Highly Divergent Actin Expressed in aChlamydomonasMutant Lacking the Conventional Actin Gene

Kato-Minoura, Takako; Uryu, Satomi; Hirono, Masafumi; Kamiya, Ritsu

doi:10.1006/bbrc.1998.9373

Cited by 48 publications

(52 citation statements)

References 19 publications

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“…The observations described above suggested that actin functions can be carried out redundantly by IDA5 and NAP1, as speculated previously (Kato-Minoura et al 1998;see Introduction). However, the absence of any means to inactivate NAP1 has prevented a direct test of this idea.…”

Section: Resultssupporting

confidence: 73%

“…One possible explanation for the surprising absence of a growth phenotype in the ida5-1 null mutant is that the upregulated expression of NAP1 in the mutant (Kato-Minoura et al 1998) allows this unconventional actin to provide any essential actin functions (see Introduction). In that case, it might be possible to investigate actin function by treating wild-type cells with LatB, a potent inhibitor of polymerization for most if not all actins that have been tested to date (see Introduction).…”

Section: Resultsmentioning

confidence: 99%

“…Although NAP1 is only $65% identical in amino acid sequence to IDA5 and other conventional actins ( Figure 1B), phylogenetic analyses consistently cluster it together with actins rather than with Arp1-type actin-related proteins ( Figure 1A; Kato-Minoura et al 2014). NAP1 is transcriptionally upregulated in the ida5-1 null mutant (Kato-Minoura et al 1998;Hirono et al 2003), so that it has been speculated that it could provide actin function under these conditions. A priori, the extensive sequence divergence of NAP1 from conventional actins provides a challenge for this model.…”

mentioning

confidence: 99%

“…It has a single gene (IDA5) encoding a conventional actin (Figure 1,A and B), and localization studies have suggested that IDA5 may have multiple roles both during vegetative growth and during mating (Harper et al 1992;Ehler and Dutcher 1998;Kato-Minoura et al 1998;Kovar et al 2001;Avasthi et al 2014). However, electron microscopy and labeling with fluorescent phallotoxin detected F-actin structures only in fertilization tubules (Detmers et al 1983;Wilson et al 1997;Hirono et al 2003), raising the surprising possibility that IDA5 might function during vegetative growth as G-actin rather than in filaments, as originally suggested by Harper et al (1992).…”

mentioning

confidence: 99%

“…and cytokinesis might be completely independent of F-actin function. However, interpretation of this surprising hypothesis has been complicated by the presence in Chlamydomonas of a gene (NAP1) that encodes an unconventional actin (Kato-Minoura et al 1998). Although NAP1 is only $65% identical in amino acid sequence to IDA5 and other conventional actins ( Figure 1B), phylogenetic analyses consistently cluster it together with actins rather than with Arp1-type actin-related proteins ( Figure 1A; Kato-Minoura et al 2014).…”

mentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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Recovery of flagellar dynein function in a Chlamydomonas actin/dynein‐deficient mutant upon introduction of muscle actin by electroporation

Hayashi

Hirono

Kamiya

2001

Cell Motil. Cytoskeleton

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Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however, the function of this actin subunit remains unknown. As a first step toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules.

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Identification of a novel actin-related protein inTetrahymena cilia

Kuribara¹,

Kato²,

Kato-Minoura³

et al. 2006

Cell Motil. Cytoskeleton

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Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In eukaryotes, its gene belongs to a highly conserved gene family, while the protein is also a member of an actin superfamily comprising various kinds of actin-related proteins (Arps). A ciliate, Tetrahymena, has a unique conventional actin. Data from the TIGR Tetrahymena genome project and our own research suggest the existence of 12 actin-like sequences: four conventional actins, two of Arp4, one each of Arp1, Arp2, Arp3, Arp5, and Arp6, and a novel actin-related protein, tArp. We cloned the entire cDNA sequences of Tetrahymena Arp2 (tArp2), Tetrahymena Arp3 (tArp3), and tArp for the work described herein. In phylogenetic analyses, tArp was not included in any Arp subfamily. Unlike other known Arps, tArp localizes in cilia, and its expression was upregulated after deciliation. To see the precise localization of tArp, cilia were fractionated and analyzed using specific antibodies. tArp was observed preferentially in the "outer-doublet" fraction, while actin was found in the "crude-dynein" fraction. In immunoelectron microscopy, most of the gold particles were found either on the outer-doublet or central-pair microtubules. These results suggest that tArp is a ciliary component and that it has a unique function in the formation and maintenance of cilia.

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Highly Divergent Actin Expressed in aChlamydomonasMutant Lacking the Conventional Actin Gene

Cited by 48 publications

References 19 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

Recovery of flagellar dynein function in a Chlamydomonas actin/dynein‐deficient mutant upon introduction of muscle actin by electroporation

Identification of a novel actin-related protein inTetrahymena cilia

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