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Differential centrifugation–based biochemical fractionation of the Drosophila adult CNS

Nature Protocols volume 9, pages 2796–2808 (2014)Cite this article

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Abstract

Drosophila is widely used as a genetic model in questions of development, cellular function and disease. Genetic screens in flies have proven to be incredibly powerful in identifying crucial components for synapse formation and function, particularly in the case of the presynaptic release machinery. Although modern biochemical methods can identify individual proteins and lipids (and their binding partners), they have typically been excluded from use in Drosophila for technical reasons. To bridge this essential gap between genetics and biochemistry, we developed a fractionation method to isolate various parts of the synaptic machinery from Drosophila, thus allowing it to be studied in unprecedented biochemical detail. This is only possible because our protocol has unique advantages in terms of enriching and preserving endogenous protein complexes. The procedure involves decapitation of adult flies, homogenization and differential centrifugation of fly heads, which allow subsequent purification of presynaptic (and to a limited degree postsynaptic) components. It is designed to require only a rudimentary knowledge of biochemical fractionation, and it takes ∼3.5 h. The yield is typically 4 mg of synaptic membrane protein per gram of Drosophila heads.

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Figure 1: Schematic workflow of the fly head fractionation with corresponding step numbers.
Figure 2: Representative illustrations from the fly head fractionation.
Figure 3: Analysis of subfractions taken during the procedure by SDS-PAGE and western blotting.

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Acknowledgements

We thank R. Jahn for the opportunity to perform experiments in his laboratory at the Max Planck Institute for Biophysical Chemistry (MPIbpc) in Göttingen, Germany. Furthermore, we thank G. Mieskes for his technical advice regarding performance and documentation of experiments. We are grateful to H. Jäckle and members of his laboratory at the MPIbpc for use of their fly facilities. In particular, we thank R. Pflanz for his help in maintaining and expanding fly stocks. We thank M. Brünner (Freie Universität Berlin) for excellent technical assistance in purifying the custom polyclonal antibodies raised against Drosophila Liprin-α and RBP, as well as in performing the respective western blots. The monoclonal antibody ab49 (against DCSP-1) was developed by E. Buchner and A. Hofbauer and was obtained from the Developmental Studies Hybridoma Bank, created by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the US National Institutes of Health and maintained at the Department of Biology, The University of Iowa. This work was funded by grants from the Deutsche Forschungsgemeinschaft to S.J.S. (SFB 958 TP A6) and by a European Research Council Starting Grant (AstroFunc: 281961 to M.G.H.).

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Authors and Affiliations

  1. Institute for Biology – Genetics, Freie Universität Berlin, Berlin, Germany

    Harald Depner, Janine Lützkendorf, Husam A Babkir & Stephan J Sigrist

  2. NeuroCure Cluster of Excellence, Charité, Berlin, Germany

    Stephan J Sigrist

  3. Laboratory of Glia Biology, Vlaams Instituut voor Biotechnologie (VIB) Center for the Biology of Disease, Katholieke Universiteit (KU) Leuven, Leuven, Belgium

    Matthew G Holt

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  1. Harald Depner
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Contributions

The experimental work was performed by H.D. and J. L., and analyzed by H.D., S.J.S. and M.G.H. H.A.B designed, cloned and purified the fusion protein construct used to generate the custom antibody against RBP that was used in this study. The project was conceived and supervised by M.G.H. and S.J.S. The manuscript was written by H.D., S.J.S and M.G.H., and commented on by all authors.

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Correspondence to Matthew G Holt.

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Integrated supplementary information

Supplementary Figure 1 Specificity test of custom polyclonal antibodies used in this study.

Western blot analysis of adult fly head extracts probed with affinity-purified (a) anti-DRBPSH3 II+III and (b) anti-DLiprin-αC-term antibodies. (a) RBP expression analysis in control (rbp heterozygous animals: S201/+) and rbp mutant lines (rbpSTOP3/S201 and rbpMinos/S201). Two exposures of the same blot to light-sensitive autoradiography films are presented: 10 seconds (upper panel) and 30 seconds (middle panel). Bands of approximately 160 kDa and 190 kDa are detected (corresponding to the predicted sizes of RBP isoforms) in control animals only. An unspecific signal at around 130 kDa is also observed (most likely due to cross-reactivity of the anti-DRBPSH3 II+III antibody with other proteins in the fly head extract). (b) Liprin-α expression analysis in the indicated wild-type control (+/+ = w1118), heterozygous liprin-α animals (lipF3ex15/+) and liprin-α mutants (lipF3ex15/lipEPexR60). A single band around 140 kDa, corresponding to the predicted size of Liprin-α, is observed in control animals and is reduced in liprin-α heterozygous animals, with only faint signal in the hypomorphic liprin-α mutant lipF3ex15/lipEPexR60. α-Tubulin is displayed as a loading control for each western blot.

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Depner, H., Lützkendorf, J., Babkir, H. et al. Differential centrifugation–based biochemical fractionation of the Drosophila adult CNS. Nat Protoc 9, 2796–2808 (2014). https://doi.org/10.1038/nprot.2014.192

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