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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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EM analysis of nuclei in multinucleated hyphae. (A) Overlay of a DIC and a fluorescence image of a young A. gossypii mycelium that was stained with Hoechst to visualize nuclei. Such mycelia with five to 10 tips and 50–100 nuclei were prepared for thin section EM analysis as described in Materials and Methods. Bar, 5 μm. (B–E) EM of nuclei in different nuclear cycle stages. The continuous nuclear membrane and nuclear pore complexes within the nuclear envelope can be seen in all images. Bars, 200 nm. (B) A single SPB (asterisk) is embedded in the nuclear envelope. A higher magnification is shown in the top right corner. (C) Duplicated SPBs (asterisks) connected by a bridge are embedded in the nuclear envelope. A higher magnification is presented in the top right corner. (D) A nucleus with spindle microtubules (arrow) and continuous nuclear membrane. The two SPBs were observed in the adjacent sections at positions marked by the asterisks. Magnifications are shown in the top and bottom right corners. (E) Montage of three EM images showing a nucleus in anaphase. The continuous nuclear envelope and spindle microtubules (arrow) are visible. The SPBs are in other sections.
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Figure 5: EM analysis of nuclei in multinucleated hyphae. (A) Overlay of a DIC and a fluorescence image of a young A. gossypii mycelium that was stained with Hoechst to visualize nuclei. Such mycelia with five to 10 tips and 50–100 nuclei were prepared for thin section EM analysis as described in Materials and Methods. Bar, 5 μm. (B–E) EM of nuclei in different nuclear cycle stages. The continuous nuclear membrane and nuclear pore complexes within the nuclear envelope can be seen in all images. Bars, 200 nm. (B) A single SPB (asterisk) is embedded in the nuclear envelope. A higher magnification is shown in the top right corner. (C) Duplicated SPBs (asterisks) connected by a bridge are embedded in the nuclear envelope. A higher magnification is presented in the top right corner. (D) A nucleus with spindle microtubules (arrow) and continuous nuclear membrane. The two SPBs were observed in the adjacent sections at positions marked by the asterisks. Magnifications are shown in the top and bottom right corners. (E) Montage of three EM images showing a nucleus in anaphase. The continuous nuclear envelope and spindle microtubules (arrow) are visible. The SPBs are in other sections.

Mentions: How is it possible that nuclear-associated SPBs organize such complex arrays of cMTs? To better understand the structural basis for the cMT arrays we decided to determine the ultrastructure of A. gossypii SPBs. Electron microscopy (EM) has provided valuable insight not only into SPB structure and assembly but also into microtubule organization in a variety of organisms, including budding and fission yeast. Here, we report the first EM study of nuclei in multinucleate hyphae of A. gossypii. To ensure that all nuclei analyzed by EM were actively dividing, we prepared young mycelium that had no >10 growing tips and contained no >100 nuclei (Figure 5A). These samples were fast-frozen and then freeze-substituted to preserve the structural integrity of the SPBs and microtubules, and serial thin sections (∼60 nm) of nuclei were examined. We found that the nuclear envelope is continuous throughout the nuclear cycle (Figure 5, B–E), indicating that A. gossypii undergoes a closed mitosis. Single SPBs (Figure 5B) or duplicated SPBs (Figure 5, C and D) were embedded in the nuclear envelope throughout the life cycle like in budding yeast.


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)

EM analysis of nuclei in multinucleated hyphae. (A) Overlay of a DIC and a fluorescence image of a young A. gossypii mycelium that was stained with Hoechst to visualize nuclei. Such mycelia with five to 10 tips and 50–100 nuclei were prepared for thin section EM analysis as described in Materials and Methods. Bar, 5 μm. (B–E) EM of nuclei in different nuclear cycle stages. The continuous nuclear membrane and nuclear pore complexes within the nuclear envelope can be seen in all images. Bars, 200 nm. (B) A single SPB (asterisk) is embedded in the nuclear envelope. A higher magnification is shown in the top right corner. (C) Duplicated SPBs (asterisks) connected by a bridge are embedded in the nuclear envelope. A higher magnification is presented in the top right corner. (D) A nucleus with spindle microtubules (arrow) and continuous nuclear membrane. The two SPBs were observed in the adjacent sections at positions marked by the asterisks. Magnifications are shown in the top and bottom right corners. (E) Montage of three EM images showing a nucleus in anaphase. The continuous nuclear envelope and spindle microtubules (arrow) are visible. The SPBs are in other sections.
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Figure 5: EM analysis of nuclei in multinucleated hyphae. (A) Overlay of a DIC and a fluorescence image of a young A. gossypii mycelium that was stained with Hoechst to visualize nuclei. Such mycelia with five to 10 tips and 50–100 nuclei were prepared for thin section EM analysis as described in Materials and Methods. Bar, 5 μm. (B–E) EM of nuclei in different nuclear cycle stages. The continuous nuclear membrane and nuclear pore complexes within the nuclear envelope can be seen in all images. Bars, 200 nm. (B) A single SPB (asterisk) is embedded in the nuclear envelope. A higher magnification is shown in the top right corner. (C) Duplicated SPBs (asterisks) connected by a bridge are embedded in the nuclear envelope. A higher magnification is presented in the top right corner. (D) A nucleus with spindle microtubules (arrow) and continuous nuclear membrane. The two SPBs were observed in the adjacent sections at positions marked by the asterisks. Magnifications are shown in the top and bottom right corners. (E) Montage of three EM images showing a nucleus in anaphase. The continuous nuclear envelope and spindle microtubules (arrow) are visible. The SPBs are in other sections.
Mentions: How is it possible that nuclear-associated SPBs organize such complex arrays of cMTs? To better understand the structural basis for the cMT arrays we decided to determine the ultrastructure of A. gossypii SPBs. Electron microscopy (EM) has provided valuable insight not only into SPB structure and assembly but also into microtubule organization in a variety of organisms, including budding and fission yeast. Here, we report the first EM study of nuclei in multinucleate hyphae of A. gossypii. To ensure that all nuclei analyzed by EM were actively dividing, we prepared young mycelium that had no >10 growing tips and contained no >100 nuclei (Figure 5A). These samples were fast-frozen and then freeze-substituted to preserve the structural integrity of the SPBs and microtubules, and serial thin sections (∼60 nm) of nuclei were examined. We found that the nuclear envelope is continuous throughout the nuclear cycle (Figure 5, B–E), indicating that A. gossypii undergoes a closed mitosis. Single SPBs (Figure 5B) or duplicated SPBs (Figure 5, C and D) were embedded in the nuclear envelope throughout the life cycle like in budding yeast.

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