title: Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies.
abstract: The unicellular green algaChlamydomonas reinhardtiiis 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. This results in a maximally homogeneous distribution of flagellar lengths at 3 h postamputation. We found 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 affecting the range of cell volumes. We also found 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.IMPORTANCECilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer.Chlamydomonas reinhardtiiis an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of small changes in ciliary length by minimizing variability in the population. We find that this method alters the key relationship between cell size and the amount of protein accumulated for flagellar growth. This provides a rapid alternative to traditional methods of cell synchronization for uncovering novel regulators of cilia.
authors.affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Soumita
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, USA; Department of Ophthalmology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Prachee
authors.initials: P
authors.lastname: Avasthi
keywords: Chlamydomonas; flagellar length; precursor pool; regeneration; synchronization