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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 GFP-Myo1p in mid-log wild-type haploid cells. (A) Differential interference contrast and GFP-Myo1p fluorescence in live cells at three different focal planes. (B–C) Stereo pairs of three-dimensional reconstructions of live cells expressing GFP-Myo1p showing localization of Myo1p patches at cell periphery. (D–E) Colocalization of GFP-Myo1p and actin in fixed cells stained with rhodamine-phalloidin shown as full thickness stack of images from three-dimensional reconstructions (D) and as stereo pair (E). Images were obtained by a conventional fluorescence microscope (A) or a deconvolution Deltavision microscope (B–E).
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Figure 3: Localization of GFP-Myo1p in mid-log wild-type haploid cells. (A) Differential interference contrast and GFP-Myo1p fluorescence in live cells at three different focal planes. (B–C) Stereo pairs of three-dimensional reconstructions of live cells expressing GFP-Myo1p showing localization of Myo1p patches at cell periphery. (D–E) Colocalization of GFP-Myo1p and actin in fixed cells stained with rhodamine-phalloidin shown as full thickness stack of images from three-dimensional reconstructions (D) and as stereo pair (E). Images were obtained by a conventional fluorescence microscope (A) or a deconvolution Deltavision microscope (B–E).

Mentions: Full-length GFP-Myo1p localized to patches in wild-type as well as Δmyo1 cells. Three-dimensional reconstructions made by deconvolution microscopy showed that all GFP-Myo1p patches were located at the periphery of living cells (Fig. 3B and Fig. C), so they appeared in different focal planes by conventional fluorescence microscopy (Fig. 3 A). Like actin patches, these Myo1p patches usually concentrated at both growing ends or in the middle of dividing cells, and were dynamic, since we observed them moving along the cell cortex. We found that GFP-Myo1p patches partially colocalized with actin patches (Fig. 3D and Fig. E). Staining of GFP-Myo1p–expressing cells with rhodamine-phalloidin revealed that ∼25% of patches contained only GFP-Myo1p (green patches) and ∼15% contained only actin (red patches). Approximately 60% of patches contained a variable ratio of actin and GFP-Myo1p, since these patches ranged in color from yellow to orange (350 patches counted). Latrunculin-A reversibly dispersed GFP-Myo1p from patches to a diffuse cytoplasmic fluorescence, indicating that cortical localization of Myo1p depended on intact actin filaments. Expression levels from the nmt1+ promoter varied from cell to cell, producing patches of different intensities but otherwise indistinguishable. Overexpression of GFP-Myo1p, while not toxic, produced uniform fluorescence throughout the cell periphery and large fluorescent aggregates in the cytoplasm. We conclude that expression of GFP-Myo1p at low levels mimics endogenous Myo1p localization.


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 GFP-Myo1p in mid-log wild-type haploid cells. (A) Differential interference contrast and GFP-Myo1p fluorescence in live cells at three different focal planes. (B–C) Stereo pairs of three-dimensional reconstructions of live cells expressing GFP-Myo1p showing localization of Myo1p patches at cell periphery. (D–E) Colocalization of GFP-Myo1p and actin in fixed cells stained with rhodamine-phalloidin shown as full thickness stack of images from three-dimensional reconstructions (D) and as stereo pair (E). Images were obtained by a conventional fluorescence microscope (A) or a deconvolution Deltavision microscope (B–E).
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Related In: Results  -  Collection

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

Figure 3: Localization of GFP-Myo1p in mid-log wild-type haploid cells. (A) Differential interference contrast and GFP-Myo1p fluorescence in live cells at three different focal planes. (B–C) Stereo pairs of three-dimensional reconstructions of live cells expressing GFP-Myo1p showing localization of Myo1p patches at cell periphery. (D–E) Colocalization of GFP-Myo1p and actin in fixed cells stained with rhodamine-phalloidin shown as full thickness stack of images from three-dimensional reconstructions (D) and as stereo pair (E). Images were obtained by a conventional fluorescence microscope (A) or a deconvolution Deltavision microscope (B–E).
Mentions: Full-length GFP-Myo1p localized to patches in wild-type as well as Δmyo1 cells. Three-dimensional reconstructions made by deconvolution microscopy showed that all GFP-Myo1p patches were located at the periphery of living cells (Fig. 3B and Fig. C), so they appeared in different focal planes by conventional fluorescence microscopy (Fig. 3 A). Like actin patches, these Myo1p patches usually concentrated at both growing ends or in the middle of dividing cells, and were dynamic, since we observed them moving along the cell cortex. We found that GFP-Myo1p patches partially colocalized with actin patches (Fig. 3D and Fig. E). Staining of GFP-Myo1p–expressing cells with rhodamine-phalloidin revealed that ∼25% of patches contained only GFP-Myo1p (green patches) and ∼15% contained only actin (red patches). Approximately 60% of patches contained a variable ratio of actin and GFP-Myo1p, since these patches ranged in color from yellow to orange (350 patches counted). Latrunculin-A reversibly dispersed GFP-Myo1p from patches to a diffuse cytoplasmic fluorescence, indicating that cortical localization of Myo1p depended on intact actin filaments. Expression levels from the nmt1+ promoter varied from cell to cell, producing patches of different intensities but otherwise indistinguishable. Overexpression of GFP-Myo1p, while not toxic, produced uniform fluorescence throughout the cell periphery and large fluorescent aggregates in the cytoplasm. We conclude that expression of GFP-Myo1p at low levels mimics endogenous Myo1p localization.

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