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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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KIP2 deletion affects Bik1 localization at cMT +tips. (A) Bik1 localization in WT (GFP-TUB1) and kip2Δ mutant. In the mutant, Bik1 accumulation is reduced or abolished at cMT +tip (*) and increased at SPB (arrowheads). (B) 1Z plane movie of Bik1-Cherry GFP-Tub1 in kip2Δ background. Numbers indicate time in seconds. Bars, 5 μm.
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Figure 6: KIP2 deletion affects Bik1 localization at cMT +tips. (A) Bik1 localization in WT (GFP-TUB1) and kip2Δ mutant. In the mutant, Bik1 accumulation is reduced or abolished at cMT +tip (*) and increased at SPB (arrowheads). (B) 1Z plane movie of Bik1-Cherry GFP-Tub1 in kip2Δ background. Numbers indicate time in seconds. Bars, 5 μm.

Mentions: In budding yeast, Bik1 is targeted to the MT +end by a kinesin-dependent transport mechanism. Bik1 forms a complex with the kinesin Kip2 and both comigrate along individual MTs (Carvalho et al., 2004). To determine the mechanism of Bik1 targeting at +end, we tested whether the absence of AgKip2 (42% identity with ScKip2) affected Bik1 localization in A. gossypii. Similar to the AgBIK1 deletion, the deletion of AgKIP2 did not affect the hyphal growth rate but led to a severe decrease in cMT length (Supplemental Figure 2 and Table 1). In kip2Δ hyphae, Bik1 accumulation at cMT +ends is strongly reduced or abolished. In contrast, the Bik1-Cherry signal is strongly increased at the SPBs (Figure 6). Therefore, like in budding yeast, AgKip2 has a role in transporting AgBik1 to the +end and probably also in maintaining Bik1 at cMT +ends. In conclusion, the A. gossypii kinesin Kip2 is also involved in the control of MT dynamics by delivering Bik1 at MT +tips. Because Kip2 has to bring Bik1 at cMT +end, its velocity along cMTs has to be higher than the cMT polymerization rate (6.5 μm/min). In budding yeast, the Bik1/Kip2 complex moves along the cMTs with an average speed of 6.6 μm/min (Carvalho et al., 2004). Finally, consistent with its role in localizing Bik1 at the cMT +ends, we showed that Kip2 is also required for nuclear dynamics. As in bik1Δ mutant, forward and backward events are strongly decreased in kip2Δ cells and bypassing events are abolished (Supplemental Figure 2 and Table 2).


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)

KIP2 deletion affects Bik1 localization at cMT +tips. (A) Bik1 localization in WT (GFP-TUB1) and kip2Δ mutant. In the mutant, Bik1 accumulation is reduced or abolished at cMT +tip (*) and increased at SPB (arrowheads). (B) 1Z plane movie of Bik1-Cherry GFP-Tub1 in kip2Δ background. Numbers indicate time in seconds. Bars, 5 μm.
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Figure 6: KIP2 deletion affects Bik1 localization at cMT +tips. (A) Bik1 localization in WT (GFP-TUB1) and kip2Δ mutant. In the mutant, Bik1 accumulation is reduced or abolished at cMT +tip (*) and increased at SPB (arrowheads). (B) 1Z plane movie of Bik1-Cherry GFP-Tub1 in kip2Δ background. Numbers indicate time in seconds. Bars, 5 μm.
Mentions: In budding yeast, Bik1 is targeted to the MT +end by a kinesin-dependent transport mechanism. Bik1 forms a complex with the kinesin Kip2 and both comigrate along individual MTs (Carvalho et al., 2004). To determine the mechanism of Bik1 targeting at +end, we tested whether the absence of AgKip2 (42% identity with ScKip2) affected Bik1 localization in A. gossypii. Similar to the AgBIK1 deletion, the deletion of AgKIP2 did not affect the hyphal growth rate but led to a severe decrease in cMT length (Supplemental Figure 2 and Table 1). In kip2Δ hyphae, Bik1 accumulation at cMT +ends is strongly reduced or abolished. In contrast, the Bik1-Cherry signal is strongly increased at the SPBs (Figure 6). Therefore, like in budding yeast, AgKip2 has a role in transporting AgBik1 to the +end and probably also in maintaining Bik1 at cMT +ends. In conclusion, the A. gossypii kinesin Kip2 is also involved in the control of MT dynamics by delivering Bik1 at MT +tips. Because Kip2 has to bring Bik1 at cMT +end, its velocity along cMTs has to be higher than the cMT polymerization rate (6.5 μm/min). In budding yeast, the Bik1/Kip2 complex moves along the cMTs with an average speed of 6.6 μm/min (Carvalho et al., 2004). Finally, consistent with its role in localizing Bik1 at the cMT +ends, we showed that Kip2 is also required for nuclear dynamics. As in bik1Δ mutant, forward and backward events are strongly decreased in kip2Δ cells and bypassing events are abolished (Supplemental Figure 2 and Table 2).

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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