Effect of lithium on flagellar length in Chlamydomonas reinhardtii.

Nakamura, Shōgo; Takino, H.; Kojima, M.

doi:10.1247/csf.12.369

Cited by 49 publications

(50 citation statements)

References 17 publications

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“…This shortening was rapidly reversed by the readdition of Ca 2ϩ to the culture medium. Finally, the elongation of flagella was induced by the treatment of cells with lithium (38,60). Taken together, these observations suggest that one or more signal transduction pathways control flagellar length in Chlamydomonas.…”

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confidence: 70%

Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii

2004

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Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12: [369][370][371][372][373][374] 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.How cells regulate the size of their organelles is poorly understood. Cilia and flagella are of particular interest, as the precise regulation of their length is critical to their function. The importance of the correct assembly of these organelles, including maintenance of the appropriate length, is illustrated by studies involving intraflagellar transport (IFT). IFT is required for the assembly and maintenance of cilia and flagella in organisms ranging from Chlamydomonas reinhardtii (where it was first identified) to sea urchins, Tetrahymena thermophila, Caenorhabditis elegans, and mice (10). In the absence of IFT, cilia and flagella fail to form correctly, resulting in various human diseases, including polycystic kidney disease and retinal degeneration, and anomalous determination of the left-right axis during development (51). Ultrastructural studies of children suffering from a particular form of primary ciliary dyskinesia revealed the presence of abnormally long cilia within their respiratory tracts, resulting in recurrent respiratory infections (1, 39). This observation suggests that not only do cilia and flagella need to assemble properly but that they also need to be of appropriate and defined lengths.The green alga Chlamydomonas reinhardtii makes an ideal model organism for studies involving ciliary and flagellar assembly and length control. Chlamydomonas has two apically localized flagella that are maintained at equal lengths. Moreover, within a population of wild-type cells, flagellar lengths fall within a very narrow range. The maintenance of flagella of equal lengths as an active process was first demonstrated in the "long-zero" experiments of Rosenbaum et al. (52). In these experiments, cells were deflagellated under conditions that amputated only one...

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confidence: 70%

Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii

2004

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“…LiCl treatment induces flagellar elongation in Chlamydomonas and in mammalian cells (Nakamura et al, 1987;Ou et al, 2009;Wilson and Lefebvre, 2004) and protein synthesis is not required (Wilson and Lefebvre, 2004). Indeed, 25 mM LiCl treatment induced flagellar elongation (Fig.…”

Section: Inhibition Of Flagellar Regeneration By Taxol Stabilization mentioning

confidence: 89%

Flagellar regeneration requires cytoplasmic microtubule depolymerization and kinesin-13

Wang

Piao

Cao

et al. 2013

Journal of Cell Science

102

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SummaryIn ciliated cells, two types of microtubules can be categorized: cytoplasmic and axonemal. It has been shown that axonemal tubulins come from a 'cytoplasmic pool' during cilia regeneration. However, the identity and regulation of this 'pool' is not understood. Previously, we have shown that Chlamydomonas kinesin-13 (CrKin13) is phosphorylated during flagellar regeneration, and required for proper flagellar assembly. In the present study, we show that CrKin13 regulates depolymerization of cytoplasmic microtubules to control flagellar regeneration. After flagellar loss and before flagellar regeneration, cytoplasmic microtubules were quickly depolymerized, which was evidenced by the appearance of sparse and shorter microtubule arrays and increased free tubulins in the cell body. Knockdown of CrKin13 expression by RNA interference inhibited depolymerization of cytoplasmic microtubules and impaired flagellar regeneration. In vitro assay showed that CrKin13 possessed microtubule depolymerization activity. CrKin13 underwent phosphorylation during microtubule depolymerization, and phosphorylation induced targeting of CrKin13 to microtubules. The phosphorylation of CrKin13 occurred at residues S100, T469 and S522 as determined by mass spectrometry. Abrogation of CrKin13 phosphorylation at S100 but not at other residues by inducing point mutation prevented CrKin13 targeting to microtubules. We propose that CrKin13 depolymerizes cytoplasmic microtubules to provide tubulin precursors for flagellar regeneration.

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“…Pharmacological and genetic perturbations modify the length-dependent injection rate but do not change injection dynamics. We used the lf4 mutation (50) (kinase) and lithium treatment (51) [GSK3 inhibitor (47)] to study the effects of long flagella on the IFT injector, and we used cycloheximide (52) (protein synthesis inhibitor) to study the effects of short flagella. (A) Examining the injection rate as a function of flagellar length showed a drastic effect on the normal length-dependent injection rate: Control trend (n = 168 flagella; blue circles, black solid line with extrapolation dashed) shows a decrease in the injection rate for longer flagella.…”

Section: Discussionmentioning

confidence: 99%

Avalanche-like behavior in ciliary import

Ludington

Wemmer

Lechtreck

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

118

201

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Cilia and flagella are microtubule-based organelles that protrude from the cell body. Ciliary assembly requires intraflagellar transport (IFT), a motile system that delivers cargo from the cell body to the flagellar tip for assembly. The process controlling injections of IFT proteins into the flagellar compartment is, therefore, crucial to ciliogenesis. Extensive biochemical and genetic analyses have determined the molecular machinery of IFT, but these studies do not explain what regulates IFT injection rate. Here, we provide evidence that IFT injections result from avalanche-like releases of accumulated IFT material at the flagellar base and that the key regulated feature of length control is the recruitment of IFT material to the flagellar base. We used total internal reflection fluorescence microscopy of IFT proteins in live cells to quantify the size and frequency of injections over time. The injection dynamics reveal a power-law tailed distribution of injection event sizes and a negative correlation between injection size and frequency, as well as rich behaviors such as quasiperiodicity, bursting, and long-memory effects tied to the size of the localized load of IFT material awaiting injection at the flagellar base, collectively indicating that IFT injection dynamics result from avalanche-like behavior. Computational models based on avalanching recapitulate observed IFT dynamics, and we further show that the flagellar Ras-related nuclear protein (Ran) guanosine 5'-triphosphate (GTP) gradient can in theory act as a flagellar length sensor to regulate this localized accumulation of IFT. These results demonstrate that a self-organizing, physical mechanism can control a biochemically complex intracellular transport pathway.Chlamydomonas | self-organization | nuclear import | long flagella mutants | power spectrum C ilia and flagella generate fluid flows and mediate cell signaling (1), and ciliary length defects cause a wide range of congenital human diseases. Many of these defects arise from mutations in intraflagellar transport (IFT) proteins, which are required to build and maintain the length of cilia and flagella (2). The IFT proteins form complexes called IFT trains that haul cargo to the ciliary tip for assembly (3-7). IFT trains first localize to the basal body (8) and then enter the cilium as a group in an injection event. Understanding the IFT injection process is critical to understanding ciliary length control because the injection rate sets the overall amount of transport that in turn determines the rate of steadystate flagellar assembly (9).A previous report indicated that entry of new IFT trains is periodic (10), suggesting that a biochemical oscillator may regulate IFT injection. However, the biochemical components of this hypothetical oscillator are currently unknown. Components of the gate controlling entry into the cilium are being identified (4, 5), but identifying the oscillating components themselves could be an extremely difficult biochemical problem because it is not obvious how to determ...

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Effect of lithium on flagellar length in Chlamydomonas reinhardtii.

Cited by 49 publications

References 17 publications

Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii

Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii

Flagellar regeneration requires cytoplasmic microtubule depolymerization and kinesin-13

Avalanche-like behavior in ciliary import

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