Science 109: 2429–2442.

Endow SA and Komma DJ (1997) Spindle dynamics during meiosis in Drosophila oocytes. Journal of Cell Biology 137: 1321–1336.

Fleming SL and Rieder CL (2003) Flattening Drosophila cells for high‐resolution light microscopic studies of mitosis in vitro. Cell Motility and the Cytoskeleton 56: 141–146.

Goshima G and Vale RD (2003) The roles of microtubule‐based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. Journal of Cell Biology 162: 1003–1016.

Goshima G, Wollman R, Goodwin SS, et al. (2007) Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417–421.

Inoue YH, Savoian MS, Suzuki T, et al. (2004) Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression. Journal of Cell Biology 166: 49–60.

Rebollo E and Gonzalez C (2000) Visualizing the spindle checkpoint in Drosophila spermatocytes. EMBO Reports 1: 65–70.

Rebollo E, Llamazares S, Reina J and Gonzalez C (2004) Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes. PLoS Biology 2: 54–64.

Savoian MS, Goldberg ML and Rieder CL (2000) The rate of poleward chromosome motion is attenuated in Drosophila zw10 and rod mutants. Nature Cell Biology 2: 948–952.

Savoian MS and Rieder CL (2002) Mitosis in primary cultures of Drosophila melanogaster larval neuroblasts. Journal of Cell Science 115: 3061–3072.

Savoian MS and Glover DM (2014) Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila. Open Biology 4: 140047. DOI: 10.1098/rsob.140047.

Siller KH, Serr M, Steward R, Hays TS and Doe CQ (2005) Live imaging of Drosophila brain neuroblasts reveals a role for lis1/dynactin in spindle assembly and mitotic checkpoint control. Molecular Biology of the Cell 16: 5127–5140.

Theurkauf WE (1994) Premature microtubule‐dependent cytoplasmic streaming in cappuccino and spire mutant oocytes. Science 265: 2093–2096.

Vazquez J, Belmont AS and Sedat JW (2002) The dynamics of homologous chromosome pairing during male Drosophila meiosis. Current Biology 12: 1473–1483.

Further Reading

Hazelrigg T (2000) GFP and other reporters. In: Sullivan W, Ashburner M and Hawley RS, (eds). Drosophila Protocols, pp. 313–343. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Khodjakov A and Rieder CL (2006) Imaging the division process in living tissue culture cells. Methods 38: 2–16.

Matthies HJG, Clarkson M, Saint RB, Namba R and Hawley RS (2000) Analysis of meiosis in fixed and live oocytes by light microscopy. In: Sullivan W, Ashburner M and Hawley RS, (eds). Drosophila Protocols, pp. 67–85. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Rebollo E and Gonzalez C (2004) Time‐lapse imaging of male meiosis by phase‐contrast and fluorescence microscopy. Methods in Molecular Biology 247: 77–87.

Shaner NC, Patterson GH and Davidson MW (2007) Advances in fluorescent protein technology. Journal of Cell Science 120: 4247–4260.

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How to Cite close
Savoian, Matthew S(Jan 2015) Methods for Live Microscopy of Drosophila Spermatocytes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0020869.pub2]