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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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Kinesin Kip3 regulates cMT length and its absence enhances nuclear dynamics in A. gossypii. (A) kip3Δ mutant grows like WT. Plates were incubated for 5 d at 30°C on full medium (AFM) containing DMSO (negative control), benomyl at 33 μM, or both. (B) Quantification of cMT length in WT and kip3Δ cells by using anti-Tub1 immunostaining pictures as shown in C. Bar, 5 μm. (D) Quantification of nuclei dynamics in kip3Δ hyphae. Distances between the center of each nucleus and a fixed point were measured for each time point. (E) Quantification of the number of steps the 35 nuclei from Table 2 could consecutively move backward or forward during the 30-min observations. (F) MT polymerization rate in kip3Δ mutant strain. Measurements were made on 6-s interval movies with cells expressing GFP-Tub1 and Bik1-cherry fusions (only 1Z plane was observed). cMTs were measured from their −end (SPB observed with GFP-Tub1) until their +tip (Bik1-cherry signal). Due to increased cMT length in kip3Δ strain, SPBs and +tips were often in different focal planes. Asterisk (*) corresponds to the actual length of the cMTs (SPB to +tip), whereas the other MTs have an underestimated size (visible part of cMT to +tip). 1 time frame corresponds to 6 s. The average WT cMT polymerization rate is shown as reference (black line).
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Figure 7: Kinesin Kip3 regulates cMT length and its absence enhances nuclear dynamics in A. gossypii. (A) kip3Δ mutant grows like WT. Plates were incubated for 5 d at 30°C on full medium (AFM) containing DMSO (negative control), benomyl at 33 μM, or both. (B) Quantification of cMT length in WT and kip3Δ cells by using anti-Tub1 immunostaining pictures as shown in C. Bar, 5 μm. (D) Quantification of nuclei dynamics in kip3Δ hyphae. Distances between the center of each nucleus and a fixed point were measured for each time point. (E) Quantification of the number of steps the 35 nuclei from Table 2 could consecutively move backward or forward during the 30-min observations. (F) MT polymerization rate in kip3Δ mutant strain. Measurements were made on 6-s interval movies with cells expressing GFP-Tub1 and Bik1-cherry fusions (only 1Z plane was observed). cMTs were measured from their −end (SPB observed with GFP-Tub1) until their +tip (Bik1-cherry signal). Due to increased cMT length in kip3Δ strain, SPBs and +tips were often in different focal planes. Asterisk (*) corresponds to the actual length of the cMTs (SPB to +tip), whereas the other MTs have an underestimated size (visible part of cMT to +tip). 1 time frame corresponds to 6 s. The average WT cMT polymerization rate is shown as reference (black line).

Mentions: Recent work has shown that the budding yeast kinesin Kip3 has unique properties. It is both a +end-directed motor and a +end-specific depolymerase that is necessary to regulate the length of MTs attached to the cell cortex and to a lesser extend interpolar MTs (Gardner et al., 2008). ScKip3 is observed as speckles that travel toward, and accumulate at, the +ends of growing MTs. However, its accumulation at MT +ends is reduced or absent from depolymerizing +ends (Gupta et al., 2006). Varga et al. (2006, 2009) proposed a model (the antenna model) in which Kip3 binds randomly along the length of the MT and its high processivity allows it to efficiently target MT +ends. The longer a MT is, the higher the concentration of Kip3 will be at the +end, and the higher the depolymerization rate will be. The dissociation of Kip3 from the +ends is greatly accelerated by incoming Kip3 molecules and is accompanied by removal of one or two tubulin dimers (Varga et al., 2006; Varga et al., 2009). Here, we tested whether AgKip3 (47% identity to ScKip3) has also a role in cMT dynamics and nuclear movements in A. gossypii. We deleted the KIP3 gene in hyphae with H4-GFP–labeled nuclei and in hyphae expressing Bik1-Cherry plus GFP-Tub1. The kip3Δ strains grew as well as control strains on full medium and did not show sensitivity or resistance to benomyl (33–132 μM; Figure 7A). However the distances between neighboring nuclei increased clearly in kip3Δ compared with WT cells. The category of N-N distances “over 9 μm” was ∼6 times more represented in kip3Δ than in WT (Supplemental Figure 3A). If AgKip3 localizes to the mitotic spindle like its budding yeast homologue, then the increase in N-N distances is probably due to a decrease of the mitotic index in kip3Δ (Miller et al., 1998; Gupta et al., 2006). Overall, this result suggests that the nuclear distance can be increased to a certain extend without affecting the growth rate.


