TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella

Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.00

The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34 degrees C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34 degrees C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited

Architecture and Assembly of Chlamydomonas Flagella

Eukaryotic cilia and flagella play crucial roles in development, signaling, and motility, and their proper function is essential to many organisms, including all vertebrates. Cilia and flagella are microtubule based, membrane bound structures that protrude from the surface of the cell, and they possess a highly stereotyped, precise internal geometry. This internal structure must be correctly built and maintained for the proper functioning of these organelles, and perturbations to the structure can result in altered functionality and disease states in humans. Their extension out away from the cell body and tendency to vary in only a single dimension - their length - also make cilia and flagella ideal model organelles for the study of organelle size control. Proper size is crucial to the functioning of a cellular organelle, however, very little is understood about size control is achieved. These structures are uniquely situated to teach us more about the mechanisms cells use to regulate the size of their organelles. This dissertation seeks to further elucidate how the structure of cilia and flagella is established and maintained using the green alga Chlamydomonas. Chlamydomonas has served as an excellent model organism for the study of flagella in the past and is amenable to a wide range of genetic and biochemical experiments. Their flagella make ideal structures to use as models as they are essentially identical to those found in vertebrates, have an highly precise geometry, and mutants strains with altered internal structure and length are readily available. In this work, I first review the state of knowledge about length control of flagella in Chlamydomonas. I use Chlamydomonas mutant strains with altered flagellar length to investigate the mechanisms that result in this phenotype, and to examine how both intrinsic and extrinsic biological noise may affect these cellular structures. Finally, I identify new components of the Chlamydomonas central pair structure. Collectively, this work furthers our understanding of flagellar architecture and motility

Analysis of biological noise in the flagellar length control system.

Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas, finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry

Analysis of biological noise in the flagellar length control system.

Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas, finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry