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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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Oscillation, rotation and bypassing of nuclei in an A. gossypii hypha. Hyphae expressing AgH4-GFP to mark nuclei (green) and AgTub4-RedStar2 to mark SPBs (red) were pregrown and mounted for fluorescence videomicroscopy at room temperature as described in Materials and Methods. GFP and red fluorescent protein (RFP) fluorescence was imaged for 500 ms each in five Z-planes, 0.75 μm apart at 1-min intervals and processed (Supplemental Movie S1). (A) Maximal projections at 11 time points showing frequent changes in the position and SPB orientation of the 11 nuclei monitored in Supplemental Movie S1. During the 110 analyzed 1-min time intervals, nuclei moved forward 34 times and backward 30 times, all of which were led by the SPB. During the remaining 46 intervals, nuclei moved only marginally. Nonmoving as well as moving nuclei rotated during 53 intervals by up to 180° as seen by changes of the SPB positions. The relative position of SPBs in all fast-moving nuclei was “head-first” (at the leading edge of nuclear movement), and inversions in the direction of nuclear movement were preceded by inversion of the SPB position. Bar, 5 μm. (B). Diagram of the position of nuclei (circles) and orientation of SPBs (red dots) at 0 and 10 min. Identical nuclei are connected by dotted lines highlighting the four bypassing events. The arrow marks the 1.5-μm elongation of the hypha during 10 min, which was measured using the overilluminated montage of A to visualize the background staining of the hyphal cytoplasm. (C). Detailed diagrammatic presentation of backward and forward as well as rotational movements of two nuclei (6, white asterisk and 7, yellow asterisk) within 10 min (movie frames 3–13 of Supplemental Movie S1). Nucleus 6 bypasses nucleus 7 by migrating 5 μm backward, inverts the position of the SPB by rotation at 4 and 5 min, and migrates 3 μm forward. Nucleus 7 represents a typical tumbling nucleus showing minor movements and small angle rotations until 8 min, at which time it undergoes a rotation and then a forward movement of 2 μm. Fast-moving nuclei in A and C seem to be stretched and sometimes display two SPBs. This does not reflect the real shape of fast nuclei or duplicated SPBs however; rather, it is due to the high migration speed and the fact that these images show projections of five focal planes taken within 6 s.
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Figure 2: Oscillation, rotation and bypassing of nuclei in an A. gossypii hypha. Hyphae expressing AgH4-GFP to mark nuclei (green) and AgTub4-RedStar2 to mark SPBs (red) were pregrown and mounted for fluorescence videomicroscopy at room temperature as described in Materials and Methods. GFP and red fluorescent protein (RFP) fluorescence was imaged for 500 ms each in five Z-planes, 0.75 μm apart at 1-min intervals and processed (Supplemental Movie S1). (A) Maximal projections at 11 time points showing frequent changes in the position and SPB orientation of the 11 nuclei monitored in Supplemental Movie S1. During the 110 analyzed 1-min time intervals, nuclei moved forward 34 times and backward 30 times, all of which were led by the SPB. During the remaining 46 intervals, nuclei moved only marginally. Nonmoving as well as moving nuclei rotated during 53 intervals by up to 180° as seen by changes of the SPB positions. The relative position of SPBs in all fast-moving nuclei was “head-first” (at the leading edge of nuclear movement), and inversions in the direction of nuclear movement were preceded by inversion of the SPB position. Bar, 5 μm. (B). Diagram of the position of nuclei (circles) and orientation of SPBs (red dots) at 0 and 10 min. Identical nuclei are connected by dotted lines highlighting the four bypassing events. The arrow marks the 1.5-μm elongation of the hypha during 10 min, which was measured using the overilluminated montage of A to visualize the background staining of the hyphal cytoplasm. (C). Detailed diagrammatic presentation of backward and forward as well as rotational movements of two nuclei (6, white asterisk and 7, yellow asterisk) within 10 min (movie frames 3–13 of Supplemental Movie S1). Nucleus 6 bypasses nucleus 7 by migrating 5 μm backward, inverts the position of the SPB by rotation at 4 and 5 min, and migrates 3 μm forward. Nucleus 7 represents a typical tumbling nucleus showing minor movements and small angle rotations until 8 min, at which time it undergoes a rotation and then a forward movement of 2 μm. Fast-moving nuclei in A and C seem to be stretched and sometimes display two SPBs. This does not reflect the real shape of fast nuclei or duplicated SPBs however; rather, it is due to the high migration speed and the fact that these images show projections of five focal planes taken within 6 s.

Mentions: Using time-lapse microscopy, we monitored hyphae expressing histone AgH4-GFP and AgTub4-RedStar2 to visualize possible connections between nuclear movements and the locations of SPBs. In total, 37 nuclei were monitored for 10 min in five hyphae growing at speeds from 0.15 to 1.28 μm/min. Table 1 summarizes the movements of all 37 nuclei examined, including the movement of nuclei and their SPBs in the slowest growing hypha (Figure 2) and hyphae with more rapid growth speeds (Supplemental Figure S1).


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)

Oscillation, rotation and bypassing of nuclei in an A. gossypii hypha. Hyphae expressing AgH4-GFP to mark nuclei (green) and AgTub4-RedStar2 to mark SPBs (red) were pregrown and mounted for fluorescence videomicroscopy at room temperature as described in Materials and Methods. GFP and red fluorescent protein (RFP) fluorescence was imaged for 500 ms each in five Z-planes, 0.75 μm apart at 1-min intervals and processed (Supplemental Movie S1). (A) Maximal projections at 11 time points showing frequent changes in the position and SPB orientation of the 11 nuclei monitored in Supplemental Movie S1. During the 110 analyzed 1-min time intervals, nuclei moved forward 34 times and backward 30 times, all of which were led by the SPB. During the remaining 46 intervals, nuclei moved only marginally. Nonmoving as well as moving nuclei rotated during 53 intervals by up to 180° as seen by changes of the SPB positions. The relative position of SPBs in all fast-moving nuclei was “head-first” (at the leading edge of nuclear movement), and inversions in the direction of nuclear movement were preceded by inversion of the SPB position. Bar, 5 μm. (B). Diagram of the position of nuclei (circles) and orientation of SPBs (red dots) at 0 and 10 min. Identical nuclei are connected by dotted lines highlighting the four bypassing events. The arrow marks the 1.5-μm elongation of the hypha during 10 min, which was measured using the overilluminated montage of A to visualize the background staining of the hyphal cytoplasm. (C). Detailed diagrammatic presentation of backward and forward as well as rotational movements of two nuclei (6, white asterisk and 7, yellow asterisk) within 10 min (movie frames 3–13 of Supplemental Movie S1). Nucleus 6 bypasses nucleus 7 by migrating 5 μm backward, inverts the position of the SPB by rotation at 4 and 5 min, and migrates 3 μm forward. Nucleus 7 represents a typical tumbling nucleus showing minor movements and small angle rotations until 8 min, at which time it undergoes a rotation and then a forward movement of 2 μm. Fast-moving nuclei in A and C seem to be stretched and sometimes display two SPBs. This does not reflect the real shape of fast nuclei or duplicated SPBs however; rather, it is due to the high migration speed and the fact that these images show projections of five focal planes taken within 6 s.
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Related In: Results  -  Collection

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Figure 2: Oscillation, rotation and bypassing of nuclei in an A. gossypii hypha. Hyphae expressing AgH4-GFP to mark nuclei (green) and AgTub4-RedStar2 to mark SPBs (red) were pregrown and mounted for fluorescence videomicroscopy at room temperature as described in Materials and Methods. GFP and red fluorescent protein (RFP) fluorescence was imaged for 500 ms each in five Z-planes, 0.75 μm apart at 1-min intervals and processed (Supplemental Movie S1). (A) Maximal projections at 11 time points showing frequent changes in the position and SPB orientation of the 11 nuclei monitored in Supplemental Movie S1. During the 110 analyzed 1-min time intervals, nuclei moved forward 34 times and backward 30 times, all of which were led by the SPB. During the remaining 46 intervals, nuclei moved only marginally. Nonmoving as well as moving nuclei rotated during 53 intervals by up to 180° as seen by changes of the SPB positions. The relative position of SPBs in all fast-moving nuclei was “head-first” (at the leading edge of nuclear movement), and inversions in the direction of nuclear movement were preceded by inversion of the SPB position. Bar, 5 μm. (B). Diagram of the position of nuclei (circles) and orientation of SPBs (red dots) at 0 and 10 min. Identical nuclei are connected by dotted lines highlighting the four bypassing events. The arrow marks the 1.5-μm elongation of the hypha during 10 min, which was measured using the overilluminated montage of A to visualize the background staining of the hyphal cytoplasm. (C). Detailed diagrammatic presentation of backward and forward as well as rotational movements of two nuclei (6, white asterisk and 7, yellow asterisk) within 10 min (movie frames 3–13 of Supplemental Movie S1). Nucleus 6 bypasses nucleus 7 by migrating 5 μm backward, inverts the position of the SPB by rotation at 4 and 5 min, and migrates 3 μm forward. Nucleus 7 represents a typical tumbling nucleus showing minor movements and small angle rotations until 8 min, at which time it undergoes a rotation and then a forward movement of 2 μm. Fast-moving nuclei in A and C seem to be stretched and sometimes display two SPBs. This does not reflect the real shape of fast nuclei or duplicated SPBs however; rather, it is due to the high migration speed and the fact that these images show projections of five focal planes taken within 6 s.
Mentions: Using time-lapse microscopy, we monitored hyphae expressing histone AgH4-GFP and AgTub4-RedStar2 to visualize possible connections between nuclear movements and the locations of SPBs. In total, 37 nuclei were monitored for 10 min in five hyphae growing at speeds from 0.15 to 1.28 μm/min. Table 1 summarizes the movements of all 37 nuclei examined, including the movement of nuclei and their SPBs in the slowest growing hypha (Figure 2) and hyphae with more rapid growth speeds (Supplemental Figure S1).

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