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Mobility, microtubule nucleation and structure of microtubule-organizing centers in multinucleated hyphae of Ashbya gossypii.

Lang C, Grava S, van den Hoorn T, Trimble R, Philippsen P, Jaspersen SL - Mol. Biol. Cell (2009)

Bottom Line: This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii.Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side.Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Biozentrum University of Basel, 4056 Basel, Switzerland.

ABSTRACT
We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 microm/min), rotations (up to 180 degrees in 30 s), and long-range nuclear bypassing (up to 9 microm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis-oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

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Localization of MTOCs in A. gossypii. (A) Images of a tip compartment expressing AgTUB4-YFP to localize MTOCs and stained with Hoechst to visualize nuclei. (B) Images of a hyphal compartment with a calcofluor stained septum and YFP-marked MTOCs. No YFP fluorescence can be seen at the septum. (C) Images of A. gossypii hyphae before and after incubation with 15 μg/ml nocodazole for 1 h and at different times after washout of the drug. Nuclei are stained with Hoechst and microtubules are labeled with anti-α-tubulin antibodies. The untreated hypha shows cMTs, one metaphase spindle before orientation along the growth axis and fluorescent foci representing SPBs. After nocodazole treatment only SPB foci are detectable with anti-α-tubulin antibodies. Twenty minutes after nocodazole washout, faintly stained short cMTs reemerged at the nuclear SPBs (arrows). By 30 min, the microtubules seemed similar to untreated cells. Bars, 5 μm (A–C).
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Figure 1: Localization of MTOCs in A. gossypii. (A) Images of a tip compartment expressing AgTUB4-YFP to localize MTOCs and stained with Hoechst to visualize nuclei. (B) Images of a hyphal compartment with a calcofluor stained septum and YFP-marked MTOCs. No YFP fluorescence can be seen at the septum. (C) Images of A. gossypii hyphae before and after incubation with 15 μg/ml nocodazole for 1 h and at different times after washout of the drug. Nuclei are stained with Hoechst and microtubules are labeled with anti-α-tubulin antibodies. The untreated hypha shows cMTs, one metaphase spindle before orientation along the growth axis and fluorescent foci representing SPBs. After nocodazole treatment only SPB foci are detectable with anti-α-tubulin antibodies. Twenty minutes after nocodazole washout, faintly stained short cMTs reemerged at the nuclear SPBs (arrows). By 30 min, the microtubules seemed similar to untreated cells. Bars, 5 μm (A–C).

Mentions: We first wanted to determine whether SPBs are the only site of microtubule nucleation in A. gossypii as they are in budding yeast or if other nonnuclear MTOCs also exist. To investigate the sites of microtubule nucleation, we fused the A. gossypii homologue of γ-tubulin with YFP because labeling of Tub4 with fluorophores has been used to visualize both nuclear and nonnuclear MTOCs in various fungi (Oakley et al., 1990; Horio et al., 1991; Stearns et al., 1991; Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al., 1996; Heitz et al., 2001). AgTub4-yellow fluorescent protein (YFP) appeared as small foci fairly evenly distributed along the length of the hypha. Colabeling of nuclei with Hoechst revealed that AgTub4-YFP localizes exclusively to one or two spots at the nuclear periphery (Figure 1A). It was never observed at other locations such as hyphal tips or septa (Figure 1B).


Mobility, microtubule nucleation and structure of microtubule-organizing centers in multinucleated hyphae of Ashbya gossypii.

Lang C, Grava S, van den Hoorn T, Trimble R, Philippsen P, Jaspersen SL - Mol. Biol. Cell (2009)

Localization of MTOCs in A. gossypii. (A) Images of a tip compartment expressing AgTUB4-YFP to localize MTOCs and stained with Hoechst to visualize nuclei. (B) Images of a hyphal compartment with a calcofluor stained septum and YFP-marked MTOCs. No YFP fluorescence can be seen at the septum. (C) Images of A. gossypii hyphae before and after incubation with 15 μg/ml nocodazole for 1 h and at different times after washout of the drug. Nuclei are stained with Hoechst and microtubules are labeled with anti-α-tubulin antibodies. The untreated hypha shows cMTs, one metaphase spindle before orientation along the growth axis and fluorescent foci representing SPBs. After nocodazole treatment only SPB foci are detectable with anti-α-tubulin antibodies. Twenty minutes after nocodazole washout, faintly stained short cMTs reemerged at the nuclear SPBs (arrows). By 30 min, the microtubules seemed similar to untreated cells. Bars, 5 μm (A–C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801712&req=5

Figure 1: Localization of MTOCs in A. gossypii. (A) Images of a tip compartment expressing AgTUB4-YFP to localize MTOCs and stained with Hoechst to visualize nuclei. (B) Images of a hyphal compartment with a calcofluor stained septum and YFP-marked MTOCs. No YFP fluorescence can be seen at the septum. (C) Images of A. gossypii hyphae before and after incubation with 15 μg/ml nocodazole for 1 h and at different times after washout of the drug. Nuclei are stained with Hoechst and microtubules are labeled with anti-α-tubulin antibodies. The untreated hypha shows cMTs, one metaphase spindle before orientation along the growth axis and fluorescent foci representing SPBs. After nocodazole treatment only SPB foci are detectable with anti-α-tubulin antibodies. Twenty minutes after nocodazole washout, faintly stained short cMTs reemerged at the nuclear SPBs (arrows). By 30 min, the microtubules seemed similar to untreated cells. Bars, 5 μm (A–C).
Mentions: We first wanted to determine whether SPBs are the only site of microtubule nucleation in A. gossypii as they are in budding yeast or if other nonnuclear MTOCs also exist. To investigate the sites of microtubule nucleation, we fused the A. gossypii homologue of γ-tubulin with YFP because labeling of Tub4 with fluorophores has been used to visualize both nuclear and nonnuclear MTOCs in various fungi (Oakley et al., 1990; Horio et al., 1991; Stearns et al., 1991; Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al., 1996; Heitz et al., 2001). AgTub4-yellow fluorescent protein (YFP) appeared as small foci fairly evenly distributed along the length of the hypha. Colabeling of nuclei with Hoechst revealed that AgTub4-YFP localizes exclusively to one or two spots at the nuclear periphery (Figure 1A). It was never observed at other locations such as hyphal tips or septa (Figure 1B).

Bottom Line: This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii.Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side.Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Biozentrum University of Basel, 4056 Basel, Switzerland.

ABSTRACT
We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 microm/min), rotations (up to 180 degrees in 30 s), and long-range nuclear bypassing (up to 9 microm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis-oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

Show MeSH
Related in: MedlinePlus