The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.
Bibliographical noteFunding Information:
We thank LeeAnn Higgins, Todd Markowski, and Sue Van Riper of the Center for Mass Spectrometry and Computational Proteomics, University of Minnesota, for assistance with iTRAQ labeling, mass spectrometry, and database management. The Center is supported by multiple grants, including National Science Foundation Major Research Instrumentation Grant 9871237 and National Science Foundation Grant DBI-0215759 as described at cbs.umn.edu/cmsp/about. We also acknowledge the Minnesota Supercomputing Institute at the University of Minnesota for software support (www.msi.umn.edu) and Mark Sanders and the University Imaging Center for assistance with imaging. We give many thanks to Katherine Augspurger and Jason Sakizadeh at the University of Minnesota for assistance with microscope assays and figure preparation. We thank William Dentler (University of Kansas, Lawrenceville, KS) for the T8D9 strain and Matt Laudon and the Chlamydomonas Genetics Center (University of Minnesota, Saint Paul, MN) for other strains used in this study, as described in Supplemental Table S2. Douglas Cole (University of Idaho, Moscow, ID), Steve King (University of Connecticut, Farmington, CT), and Dennis Diener (Yale University, New Haven, CT) generously supplied antibodies, as listed in Supplemental Table S3. Preliminary reports of this work were presented at American Society for Cell Biology meetings in 2006, 2012, and 2013. This work was supported by National Institutes of Health Grant GM-055667 to M.E.P. K.V.M. was supported in part by an American Heart Association Pre-Doctoral Fellowship, a University of Minnesota Graduate School Fellowship, and a University of Minnesota Graduate School Grant to M.E.P.
© 2016 Reck et al.