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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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Localization of Myo1p head and/or tail constructs fused to GFP. Live mid-log cells expressing indicated constructs at 25°C in Δmyo1 cells. All images were obtained by a conventional fluorescence microscope.
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Figure 4: Localization of Myo1p head and/or tail constructs fused to GFP. Live mid-log cells expressing indicated constructs at 25°C in Δmyo1 cells. All images were obtained by a conventional fluorescence microscope.

Mentions: GFP-H/1, a construct that rescued Δmyo1 defects, localized to discrete patches similar to GFP-Myo1p in Δmyo1 (Fig. 4) and wild-type cells. However, the level of expression was more variable from cell to cell and cytoplasmic fluorescence was greater for GFP-H/1 than for GFP-Myo1p. GFP fusions that failed to complement Δmyo1 were either mislocalized to the nucleus (Fig. 4, GFP-1 and GFP-1/2/3/A), aggregated (GFP-H), or diffuse in the cytoplasm (GFP-2/3/A). Nuclear localization of GFP-1 and GFP-1/2/3/A were confirmed by staining with DAPI.


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)

Localization of Myo1p head and/or tail constructs fused to GFP. Live mid-log cells expressing indicated constructs at 25°C in Δmyo1 cells. All images were obtained by a conventional fluorescence microscope.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169449&req=5

Figure 4: Localization of Myo1p head and/or tail constructs fused to GFP. Live mid-log cells expressing indicated constructs at 25°C in Δmyo1 cells. All images were obtained by a conventional fluorescence microscope.
Mentions: GFP-H/1, a construct that rescued Δmyo1 defects, localized to discrete patches similar to GFP-Myo1p in Δmyo1 (Fig. 4) and wild-type cells. However, the level of expression was more variable from cell to cell and cytoplasmic fluorescence was greater for GFP-H/1 than for GFP-Myo1p. GFP fusions that failed to complement Δmyo1 were either mislocalized to the nucleus (Fig. 4, GFP-1 and GFP-1/2/3/A), aggregated (GFP-H), or diffuse in the cytoplasm (GFP-2/3/A). Nuclear localization of GFP-1 and GFP-1/2/3/A were confirmed by staining with DAPI.

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