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)

Kinesin Kip3 regulates cMT length and its absence enhances nuclear dynamics in A. gossypii. (A) kip3Δ mutant grows like WT. Plates were incubated for 5 d at 30°C on full medium (AFM) containing DMSO (negative control), benomyl at 33 μM, or both. (B) Quantification of cMT length in WT and kip3Δ cells by using anti-Tub1 immunostaining pictures as shown in C. Bar, 5 μm. (D) Quantification of nuclei dynamics in kip3Δ hyphae. Distances between the center of each nucleus and a fixed point were measured for each time point. (E) Quantification of the number of steps the 35 nuclei from Table 2 could consecutively move backward or forward during the 30-min observations. (F) MT polymerization rate in kip3Δ mutant strain. Measurements were made on 6-s interval movies with cells expressing GFP-Tub1 and Bik1-cherry fusions (only 1Z plane was observed). cMTs were measured from their −end (SPB observed with GFP-Tub1) until their +tip (Bik1-cherry signal). Due to increased cMT length in kip3Δ strain, SPBs and +tips were often in different focal planes. Asterisk (*) corresponds to the actual length of the cMTs (SPB to +tip), whereas the other MTs have an underestimated size (visible part of cMT to +tip). 1 time frame corresponds to 6 s. The average WT cMT polymerization rate is shown as reference (black line).
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Figure 7: Kinesin Kip3 regulates cMT length and its absence enhances nuclear dynamics in A. gossypii. (A) kip3Δ mutant grows like WT. Plates were incubated for 5 d at 30°C on full medium (AFM) containing DMSO (negative control), benomyl at 33 μM, or both. (B) Quantification of cMT length in WT and kip3Δ cells by using anti-Tub1 immunostaining pictures as shown in C. Bar, 5 μm. (D) Quantification of nuclei dynamics in kip3Δ hyphae. Distances between the center of each nucleus and a fixed point were measured for each time point. (E) Quantification of the number of steps the 35 nuclei from Table 2 could consecutively move backward or forward during the 30-min observations. (F) MT polymerization rate in kip3Δ mutant strain. Measurements were made on 6-s interval movies with cells expressing GFP-Tub1 and Bik1-cherry fusions (only 1Z plane was observed). cMTs were measured from their −end (SPB observed with GFP-Tub1) until their +tip (Bik1-cherry signal). Due to increased cMT length in kip3Δ strain, SPBs and +tips were often in different focal planes. Asterisk (*) corresponds to the actual length of the cMTs (SPB to +tip), whereas the other MTs have an underestimated size (visible part of cMT to +tip). 1 time frame corresponds to 6 s. The average WT cMT polymerization rate is shown as reference (black line).
Mentions: Recent work has shown that the budding yeast kinesin Kip3 has unique properties. It is both a +end-directed motor and a +end-specific depolymerase that is necessary to regulate the length of MTs attached to the cell cortex and to a lesser extend interpolar MTs (Gardner et al., 2008). ScKip3 is observed as speckles that travel toward, and accumulate at, the +ends of growing MTs. However, its accumulation at MT +ends is reduced or absent from depolymerizing +ends (Gupta et al., 2006). Varga et al. (2006, 2009) proposed a model (the antenna model) in which Kip3 binds randomly along the length of the MT and its high processivity allows it to efficiently target MT +ends. The longer a MT is, the higher the concentration of Kip3 will be at the +end, and the higher the depolymerization rate will be. The dissociation of Kip3 from the +ends is greatly accelerated by incoming Kip3 molecules and is accompanied by removal of one or two tubulin dimers (Varga et al., 2006; Varga et al., 2009). Here, we tested whether AgKip3 (47% identity to ScKip3) has also a role in cMT dynamics and nuclear movements in A. gossypii. We deleted the KIP3 gene in hyphae with H4-GFP–labeled nuclei and in hyphae expressing Bik1-Cherry plus GFP-Tub1. The kip3Δ strains grew as well as control strains on full medium and did not show sensitivity or resistance to benomyl (33–132 μM; Figure 7A). However the distances between neighboring nuclei increased clearly in kip3Δ compared with WT cells. The category of N-N distances “over 9 μm” was ∼6 times more represented in kip3Δ than in WT (Supplemental Figure 3A). If AgKip3 localizes to the mitotic spindle like its budding yeast homologue, then the increase in N-N distances is probably due to a decrease of the mitotic index in kip3Δ (Miller et al., 1998; Gupta et al., 2006). Overall, this result suggests that the nuclear distance can be increased to a certain extend without affecting the growth rate.

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
Related in: MedlinePlus