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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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Model of A. gossypii SPB and cMT arrays emanating at SPBs. (A) Model of three nuclei with independent cMT arrays in a hypha. The most apical nucleus has close contact to the growing tip via its short cMTs. Loss of cMTs will increase the distance between the first nucleus and the tip as shown in Figure 3. The two other nuclei are connected with the hyphal cortex via short cMTs and most likely also long cMTs. Growth and shrinkage of these cMTs provides pushing and pulling forces for short-range nuclear oscillations. The most likely pulling force in the direction of a single short cortex-connected cMT was discussed in Figure 4A. Nuclear bypassing is a long-range movement and is very likely achieved by pulling forces of a long cMT when the cortex connection of short cMTs is reduced or absent. Nuclei cannot oscillate or bypass in hyphae lacking short and long cMTs as shown in Figure 3. Nuclei with mutant SPBs able to emanate mainly/only long cMTs cannot oscillate but are still able to bypass other nuclei (unpublished observations). (B) Schematic representation of an A. gossypii SPB and associated microtubules.
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Figure 9: Model of A. gossypii SPB and cMT arrays emanating at SPBs. (A) Model of three nuclei with independent cMT arrays in a hypha. The most apical nucleus has close contact to the growing tip via its short cMTs. Loss of cMTs will increase the distance between the first nucleus and the tip as shown in Figure 3. The two other nuclei are connected with the hyphal cortex via short cMTs and most likely also long cMTs. Growth and shrinkage of these cMTs provides pushing and pulling forces for short-range nuclear oscillations. The most likely pulling force in the direction of a single short cortex-connected cMT was discussed in Figure 4A. Nuclear bypassing is a long-range movement and is very likely achieved by pulling forces of a long cMT when the cortex connection of short cMTs is reduced or absent. Nuclei cannot oscillate or bypass in hyphae lacking short and long cMTs as shown in Figure 3. Nuclei with mutant SPBs able to emanate mainly/only long cMTs cannot oscillate but are still able to bypass other nuclei (unpublished observations). (B) Schematic representation of an A. gossypii SPB and associated microtubules.

Mentions: Earlier studies of fixed A. gossypii cells stained with anti-α-tubulin suggested a complex microtubule network within hyphae (Alberti-Segui et al., 2001). Long cMTs anchored in the nuclear membrane were proposed to interact with microtubules from other SPBs, the cell wall or nonnuclear MTOCs to provide forces that could explain the distinct nuclear dynamics of A. gossypii. Our data suggest that a nuclear-associated SPB in growing A. gossypii hyphae is the sole site of microtubule nucleation with each SPB forming its own cytoskeletal subdomain consisting of short and long cMTs, the short ones often reaching the cortex and the long ones frequently growing far beyond adjacent nuclei (Figure 9A). Interactions between microtubules from adjacent nuclei are, if anything, transient. The ability of nuclei in multinucleate hyphae to form distinct cytoskeletal domains is likely essential for their autonomous dynamic behavior, which is independent from the dynamics of adjacent nuclei. A unique cytoskeletal domain for each nucleus would also permit nonsynchronized oscillatory movements and facilitate orientation of the spindle axis and nuclear bypassing. If we assume that long microtubules are important for nuclear migration along a hypha, the different growth-mode of budding yeast could explain why the ability to nucleate this class of microtubules may have disappeared in the 100 million y between the divergence of A. gossypii and S. cerevisiae. However, our current work does not address the relative contribution of long and short cMTs to nuclear dynamics. Nor does it exclude the possibility that short and long microtubules can interconvert through microtubule growth or shrinkage. Analysis of proteins involved in microtubule nucleation and anchoring will shed light on these questions.


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)

Model of A. gossypii SPB and cMT arrays emanating at SPBs. (A) Model of three nuclei with independent cMT arrays in a hypha. The most apical nucleus has close contact to the growing tip via its short cMTs. Loss of cMTs will increase the distance between the first nucleus and the tip as shown in Figure 3. The two other nuclei are connected with the hyphal cortex via short cMTs and most likely also long cMTs. Growth and shrinkage of these cMTs provides pushing and pulling forces for short-range nuclear oscillations. The most likely pulling force in the direction of a single short cortex-connected cMT was discussed in Figure 4A. Nuclear bypassing is a long-range movement and is very likely achieved by pulling forces of a long cMT when the cortex connection of short cMTs is reduced or absent. Nuclei cannot oscillate or bypass in hyphae lacking short and long cMTs as shown in Figure 3. Nuclei with mutant SPBs able to emanate mainly/only long cMTs cannot oscillate but are still able to bypass other nuclei (unpublished observations). (B) Schematic representation of an A. gossypii SPB and associated microtubules.
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Figure 9: Model of A. gossypii SPB and cMT arrays emanating at SPBs. (A) Model of three nuclei with independent cMT arrays in a hypha. The most apical nucleus has close contact to the growing tip via its short cMTs. Loss of cMTs will increase the distance between the first nucleus and the tip as shown in Figure 3. The two other nuclei are connected with the hyphal cortex via short cMTs and most likely also long cMTs. Growth and shrinkage of these cMTs provides pushing and pulling forces for short-range nuclear oscillations. The most likely pulling force in the direction of a single short cortex-connected cMT was discussed in Figure 4A. Nuclear bypassing is a long-range movement and is very likely achieved by pulling forces of a long cMT when the cortex connection of short cMTs is reduced or absent. Nuclei cannot oscillate or bypass in hyphae lacking short and long cMTs as shown in Figure 3. Nuclei with mutant SPBs able to emanate mainly/only long cMTs cannot oscillate but are still able to bypass other nuclei (unpublished observations). (B) Schematic representation of an A. gossypii SPB and associated microtubules.
Mentions: Earlier studies of fixed A. gossypii cells stained with anti-α-tubulin suggested a complex microtubule network within hyphae (Alberti-Segui et al., 2001). Long cMTs anchored in the nuclear membrane were proposed to interact with microtubules from other SPBs, the cell wall or nonnuclear MTOCs to provide forces that could explain the distinct nuclear dynamics of A. gossypii. Our data suggest that a nuclear-associated SPB in growing A. gossypii hyphae is the sole site of microtubule nucleation with each SPB forming its own cytoskeletal subdomain consisting of short and long cMTs, the short ones often reaching the cortex and the long ones frequently growing far beyond adjacent nuclei (Figure 9A). Interactions between microtubules from adjacent nuclei are, if anything, transient. The ability of nuclei in multinucleate hyphae to form distinct cytoskeletal domains is likely essential for their autonomous dynamic behavior, which is independent from the dynamics of adjacent nuclei. A unique cytoskeletal domain for each nucleus would also permit nonsynchronized oscillatory movements and facilitate orientation of the spindle axis and nuclear bypassing. If we assume that long microtubules are important for nuclear migration along a hypha, the different growth-mode of budding yeast could explain why the ability to nucleate this class of microtubules may have disappeared in the 100 million y between the divergence of A. gossypii and S. cerevisiae. However, our current work does not address the relative contribution of long and short cMTs to nuclear dynamics. Nor does it exclude the possibility that short and long microtubules can interconvert through microtubule growth or shrinkage. Analysis of proteins involved in microtubule nucleation and anchoring will shed light on these questions.

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