Takako Kato-Minoura | Chuo University (original) (raw)

Papers by Takako Kato-Minoura

Cell Structure and Function, 1993

Two novel Chlamydomonas mutants, idaS and ida69 that lack subsets of inner-arm dynein have been i... more Two novel Chlamydomonas mutants, idaS and ida69 that lack subsets of inner-arm dynein have been isolated and mapped to discrete loci on the right arm of linkage group XIV. Of the seven different innerarm dynein subspecies (a, b, c, d, e, /and g) identified by ion-exchange chromatography, idaS lacks a, c, d and e, while ida6 lacks e alone; these are the only mutants that have been shown to lack subspecies e. Both strains can swim, albeit more slowly than the wild type. Hence, subspecies e must contribute to flagellar movement although it is unnecessary for the generation of undulating movement. Inner and outer dynein arms in cilia and flagella are molecular assemblies responsible for force production. Both arms contain several heavy chains (Mr>400,000) that have ATP-hydrolyzing activities. Although the two kinds of arms are similar in gross appearance in the cross section of the axoneme, recent studies using Chlamydomonas mutants have revealed that they differ rather strikingly in composition and organization. In particular, it has been proposed that the inner arm comprises three or more heterologous species each containing multiple heavy chains, whereas the outer dynein arm comprises a single species composed of two or three heavy chains and several smaller polypeptides. Piperno et al. (21) suggested that the inner arms of Chlamydomonas comprise three sub forms (II, 12 and 13), each containing two heavy chains, and the unit of the three sub forms repeats every 96 nm along the outer doublet. Moreover, the 12 and 13 species possibly contain different heavy chains depending on their position along the flagellum (20). These studies have identified a total of six different inner-arm heavy chains. Kagami and Kamiya (7), on the other hand, found that ionexchange chromatography can separate the inner-arm dynein into seven discrete subspecies designated a-g9 and that the total number of different inner-arm heavy c hains is as many as eight, rather than six. The classification of the inner arms into II, 12 and 13, however, has gained little support from recent studies. The classification is based on the analysis of inner-arm composition by sucrose density gradient centrifugation * Address for correspondence. and electron microscopic observation on mutant axonemes lacking subsets of inner arms (21). However, recent electron microscopic studies have indicated that the inner arms are arranged in a more complex manner than has previously been thought; the inner arms do not appear to be grouped in three entities within the repeating unit distance of 96 nm (16, 17). More specifical

bioRxiv (Cold Spring Harbor Laboratory), Apr 8, 2020

The single-cell green alga Chlamydomonas reinhardtii possesses two -tubulin genes (tua1 and tua2... more The single-cell green alga Chlamydomonas reinhardtii possesses two -tubulin genes (tua1 and tua2) and two -tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different -tubulin mutations and 24 strains bearing 12 different -tubulin mutations. Some of these mutations are known to confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an efficient means of isolating novel C. reinhardtii tubulin mutants. IMPORTANCE: Chlamydomonas reinhardtii is a useful organism for the study of tubulin function; however, only five kinds of tubulin mutations have been reported to date. This scarcity is partly due to C. reinhardtii possessing two tubulin genes each for and -tubulin. Here, we obtained several strains in which one of the or -tubulin genes was disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel tubulin mutations that conferred differing degrees of resistance to two anti-tubulin compounds. The majority of the tubulin mutations were located outside of the drug-binding sites in the three-dimensional tubulin structure, suggesting that structural changes underlie (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. the drug resistance conferred by these mutations. Thus, single-tubulin-gene disruptants are an efficient means of generating tubulin mutants for the study of the structure-function relationship of tubulin and for the development of novel therapies based on anti-tubulin agents.

Cell Structure and Function, 2023

Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and lig... more Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein. This mutation, E279K, is in the third of the seven WD repeat domains. No apparent abnormality was observed in electron microscope observations of axonemes or in SDS-PAGE analyses of dynein subunits. To explore the reason for the lowered motility in this mutant, in vitro microtubule sliding experiments were performed, which revealed that the motor activity of the mutant OAD was lowered. In particular, a large difference was observed between wild type (WT) and the mutant in the microtubule sliding velocity in microtubule bundles formed with the addition of OAD: ~35.3 μm/sec (WT) and ~4.3 μm/sec (mutant). From this and other results, we propose that IC2 in an OAD interacts with the β HC of the adjacent OAD, and that an OAD-OAD interaction is important for efficient beating of cilia and flagella.

