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Dynamics of multiple nuclei in Ashbya gossypii hyphae depend on the control of cytoplasmic microtubules length by Bik1, Kip2, Kip3, and not on a capture/shrinkage mechanism.

Grava S, Philippsen P - Mol. Biol. Cell (2010)

Bottom Line: Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei.Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips.Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.

View Article: PubMed Central - PubMed

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

ABSTRACT
Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.

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Nuclear distribution and dynamics in wild-type and bik1Δ cells. (A) Radial growth of wild type and bik1Δ mutant after 4 d of incubation on full medium (AFM) at 30°C. Plates containing DMSO are negative controls for benomyl plates. (B) Observation of nuclear dynamics by time-lapse microscopy in wild-type and bik1Δ H4-GFP hyphae. Pictures are overlays of DIC (red) and histone 4-GFP signal. The circles mark bypassing events. Numbers indicate time in min. Bars, 5 μm. (C) Quantification of nuclear oscillation and bypassing in WT and bik1Δ mutant. Distances between a fixed point set at time t0 and the center of the nuclei were measured at each time point. The first five nuclei shown in B were tracked.
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Figure 5: Nuclear distribution and dynamics in wild-type and bik1Δ cells. (A) Radial growth of wild type and bik1Δ mutant after 4 d of incubation on full medium (AFM) at 30°C. Plates containing DMSO are negative controls for benomyl plates. (B) Observation of nuclear dynamics by time-lapse microscopy in wild-type and bik1Δ H4-GFP hyphae. Pictures are overlays of DIC (red) and histone 4-GFP signal. The circles mark bypassing events. Numbers indicate time in min. Bars, 5 μm. (C) Quantification of nuclear oscillation and bypassing in WT and bik1Δ mutant. Distances between a fixed point set at time t0 and the center of the nuclei were measured at each time point. The first five nuclei shown in B were tracked.

Mentions: To know whether the loss of Bik1 not only affects cMT dynamics and interactions with the cortex but also nuclear distribution, we deleted BIK1 in a strain in which nuclei were labeled with histone H4-GFP (considered here as a wild-type strain). As shown in Figure 5A, wild-type and bik1Δ strains had similar radial growth after 4 d at 30°C on full medium (AFM). Similar to wild type, bik1Δ spores were able to germinate after 7 h of incubation on AFM at 30°C and displayed a normal cell morphology. The presence of 33 μM of the MT-destabilizing drug benomyl did not affect the growth rate of bik1Δ strains. We observed similar results with bik1Δ GFP-TUB1 strains (data not shown). To measure the general spacing of nuclei in wild-type and mutant cells, we classified the distances between two neighboring nuclei (N-N distances) into four categories (Figure 5B). The proportion of 3- to 6-μm N-N distances was twice lower in bik1Δ mutant compared with wild type and almost 20% of the N-N distances were longer than 9 μm in the mutant (4% in WT). Because Bik1 also localized to the central spindle (Supplemental Figure 1), the increased N-N distances in bik1Δ also could result from a lower mitotic index. If anaphases get delayed due to spindle defects, less mitotic events would occur and therefore nuclear density would decrease. Together, our results suggest that the absence of long MTs may only slightly affect the control of nuclear spacing.


Dynamics of multiple nuclei in Ashbya gossypii hyphae depend on the control of cytoplasmic microtubules length by Bik1, Kip2, Kip3, and not on a capture/shrinkage mechanism.

Grava S, Philippsen P - Mol. Biol. Cell (2010)

Nuclear distribution and dynamics in wild-type and bik1Δ cells. (A) Radial growth of wild type and bik1Δ mutant after 4 d of incubation on full medium (AFM) at 30°C. Plates containing DMSO are negative controls for benomyl plates. (B) Observation of nuclear dynamics by time-lapse microscopy in wild-type and bik1Δ H4-GFP hyphae. Pictures are overlays of DIC (red) and histone 4-GFP signal. The circles mark bypassing events. Numbers indicate time in min. Bars, 5 μm. (C) Quantification of nuclear oscillation and bypassing in WT and bik1Δ mutant. Distances between a fixed point set at time t0 and the center of the nuclei were measured at each time point. The first five nuclei shown in B were tracked.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Nuclear distribution and dynamics in wild-type and bik1Δ cells. (A) Radial growth of wild type and bik1Δ mutant after 4 d of incubation on full medium (AFM) at 30°C. Plates containing DMSO are negative controls for benomyl plates. (B) Observation of nuclear dynamics by time-lapse microscopy in wild-type and bik1Δ H4-GFP hyphae. Pictures are overlays of DIC (red) and histone 4-GFP signal. The circles mark bypassing events. Numbers indicate time in min. Bars, 5 μm. (C) Quantification of nuclear oscillation and bypassing in WT and bik1Δ mutant. Distances between a fixed point set at time t0 and the center of the nuclei were measured at each time point. The first five nuclei shown in B were tracked.
Mentions: To know whether the loss of Bik1 not only affects cMT dynamics and interactions with the cortex but also nuclear distribution, we deleted BIK1 in a strain in which nuclei were labeled with histone H4-GFP (considered here as a wild-type strain). As shown in Figure 5A, wild-type and bik1Δ strains had similar radial growth after 4 d at 30°C on full medium (AFM). Similar to wild type, bik1Δ spores were able to germinate after 7 h of incubation on AFM at 30°C and displayed a normal cell morphology. The presence of 33 μM of the MT-destabilizing drug benomyl did not affect the growth rate of bik1Δ strains. We observed similar results with bik1Δ GFP-TUB1 strains (data not shown). To measure the general spacing of nuclei in wild-type and mutant cells, we classified the distances between two neighboring nuclei (N-N distances) into four categories (Figure 5B). The proportion of 3- to 6-μm N-N distances was twice lower in bik1Δ mutant compared with wild type and almost 20% of the N-N distances were longer than 9 μm in the mutant (4% in WT). Because Bik1 also localized to the central spindle (Supplemental Figure 1), the increased N-N distances in bik1Δ also could result from a lower mitotic index. If anaphases get delayed due to spindle defects, less mitotic events would occur and therefore nuclear density would decrease. Together, our results suggest that the absence of long MTs may only slightly affect the control of nuclear spacing.

Bottom Line: Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei.Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips.Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.

View Article: PubMed Central - PubMed

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

ABSTRACT
Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.

Show MeSH