Flagellar synchronization is a simple alternative to cell cycle synchronization for ciliary and flagellar studies

Abstract

AbstractThe unicellular green alga,Chlamydomonas reinhardtii, is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle. The method requires inducing flagellar regeneration by amputation of the entire cell population and limiting regeneration time, which results in a maximally homogeneous distribution of flagellar length at three hours post-amputation. We find that time-limiting new protein synthesis during flagellar synchronization limits variability in the unassembled pool of limiting flagellar protein and variability in flagellar length without scaling the cell volume range. We also find that long and short flagella mutants that regenerate normally require longer and shorter synchronization times, respectively. By minimizing flagellar length variability using a simple method requiring only hours and no changes in media, flagellar synchronization facilitates the detection of small changes in flagellar length resulting from both chemical and genetic perturbations inChlamydomonas. This method increases our ability to probe the basic biology of ciliary size regulation and related disease etiologies.