Radial Profile-Based Quantification of Centrosomal Proteins.

Abstract

Centrosomes are dynamic organelles critical for mitotic spindle assembly and cilia formation. Here, I describe a protocol for quantifying relative centrosomal protein abundance in <i>Drosophila melanogaster</i> embryos using radial profile analysis of fluorescence intensity. The method involves embryo collection, manual dechorionation, mounting for live imaging, confocal microscopy, and subsequent image analysis. Radial profiling allows quantification of relative protein abundance together with its spatial distribution at the centrosome, providing either relative or normalized intensity profiles. I then outline how this approach can be integrated with complementary techniques such as fluorescence recovery after photobleaching (FRAP) and super-resolution imaging, in this case, three-dimensional structured illumination microscopy (3D-SIM). Combining radial fluorescence profiling with these imaging modalities enables high-resolution, quantitative analysis of dynamic centrosome assembly in a genetically tractable system. Key features • Manual embryo dechorionation and heptane glue mounting enable reliable preparation for high-quality live imaging. • Radial profile analysis provides quantitative measurement of centrosomal protein abundance and spatial distribution. • The protocol is compatible with both confocal and 3D-SIM super-resolution microscopy, offering complementary spatial and temporal resolution.