Zoological Science, Dec 25, 2005

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In euk... more 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.

Biochemical and Biophysical Research Communications, 2011

Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its l... more Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.

SAXS signals from the DNA of salmon spermatozoa in an aqueous solution were analyzed using a rheo... more SAXS signals from the DNA of salmon spermatozoa in an aqueous solution were analyzed using a rheometer-device, which controlled the rate of shearing during SAXS observations. When shearing rate > 1000 s was applied to the DNA suspension, DNA strands were not aligned as observed with other biological filaments, but a stable diffraction peak approximately ranging 1.7–1.8 [nm] of Q-values were observed. We compared our observation with simulated SAXS signals.

ABSTRACTThe single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 ... more ABSTRACTThe single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two β-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 24 strains bearing 12 different β-tubulin mutations. Some of these mutations are known to confer drug resistance in human can...

Zoological Science, 2018

Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) ... more Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.

Zoological Science, Dec 25, 2003

Zoological Science, Dec 25, 2002

Journal of Cell Biology, 1997

Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f c... more Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5′ end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = ∼5.3) but had two novel spots with pIs of ∼5.6 and ∼5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional a...

Plant Systematics and Evolution, 2015

The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventi... more The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventional actin and the other an unconventional actin called novel actin-like protein (NAP). These actins apparently differ in their ability to polymerize, but their specific functions in the cell are unknown. To understand their evolutionary relationship, we investigated the molecular phylogeny of the actin/NAP family in Volvocales, using fully identified sequences (Chlamydomonas moewusii, Gonium pectorale, and Volvox carteri) and newly determined partial sequences (Eudorina elegans, and Volvulina steinii). Although the origin of the NAP clade remains ambiguous, the inferred phylogenetic trees strongly support the monophyly of NAP genes and show that only the genomes of volvocine species contain NAP genes. The nonsynonymous substitution rate of the NAP gene is, in consistence with its long branch length in the phylogeny, relatively high compared with that of the actin gene. NAP is thus apparently unique to volvocine species and most likely performs a cellular function with fewer constraints.

Cell Motility and the Cytoskeleton, 2006

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In euk... more 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.

Zoological Science, 2005

BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access t... more BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses.

Cell Structure and Function, 1993

Two novel Chlamydomonas mutants, idaS and ida69 that lack subsets of inner-arm dynein have been i... more Two novel Chlamydomonas mutants, idaS and ida69 that lack subsets of inner-arm dynein have been isolated and mapped to discrete loci on the right arm of linkage group XIV. Of the seven different innerarm dynein subspecies (a, b, c, d, e, /and g) identified by ion-exchange chromatography, idaS lacks a, c, d and e, while ida6 lacks e alone; these are the only mutants that have been shown to lack subspecies e. Both strains can swim, albeit more slowly than the wild type. Hence, subspecies e must contribute to flagellar movement although it is unnecessary for the generation of undulating movement. Inner and outer dynein arms in cilia and flagella are molecular assemblies responsible for force production. Both arms contain several heavy chains (Mr>400,000) that have ATP-hydrolyzing activities. Although the two kinds of arms are similar in gross appearance in the cross section of the axoneme, recent studies using Chlamydomonas mutants have revealed that they differ rather strikingly in composition and organization. In particular, it has been proposed that the inner arm comprises three or more heterologous species each containing multiple heavy chains, whereas the outer dynein arm comprises a single species composed of two or three heavy chains and several smaller polypeptides. Piperno et al. (21) suggested that the inner arms of Chlamydomonas comprise three sub forms (II, 12 and 13), each containing two heavy chains, and the unit of the three sub forms repeats every 96 nm along the outer doublet. Moreover, the 12 and 13 species possibly contain different heavy chains depending on their position along the flagellum (20). These studies have identified a total of six different inner-arm heavy chains. Kagami and Kamiya (7), on the other hand, found that ionexchange chromatography can separate the inner-arm dynein into seven discrete subspecies designated a-g9 and that the total number of different inner-arm heavy c hains is as many as eight, rather than six. The classification of the inner arms into II, 12 and 13, however, has gained little support from recent studies. The classification is based on the analysis of inner-arm composition by sucrose density gradient centrifugation * Address for correspondence. and electron microscopic observation on mutant axonemes lacking subsets of inner arms (21). However, recent electron microscopic studies have indicated that the inner arms are arranged in a more complex manner than has previously been thought; the inner arms do not appear to be grouped in three entities within the repeating unit distance of 96 nm (16, 17). More specifical

