title: Quantitative analysis of flagellar proteins in Drosophila sperm tails.
abstract: The cilium has a well-defined structure, which can still accommodate some morphological and molecular composition diversity to suit the functional requirements of different cell types. The sperm flagellum of the fruit fly Drosophila melanogaster appears as a good model to study the genetic regulation of axoneme assembly and motility, due to the wealth of genetic tools publically available for this organism. In addition, the fruit fly's sperm flagellum displays quite a long axoneme (∼1.8mm), which may facilitate both histological and biochemical analyses. Here, we present a protocol for imaging and quantitatively analyze proteins, which associate with the fly differentiating, and mature sperm flagella. We will use as an example the quantification of tubulin polyglycylation in wild-type testes and in Bug22 mutant testes, which present defects in the deposition of this posttranslational modification. During sperm biogenesis, flagella appear tightly bundled, which makes it more challenging to get accurate measurements of protein levels from immunostained specimens. The method we present is based on the use of a novel semiautomated, macro installed in the image processing software ImageJ. It allows to measure fluorescence levels in closely associated sperm tails, through an exact distinction between positive and background signals, and provides background-corrected pixel intensity values that can directly be used for data analysis.
authors.affiliation: Cell Biology, Institut Curie, UMR 144 CNRS, Paris, France.
authors.forename: Teresa
authors.initials: T
authors.lastname: Mendes Maia
authors.affiliation: Cell Biology, Institut Curie, UMR 144 CNRS, Paris, France.
authors.forename: Perrine
authors.initials: P
authors.lastname: Paul-Gilloteaux
authors.affiliation: Cell Biology, Institut Curie, UMR 144 CNRS, Paris, France.
authors.forename: Renata
authors.initials: R
authors.lastname: Basto
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics; D005407:Flagella / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008297:Male; D061848:Optical Imaging / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000235:genetics; D020543:Proteome / Q000032:analysis*; D013082:Sperm Tail / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D020543:Proteome; D014404:Tubulin
keywords: Drosophila; Flagella; Sperm individualization; fluorescence quantification; motile sperm; tubulin post-translation modifications