A motility in the eukaryotic flagellum unrelated to flagellar beating.

Abstract

We report a motiliy in the flagella of the green alga Chlamydomonas that is unrelated to dynein-based flgelar beating. This motility, referred to as intraflageUar transport, was observed as the rapid bidirectional movement of granule-like particles along the length of the ftgella. IntraflageUar transport could be experimentally separated from other, previously reported, nonbeat fgellar motilities. EM of flageUla showed groups of nonvesicular, lollipop-shaped structures positioned between the outer doublet microtubules and the flagellar membrane. Movement of these complexes along the length of the flagella may be responsible for intraflagellar transport. While reexamining these three motilities with videoenhanced differential interference-contrast (DIC) microscopy, we were surprised to find a fourth, previously unobserved, nonbeat motility within the flagella of Chlamydomonas. In prior light microscopic studies, the flagella of Chlamydomonas have always been observed as relatively featureless high-contrast rods. Therefore, the visualization of granule-like particles moving bidirectionally along the length ofthe flagella, apparently between the microtubular axoneme and flagellar membrane, was striking. In addition, improved fixation methods for EM have allowed for the routine observation, in thin section, of complexes between the flagellar membrane and the axonemal microtubules. The movement of these complexes may account for this motility, referred to as intraflagellar transport (IFT). for video-enhanced DIC microscopy because their flagella are =50%o longer than those of C. reinhardtii. The paralyzed flagellar dynein triple-mutant ida2 ida4 oda6 of C. reinhardtii was from R. Kamiya (Nagoya University). Logarithmicphase, synchronously dividing cultures were grown in minimal medium (MI) (8) on a 12 hr:12 hr light/dark cycle with continuous aeration. MATERIALS AND METHODSFlagellar Regeneration and Resorption. Flagellar regeneration was induced by pH-shock (9). After deflagellation, the cells were washed with fresh MI medium. Flagellar resorption was induced by placing the cells in a low-Ca2+, high-Na+ medium (10) or also, with C. reinhardtii, by adding 3-isobutyl-l-methylxanthine to MI medium at a final concentration of 0.5 mM (11).Video Microscopy. For all experiments, cultures were diluted 1:5 with double-distilled water and placed between two acid-washed no. 1 coverslips (Corning) supported with 1-mm plastic shims affixed with Vaseline. When required, polystyrene beads with a 0.3-,&m diameter (Polysciences), washed four times with double-distilled water, were mixed with the cells to a final dilution of 1:500. Dilution of the culture medium enhanced the attachment of beads to the flagellar membrane. All experiments were done at room temperature using a Zeiss Axiovert microscope. The optics and method ofdigital data acquisition have been detailed (12), except that the light source used in this study was passed through a fiber optic scrambler to obtain full and even illumination of the condenser (1.4 N.A.) ...

Three types of cilia including a novel 9+4 axoneme on the notochordal plate of the rabbit embryo

“…The cilium is built and maintained by an evolutionarily conserved process called intraflagellar transport (IFT). This mechanism has been first detected in the biflagellated green alga Chlamydomonas, in which particles moving up and down the axoneme were visualized (Kozminski et al, 1993). For ciliogenesis, preassembled protein complexes dock to an IFT raft, a multiprotein platform, which is transported into the cilium by heterotrimeric kinesin II motor proteins, whereas retrograde transport is facilitated by cytoplasmic dynein (Rosenbaum and Witman, 2002).…”

Eukaryotic Flagella: Variations in Form, Function, and Composition during Evolution

“…Since the discovery of IFT in Chlamydomonas just over twenty years ago (Kozminski et al 1993), the IFT pathway has been studied in many …”

IFT‐81 and IFT‐74 are required for intraflagellar transport in C. elegans

“…Cilium biogenesis and maintenance depend on a microtuble associated transport machinery: intraflagellar transport (IFT) (Kozminski et al 1993;Rosenbaum & Witman 2002;Scholey 2003). The IFT machinery conveys cargo, such as receptors and channels, to the ciliary tip by kinesin-II motors and recycles components to the basal body by a dynein motor (Rosenbaum & Witman 2002;Cole 2003;Scholey 2003;Inglis et al 2006).…”