bioRxiv (Cold Spring Harbor Laboratory), Apr 8, 2020

The single-cell green alga Chlamydomonas reinhardtii possesses two -tubulin genes (tua1 and tua2... more The single-cell green alga Chlamydomonas reinhardtii possesses two -tubulin genes (tua1 and tua2) and two -tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different -tubulin mutations and 24 strains bearing 12 different -tubulin mutations. Some of these mutations are known to confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an efficient means of isolating novel C. reinhardtii tubulin mutants. IMPORTANCE: Chlamydomonas reinhardtii is a useful organism for the study of tubulin function; however, only five kinds of tubulin mutations have been reported to date. This scarcity is partly due to C. reinhardtii possessing two tubulin genes each for and -tubulin. Here, we obtained several strains in which one of the or -tubulin genes was disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel tubulin mutations that conferred differing degrees of resistance to two anti-tubulin compounds. The majority of the tubulin mutations were located outside of the drug-binding sites in the three-dimensional tubulin structure, suggesting that structural changes underlie (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. the drug resistance conferred by these mutations. Thus, single-tubulin-gene disruptants are an efficient means of generating tubulin mutants for the study of the structure-function relationship of tubulin and for the development of novel therapies based on anti-tubulin agents.

Cell Structure and Function, 2023

Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and lig... more Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein. This mutation, E279K, is in the third of the seven WD repeat domains. No apparent abnormality was observed in electron microscope observations of axonemes or in SDS-PAGE analyses of dynein subunits. To explore the reason for the lowered motility in this mutant, in vitro microtubule sliding experiments were performed, which revealed that the motor activity of the mutant OAD was lowered. In particular, a large difference was observed between wild type (WT) and the mutant in the microtubule sliding velocity in microtubule bundles formed with the addition of OAD: ~35.3 μm/sec (WT) and ~4.3 μm/sec (mutant). From this and other results, we propose that IC2 in an OAD interacts with the β HC of the adjacent OAD, and that an OAD-OAD interaction is important for efficient beating of cilia and flagella.

Zoological Science, Dec 25, 2005

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In euk... more 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.

Biochemical and Biophysical Research Communications, 2011

Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its l... more Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.

SAXS signals from the DNA of salmon spermatozoa in an aqueous solution were analyzed using a rheo... more SAXS signals from the DNA of salmon spermatozoa in an aqueous solution were analyzed using a rheometer-device, which controlled the rate of shearing during SAXS observations. When shearing rate > 1000 s was applied to the DNA suspension, DNA strands were not aligned as observed with other biological filaments, but a stable diffraction peak approximately ranging 1.7–1.8 [nm] of Q-values were observed. We compared our observation with simulated SAXS signals.

ABSTRACTThe single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 ... more ABSTRACTThe single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two β-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 24 strains bearing 12 different β-tubulin mutations. Some of these mutations are known to confer drug resistance in human can...

Zoological Science, 2018

Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) ... more Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.

Zoological Science, Dec 25, 2003

Zoological Science, Dec 25, 2002

Journal of Cell Biology, 1997

Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f c... more Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5′ end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = ∼5.3) but had two novel spots with pIs of ∼5.6 and ∼5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional a...

Plant Systematics and Evolution, 2015

The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventi... more The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventional actin and the other an unconventional actin called novel actin-like protein (NAP). These actins apparently differ in their ability to polymerize, but their specific functions in the cell are unknown. To understand their evolutionary relationship, we investigated the molecular phylogeny of the actin/NAP family in Volvocales, using fully identified sequences (Chlamydomonas moewusii, Gonium pectorale, and Volvox carteri) and newly determined partial sequences (Eudorina elegans, and Volvulina steinii). Although the origin of the NAP clade remains ambiguous, the inferred phylogenetic trees strongly support the monophyly of NAP genes and show that only the genomes of volvocine species contain NAP genes. The nonsynonymous substitution rate of the NAP gene is, in consistence with its long branch length in the phylogeny, relatively high compared with that of the actin gene. NAP is thus apparently unique to volvocine species and most likely performs a cellular function with fewer constraints.

Cell Motility and the Cytoskeleton, 2006

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In euk... more 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.

Zoological Science, 2005

BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access t... more BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses.