Witman Lan

A unified taxonomy for ciliary dyneins

Erik F. Y. Hom et al. — Fri, 04 May 2012 08:49:14 PDT

The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, 54 proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologs of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles, and orthologs has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.

High-speed digital imaging of ependymal cilia in the murine brain

Karl-Ferdinand Lechtreck et al. — Fri, 21 Jan 2011 08:22:57 PST

The development and health of mammals requires proper ciliary motility. Ciliated epithelia are found in the airways, the uterus and Fallopian tubes, the efferent ducts of the testes, and the ventricular system of the brain. A technique is described for the motion analysis of ependymal cilia in the murine brain. Vibratome sections of the brain are imaged by differential interference contrast microscopy and recorded by high-speed digital imaging. Side views of individual cilia are traced to establish their bending pattern. Tracking of individual cilia recorded in top view allows determination of bend planarity and beat direction. Ciliary beat frequency is determined from line scans of image sequences. The capacity of the epithelium to move fluid and objects is revealed by analyzing the velocity of polystyrene beads added to brain sections. The technique is useful for detailed assessment of how various conditions or mutations affect the fidelity of ciliary motility at the ependyma. The methods are also applicable to other ciliated epithelia, for example, in airways.

Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella

Benjamin D. Engel et al. — Fri, 21 Jan 2011 08:22:56 PST

The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies.

IC97 is a novel intermediate chain of I1 dynein that interacts with tubulin and regulates interdoublet sliding

Maureen Wirschell et al. — Fri, 21 Jan 2011 08:22:55 PST

Our goal is to understand the assembly and regulation of flagellar dyneins, particularly the Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on the uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel IC without notable structural domains. IC97 shares homology with the murine lung adenoma susceptibility 1 (Las1) protein--a candidate tumor suppressor gene implicated in lung tumorigenesis. Multiple, independent biochemical assays determined that IC97 interacts with both alpha- and beta-tubulin subunits within the axoneme. I1-dynein assembly mutants suggest that IC97 interacts with both the IC138 and IC140 subunits within the I1-dynein motor complex and that IC97 is part of a regulatory complex that contains IC138. Microtubule sliding assays, using axonemes containing I1 dynein but devoid of IC97, show reduced microtubule sliding velocities that are not rescued by kinase inhibitors, revealing a critical role for IC97 in I1-dynein function and control of dynein-driven motility.

HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B

Karl-Ferdinand Lechtreck et al. — Fri, 21 Jan 2011 08:22:54 PST

Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii. Expression was confirmed by western blotting. Indirect immunofluorescence located all 10 fusion proteins in the flagellum; PACRG was localized to a subset of outer doublet microtubules. For some proteins, additional signal was observed in the cell body. Among the latter was FAP232-HA, which showed a spotted distribution along the flagella and an accumulation at the basal bodies. This pattern is characteristic for intraflagellar transport (IFT) proteins. FAP232-HA co-localized with the IFT protein IFT46 and co-sedimented with IFT particles in sucrose gradients. Furthermore, it co-immunoprecipitated with IFT complex B protein IFT46, but not with IFT complex A protein IFT139. We conclude that FAP232 is a novel component of IFT complex B and rename it IFT25. Homologues of IFT25 are encoded in the genomes of a subset of organisms that assemble cilia or flagella; C. reinhardtii IFT25 is 37% identical to the corresponding human protein. Genes encoding IFT25 homologues are absent from the genomes of organisms that lack cilia and flagella and, interestingly, also from those of Drosophila melanogaster and Caenorhabditis elegans, suggesting that IFT25 has a specialized role in IFT that is not required for the assembly of cilia or flagella in the worm and fly. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella

Karl-Ferdinand Lechtreck et al. — Fri, 21 Jan 2011 08:22:53 PST

In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Branch Craige et al. — Fri, 21 Jan 2011 08:22:52 PST

Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule-membrane links there. Ultrastructural analysis revealed defects in these microtubule-membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule-membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.