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Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp.

Lee WL, Bezanilla M, Pollard TD - J. Cell Biol. (2000)

Bottom Line: Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is.Additional tail domains, TH2 and TH3, are required to complement the double mutant.Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Biochemistry, Cellular and Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1(+) is not lethal, Deltamyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1(+) is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Deltamyo1 defects. However, it fails to rescue the Deltamyo1 Deltawsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.

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Targeted disruption of wsp1+. (A) Scale drawing of the wsp1+ locus showing exons (box) and introns (line). Sequences coding for various domains are filled with different shades. Black vertical bars within exon 2 indicate strings of six to seven proline residues. wsp1+ was disrupted in a leu1− diploid by replacing a BglII–BglII fragment with an oppositely oriented leu1+ gene. Agarose gel of PCR amplification from the disrupted locus of viable Leu+ haploid is shown. (B) Viable Leu+ haploids were transformed with plasmids and streaked on EMM-Leu ± 1 M KCl at 32°C. Δwsp1 cells formed no colonies on 1 M KCl when transformed with an empty vector (pUR19), but were able to grow when transformed with a wsp1+ genomic clone (pUR-wsp1). (C) Wild-type and Δwsp1 cells grown to log phase in EMM-Leu at 32°C were stained with rhodamine-phalloidin and calcofluor. Scale bars, 10 μm.
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Figure 6: Targeted disruption of wsp1+. (A) Scale drawing of the wsp1+ locus showing exons (box) and introns (line). Sequences coding for various domains are filled with different shades. Black vertical bars within exon 2 indicate strings of six to seven proline residues. wsp1+ was disrupted in a leu1− diploid by replacing a BglII–BglII fragment with an oppositely oriented leu1+ gene. Agarose gel of PCR amplification from the disrupted locus of viable Leu+ haploid is shown. (B) Viable Leu+ haploids were transformed with plasmids and streaked on EMM-Leu ± 1 M KCl at 32°C. Δwsp1 cells formed no colonies on 1 M KCl when transformed with an empty vector (pUR19), but were able to grow when transformed with a wsp1+ genomic clone (pUR-wsp1). (C) Wild-type and Δwsp1 cells grown to log phase in EMM-Leu at 32°C were stained with rhodamine-phalloidin and calcofluor. Scale bars, 10 μm.

Mentions: We made a wsp1+ disruption strain (Δwsp1), which would produce a COOH-terminally truncated Wsp1p protein (Fig. 6 A). Tetrad dissection of individual asci and random spore analysis of a Δwsp1/wsp1+ diploid revealed that COOH-terminal truncation of Wsp1p is not lethal. Amplification with specific primers from the genomic locus verified that wsp1+ was disrupted in Leu+ haploids (Fig. 6 A). Δwsp1 cells formed smaller colonies than wild-type in selective EMM, were sensitive to 1 M KCl (Fig. 6 B) and mated inefficiently. A genomic clone of wsp1+ (pUR19-wsp1) fully complemented the salt phenotype and mating defects. Like Δmyo1 cells, Δwsp1 cells had depolarized actin patches and morphological defects (Fig. 6 C). Disruption of wsp1+ did not cause faulty targeting of septal material, but mid-log Δwsp1 cells grown at 32°C did have more uniseptated cells than wild-type (Table II; Fig. 6 C). The lack of aberrant septal targeting suggests that, unlike Myo1p, Wsp1p is not involved in proper septal deposition.


Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp.

Lee WL, Bezanilla M, Pollard TD - J. Cell Biol. (2000)

Targeted disruption of wsp1+. (A) Scale drawing of the wsp1+ locus showing exons (box) and introns (line). Sequences coding for various domains are filled with different shades. Black vertical bars within exon 2 indicate strings of six to seven proline residues. wsp1+ was disrupted in a leu1− diploid by replacing a BglII–BglII fragment with an oppositely oriented leu1+ gene. Agarose gel of PCR amplification from the disrupted locus of viable Leu+ haploid is shown. (B) Viable Leu+ haploids were transformed with plasmids and streaked on EMM-Leu ± 1 M KCl at 32°C. Δwsp1 cells formed no colonies on 1 M KCl when transformed with an empty vector (pUR19), but were able to grow when transformed with a wsp1+ genomic clone (pUR-wsp1). (C) Wild-type and Δwsp1 cells grown to log phase in EMM-Leu at 32°C were stained with rhodamine-phalloidin and calcofluor. Scale bars, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Targeted disruption of wsp1+. (A) Scale drawing of the wsp1+ locus showing exons (box) and introns (line). Sequences coding for various domains are filled with different shades. Black vertical bars within exon 2 indicate strings of six to seven proline residues. wsp1+ was disrupted in a leu1− diploid by replacing a BglII–BglII fragment with an oppositely oriented leu1+ gene. Agarose gel of PCR amplification from the disrupted locus of viable Leu+ haploid is shown. (B) Viable Leu+ haploids were transformed with plasmids and streaked on EMM-Leu ± 1 M KCl at 32°C. Δwsp1 cells formed no colonies on 1 M KCl when transformed with an empty vector (pUR19), but were able to grow when transformed with a wsp1+ genomic clone (pUR-wsp1). (C) Wild-type and Δwsp1 cells grown to log phase in EMM-Leu at 32°C were stained with rhodamine-phalloidin and calcofluor. Scale bars, 10 μm.
Mentions: We made a wsp1+ disruption strain (Δwsp1), which would produce a COOH-terminally truncated Wsp1p protein (Fig. 6 A). Tetrad dissection of individual asci and random spore analysis of a Δwsp1/wsp1+ diploid revealed that COOH-terminal truncation of Wsp1p is not lethal. Amplification with specific primers from the genomic locus verified that wsp1+ was disrupted in Leu+ haploids (Fig. 6 A). Δwsp1 cells formed smaller colonies than wild-type in selective EMM, were sensitive to 1 M KCl (Fig. 6 B) and mated inefficiently. A genomic clone of wsp1+ (pUR19-wsp1) fully complemented the salt phenotype and mating defects. Like Δmyo1 cells, Δwsp1 cells had depolarized actin patches and morphological defects (Fig. 6 C). Disruption of wsp1+ did not cause faulty targeting of septal material, but mid-log Δwsp1 cells grown at 32°C did have more uniseptated cells than wild-type (Table II; Fig. 6 C). The lack of aberrant septal targeting suggests that, unlike Myo1p, Wsp1p is not involved in proper septal deposition.

Bottom Line: Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is.Additional tail domains, TH2 and TH3, are required to complement the double mutant.Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Biochemistry, Cellular and Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1(+) is not lethal, Deltamyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1(+) is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Deltamyo1 defects. However, it fails to rescue the Deltamyo1 Deltawsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.

Show MeSH
Related in: MedlinePlus