Dennis R. Diener

Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles

Intraflagellar transport (IFT) is a motility in which particles composed of at least 17 polypeptides move underneath the flagellar membrane. Anterograde (outward) and retrograde (inward) movements of these IFT particles are mediated by FLA10 kinesin-II and cytoplasmic dynein DHC1b, respectively. Mutations affecting IFT particle polypeptides or motors result in the inability to assemble flagella. IFT particles and the motors moving them are located principally around the basal bodies as well as in the flagella. Here, we clone the cDNA encoding one of the IFT particle proteins, IFT52, and show by immunofluorescence that while some IFT52 is in the flagella, the majority is found in two horseshoe-shaped rings around the basal bodies. Immunoelectron microscopy indicates that IFT52 is associated with the periphery of the transitional fibers, which extend from the distal portion of the basal body to the cell membrane and demarcate the entrance to the flagellar compartment. This localization suggests that the transitional fibers form a docking complex for the IFT particles destined for the flagellum. Finally, the flagellaless mutant bld1 completely lacks IFT52 due to a deletion in the gene encoding IFT52.

Intraflagellar transport (IFT) cargo IFT transports flagellar precursors to the tip and turnover products to the cell body

Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.

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

Entry and exit of proteins into flagella is gauged by CEP290 in the transition zone.

Intraflagellar Transport Protein 27 Is a Small G Protein Involved in Cell-Cycle Control

BACKGROUND Intraflagellar transport (IFT) is a motility process operating between the ciliary/flagellar (interchangeable terms) membrane and the microtubular axoneme of motile and sensory cilia. Multipolypeptide IFT particles, composed of complexes A and B, carry flagellar precursors to their assembly site at the flagellar tip (anterograde) powered by kinesin, and turnover products from the tip back to the cytoplasm (retrograde) driven by cytoplasmic dynein. IFT is essential for the assembly and maintenance of almost all eukaryotic cilia and flagella, and mutations affecting either the IFT motors or the IFT particle polypeptides result in the inability to assemble normal flagella or in defects in the sensory functions of cilia. RESULTS We found that the IFT complex B polypeptide, IFT27, is a Rab-like small G protein. Reduction of the level of IFT27 by RNA interference reduces the levels of other complex A and B proteins, suggesting that this protein is instrumental in maintaining the stability of both IFT complexes. Furthermore, in addition to its role in flagellar assembly, IFT27 is unique among IFT polypeptides in that its partial knockdown results in defects in cytokinesis and elongation of the cell cycle and a more complete knockdown is lethal. CONCLUSION IFT27, a small G protein, is one of a growing number of flagellar proteins that are now known to have a role in cell-cycle control.

Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella

To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2–GFP, about 10% of flagellar CrPKD2–GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2–GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.

Electron-tomographic analysis of intraflagellar transport particle trains in situ

Ultrastructural study of Chlamydomonas cilia shows that anterograde IFT particles form trains that are long and narrow, while retrograde IFT form short, compact particle trains.

The Cilium Secretes Bioactive Ectosomes

The release of membrane vesicles from the surface of cells into their surrounding environment is now recognized as an important pathway for the delivery of proteins to extracellular sites of biological function. Membrane vesicles of this kind, termed exosomes and ectosomes, are the result of active processes and have been shown to carry a wide array of biological effector molecules that can play roles in cell-to-cell communication and remodeling of the extracellular space. Degradation of the extracellular matrix (ECM) through the regulated release of proteolytic enzymes is a key process for development, morphogenesis, and cell migration in animal and plant cells. Here we show that the unicellular alga Chlamydomonas achieves the timely degradation of its mother cell wall, a type of ECM, through the budding of ectosomes directly from the membranes of its flagella. Using a combination of immunoelectron microscopy, immunofluorescence microscopy, and functional analysis, we demonstrate that these vesicles, which we term ciliary ectosomes, act as carriers of the proteolytic enzyme necessary for the liberation of daughter cells following mitosis. Chlamydomonas has proven to be the key unicellular model for the highly conserved mechanisms of mammalian cilia, and our results suggest that cilia may be an underappreciated source of bioactive, extracellular membrane vesicles.

Cryoelectron tomography of radial spokes in cilia and flagella

Cryo-EM tomography of wild-type and mutant cilia and flagella from Tetrahymena and Chlamydomonas reveals new information on the substructure of radial spokes.

The ubiquitin conjugation system is involved in the disassembly of cilia and flagella

The disassembly of cilia and flagella is linked to the cell cycle and environmental cues. We have found that ubiquitination of flagellar proteins is an integral part of flagellar disassembly. Free ubiquitin and the ubiquitin-conjugating enzyme CrUbc13 are detected in flagella, and several proteins are ubiquitinated in isolated flagella when exogenous ubiquitin and adenosine triphosphatase are added, suggesting that the ubiquitin conjugation system operates in flagella. Levels of ubiquitinated flagellar proteins increase during flagellar resorption, especially in intraflagellar transport (IFT) mutants, suggesting that disassembly products are labeled with ubiquitin and transported to the cell body by IFT. Substrates of the ubiquitin conjugation system include α-tubulin (but not β-tubulin), a dynein subunit (IC2), two signaling proteins involved in the mating process, cyclic guanosine monophosphate–dependent kinase, and the cation channel polycystic kidney disease 2. Ubiquitination of flagellar proteins is enhanced early in mating, suggesting that ubiquitination also plays an active role in regulating signaling pathways in flagella.

Target-of-rapamycin complex 1 (Torc1) signaling modulates cilia size and function through protein synthesis regulation

The cilium serves as a cellular antenna by coordinating upstream environmental cues with numerous downstream signaling processes that are indispensable for the function of the cell. This role is supported by the revelation that defects of the cilium underlie an emerging class of human disorders, termed “ciliopathies.” Although mounting interest in the cilium has demonstrated the essential role that the organelle plays in vertebrate development, homeostasis, and disease pathogenesis, the mechanisms regulating cilia morphology and function remain unclear. Here, we show that the target-of-rapamycin (TOR) growth pathway modulates cilia size and function during zebrafish development. Knockdown of tuberous sclerosis complex 1a (tsc1a), which encodes an upstream inhibitor of TOR complex 1 (Torc1), increases cilia length. In contrast, treatment of embryos with rapamycin, an inhibitor of Torc1, shortens cilia length. Overexpression of ribosomal protein S6 kinase 1 (S6k1), which encodes a downstream substrate of Torc1, lengthens cilia. Furthermore, we provide evidence that TOR-mediated cilia assembly is evolutionarily conserved and that protein synthesis is essential for this regulation. Finally, we demonstrate that TOR signaling and cilia length are pivotal for a variety of downstream ciliary functions, such as cilia motility, fluid flow generation, and the establishment of left-right body asymmetry. Our findings reveal a unique role for the TOR pathway in regulating cilia size through protein synthesis and suggest that appropriate and defined lengths are necessary for proper function of the cilium.