Flagellar microtubule dynamics in Chlamydomonas: cytochalasin D induces periods of microtubule shortening and elongation; and colchicine induces disassembly of the distal, but not proximal, half of the flagellum

Dentler, William L.; Adams, Christopher

doi:10.1083/jcb.117.6.1289

Cited by 57 publications

(56 citation statements)

References 29 publications

(49 reference statements)

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“…This mutation has essentially no effect on cell proliferation, although it does affect intraflagellar transport, flagellar regeneration, and fertilization-tubule formation (Kato et al 1993;Kato-Minoura et al 1997;Avasthi et al 2014). Moreover, although very high doses of cytochalasin D caused temporary shortening of the flagella (Dentler and Adams 1992), they had no effect on proliferation (Harper et al 1992).…”

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confidence: 99%

Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity inChlamydomonas

2015

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Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses.

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confidence: 99%

Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity inChlamydomonas

2015

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“…Although colchicine blocked flagellar growth and did not stimulate shortening, it did not affect the exchange of other axonemal or membrane plus matrix components. Colchicine induces flagellar shortening in pf18 cells treated with low levels of red light and in pf18 and wild-type cells treated with cytochalasin D (14), but these appear to reflect unique properties of pf18 (13,14) and a yet unexplained effect of cytochalasin D on flagellar length. Axonemal protein exchange may make the axonemes more responsive to signals that regulate length, but turnover alone does not appear to drive flagellar length changes.…”

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confidence: 99%

“…2 and Rosenbaum (36) reported the incorporation of epitopetagged tubulin in nongrowing flagella, but the exchange of other flagellar components was not examined. Recent discoveries of the importance of the IFT motors kinesin-II and cytoplasmic dynein in flagellar assembly and maintenance in a variety of organisms (16,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) combined with earlier studies showing that flagellar growth and/or shortening can be induced by light (13), various drugs and ions (12)(13)(14), and temperature (15,24), prompted a careful examination of protein turnover in steady state Chlamydomonas flagella. Our experiments confirm that more than 80 different polypeptides are exchanged with a cytoplasmic protein pool in steady state flagella.…”

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confidence: 99%

“…If one flagellum is amputated, the remaining flagellum shortens and then elongates once a new flagellum has started to grow on the "amputated" basal body (11). Chlamydomonas flagellar growth can be stimulated with lithium (12,13), induced to disassemble by IBMX and other agents known to be involved with signal transduction pathways (13), and destabilized by cytochalasin D, which induces periods of rapid shortening and elongation (14). A temperaturesensitive mutant, fla10, shortens its flagella at 33°C but not at 22°C (15,16).…”

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confidence: 99%

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Flagellar Protein Dynamics in Chlamydomonas

Song

Dentler

2001

Journal of Biological Chemistry

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Cilia and flagella appear to be stable, terminal, microtubule-containing organelles, but they also elongate and shorten in response to a variety of signals. To understand mechanisms that regulate flagellar dynamics, Chlamydomonas cells with nongrowing flagella were labeled with 35 S, and flagella and basal body components were examined for labeled polypeptides. Maximal incorporation of label into the flagella occurred within 3 h. Twenty percent of the flagellar polypeptides were exchanged. These included tubulins, dyneins, and 80 other axonemal and membrane plus matrix polypeptides. The most stable flagellar structure is the PF-ribbon, which comprises part of the wall of each doublet microtubule and is composed of tubulin and three other polypeptides. Most 35 S was incorporated into the high molecular weight ribbon polypeptide, rib240, and little, if any, 35 S is incorporated into PF-ribbon-associated tubulin. Both wild-type (9 ؉ 2) and 9 ؉ 0 flagella, which lack central microtubules, exhibited nearly identical exchange patterns, so labeling is not due to turnover of relatively labile central microtubules. To determine if flagellar length is balanced by protein exchange, 35 S incorporation into disassembling flagella was examined, as was exchange in flagella in which microtubule assembly was blocked by colchicine. Incorporation of 35 S-labeled polypeptides was found to occur into flagellar axonemes during wavelength-dependent shortening in pf18 and in fla10 cells induced to shorten flagella by incubation at 33°C. Colchicine blocked tubulin addition but did not affect the exchange of the other exchangeable polypeptides; nor did it induce any change in flagellar length. Basal bodies also incorporated newly synthesized proteins. These data reveal that Chlamydomonas flagella are dynamic structures that incorporate new protein both during steady state and as flagella shorten and that protein exchange does not, alone, explain length regulation.

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“…The abundant presence of Bug22 in the axoneme and its tight association with axonemal microtubules might provide stability and stiffness to the axoneme. It has been shown that the proximal half to two-thirds of the flagellum is more stable and resistant to drug-induced flagellar shortening (Dentler and Adams, 1992). It has also been proposed that Bug22 in Paramecium might regulate cilia rigidity (Laligné et al, 2010).…”

Section: Bug22 Associates With All Axonemal Microtubulesmentioning

confidence: 99%

The conserved ciliary protein Bug22 controls planar beating ofChlamydomonasflagella

Meng

Cao

Oda

et al. 2013

Journal of Cell Science

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Eukaryotic flagella and cilia can exhibit planar and non-planar beating, and the mechanism controlling these beating patterns is not well understood. Chlamydomonas reinhardtii flagella beat in approximately the same plane with either an asymmetric ciliary-type or symmetric flagellar-type waveform. Each B-tubule of the number 1, 5 and 6 doublets of the flagellar axoneme possesses a beak-like structure. The number 5 and 6 beak structures are implicated in conversion of ciliary motion into flagellar motion. Here, we show that in a null mutant of Bug22, the asymmetric ciliary waveform is converted into a three-dimensional (non-planar) symmetric flagellar waveform. Bug22 is localized to approximately the proximal half to two-thirds of the flagellum, similar to localization of beak-like structures. However, as shown by immunogold labeling, Bug22 associates with axonemal microtubules without apparent preference for any particular doublets. Interestingly, bug22 mutants lack all beaklike structures. We propose that one function of Bug22 is to regulate the anchoring of the beak-like structures to the doublet microtubules and confine flagellar beating to a plane.

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Flagellar microtubule dynamics in Chlamydomonas: cytochalasin D induces periods of microtubule shortening and elongation; and colchicine induces disassembly of the distal, but not proximal, half of the flagellum

Cited by 57 publications

References 29 publications

Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity inChlamydomonas

Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity inChlamydomonas

Flagellar Protein Dynamics in Chlamydomonas

The conserved ciliary protein Bug22 controls planar beating ofChlamydomonasflagella

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