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The Saccharomyces cerevisiae kinesin-related motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules.

Saunders W, Hornack D, Lengyel V, Deng C - J. Cell Biol. (1997)

Bottom Line: We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner.Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization.These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA. wsaund@vms.cis.pitt.edu

ABSTRACT
The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

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Model for Kar3p function in microtubule arrays. The Kar3p motor (shown as shaded circles) is proposed to function predominantly at spindle poles to stimulate microtubule turnover. At START of the cell cycle, the activity of Kar3p is required to limit microtubule number and length at the single spindle pole body (spb). During spindle assembly, the now duplicated spindle poles become linked  by microtubule crosslinking and chromosome attachment (latter not shown). Depolymerization of the microtubules linking the spindle  poles would now have the effect of pulling the poles together. Cin8p and Kip1p (dark circles) apparently function to counter this inwardly directed force, most likely by crosslinking and sliding the nuclear microtubules, using the microtubules to push out on the spindle  poles (Hoyt et al., 1992) though other mechanisms are also possible. The bipolar spindle now has two types of forces acting in opposition  on the spindle poles, represented by open arrows. Once anaphase begins, the antagonistic relationship between Cin8p and Kar3p  changes. Kar3p-driven spindle collapse no longer occurs in cin8 kip1 mutants (Saunders et al., 1992); Kar3p is no longer found at the  spindle poles, and there is a net polymerization of the spindle microtubules (and to a lesser extent the cytoplasmic microtubules) resulting in a great increase in spindle length. Thus the outwardly directed force of Cin8p and Kip1p are retained, but the inward pull of  Kar3p is lost. It is proposed that this transition is a major contributor to the onset of anaphase B and can occur in the absence of chromosomes (Zhang and Nicklas, 1996). Spindle pole bodies are shown as discs embedded in a nuclear envelope, small open circles represent  microtubule depolymerization, and dark arrows the presumed direction of Kar3p movement.
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Figure 9: Model for Kar3p function in microtubule arrays. The Kar3p motor (shown as shaded circles) is proposed to function predominantly at spindle poles to stimulate microtubule turnover. At START of the cell cycle, the activity of Kar3p is required to limit microtubule number and length at the single spindle pole body (spb). During spindle assembly, the now duplicated spindle poles become linked by microtubule crosslinking and chromosome attachment (latter not shown). Depolymerization of the microtubules linking the spindle poles would now have the effect of pulling the poles together. Cin8p and Kip1p (dark circles) apparently function to counter this inwardly directed force, most likely by crosslinking and sliding the nuclear microtubules, using the microtubules to push out on the spindle poles (Hoyt et al., 1992) though other mechanisms are also possible. The bipolar spindle now has two types of forces acting in opposition on the spindle poles, represented by open arrows. Once anaphase begins, the antagonistic relationship between Cin8p and Kar3p changes. Kar3p-driven spindle collapse no longer occurs in cin8 kip1 mutants (Saunders et al., 1992); Kar3p is no longer found at the spindle poles, and there is a net polymerization of the spindle microtubules (and to a lesser extent the cytoplasmic microtubules) resulting in a great increase in spindle length. Thus the outwardly directed force of Cin8p and Kip1p are retained, but the inward pull of Kar3p is lost. It is proposed that this transition is a major contributor to the onset of anaphase B and can occur in the absence of chromosomes (Zhang and Nicklas, 1996). Spindle pole bodies are shown as discs embedded in a nuclear envelope, small open circles represent microtubule depolymerization, and dark arrows the presumed direction of Kar3p movement.

Mentions: The results reported here support the following model (Fig. 9): Kar3p is proposed to use its minus end–directed motor capacity to move to the spindle pole region of the cells before or at the START point of the cell cycle. At this time Kar3p functions to stimulate microtubule depolymerization at the minus ends associated with the spindle pole. As the bipolar spindle forms, the two poles become physically linked by intrapolar microtubules via kinetochore attachments and polar microtubule crosslinking. Microtubule depolymerization now would have the consequence of pulling the linked spindle poles together. (This model assumes a net polymerization at the kinetochore or plus end of the spindle microtubules and a net depolymerization at the minus end. This type of spindle flux has been observed in spindles assembled in Xenopus oocyte extracts [Sawin and Mitchison, 1991] but has not yet been documented in the smaller yeast spindles.) In the normal spindle the assembly motors Cin8p and Kip1p resist this collapsing action producing a balanced level of motor function in the spindle. As anaphase begins, the microtubule dynamics in the cell change. Now there is a net polymerization of the nuclear microtubules (and to a lesser extent of the cytoplasmic microtubules, as shown here) to produce the long anaphase spindle necessary for proper chromosome segregation. During this time of net microtubule polymerization, the depolymerizing activity of Kar3p is no longer useful, and Kar3p is lost from the spindle poles. The activity of Cin8p and Kip1p is retained, and this shift in the balance of motor activity is proposed to make a major contribution to the timing of the onset of anaphase B. Further experimentation will be required to confirm and expand upon this model. For example, it is currently unknown whether Kar3p acts directly to depolymerize microtubules in vivo and whether it acts on all microtubules as shown, or on a more limited set.


The Saccharomyces cerevisiae kinesin-related motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules.

Saunders W, Hornack D, Lengyel V, Deng C - J. Cell Biol. (1997)

Model for Kar3p function in microtubule arrays. The Kar3p motor (shown as shaded circles) is proposed to function predominantly at spindle poles to stimulate microtubule turnover. At START of the cell cycle, the activity of Kar3p is required to limit microtubule number and length at the single spindle pole body (spb). During spindle assembly, the now duplicated spindle poles become linked  by microtubule crosslinking and chromosome attachment (latter not shown). Depolymerization of the microtubules linking the spindle  poles would now have the effect of pulling the poles together. Cin8p and Kip1p (dark circles) apparently function to counter this inwardly directed force, most likely by crosslinking and sliding the nuclear microtubules, using the microtubules to push out on the spindle  poles (Hoyt et al., 1992) though other mechanisms are also possible. The bipolar spindle now has two types of forces acting in opposition  on the spindle poles, represented by open arrows. Once anaphase begins, the antagonistic relationship between Cin8p and Kar3p  changes. Kar3p-driven spindle collapse no longer occurs in cin8 kip1 mutants (Saunders et al., 1992); Kar3p is no longer found at the  spindle poles, and there is a net polymerization of the spindle microtubules (and to a lesser extent the cytoplasmic microtubules) resulting in a great increase in spindle length. Thus the outwardly directed force of Cin8p and Kip1p are retained, but the inward pull of  Kar3p is lost. It is proposed that this transition is a major contributor to the onset of anaphase B and can occur in the absence of chromosomes (Zhang and Nicklas, 1996). Spindle pole bodies are shown as discs embedded in a nuclear envelope, small open circles represent  microtubule depolymerization, and dark arrows the presumed direction of Kar3p movement.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139775&req=5

Figure 9: Model for Kar3p function in microtubule arrays. The Kar3p motor (shown as shaded circles) is proposed to function predominantly at spindle poles to stimulate microtubule turnover. At START of the cell cycle, the activity of Kar3p is required to limit microtubule number and length at the single spindle pole body (spb). During spindle assembly, the now duplicated spindle poles become linked by microtubule crosslinking and chromosome attachment (latter not shown). Depolymerization of the microtubules linking the spindle poles would now have the effect of pulling the poles together. Cin8p and Kip1p (dark circles) apparently function to counter this inwardly directed force, most likely by crosslinking and sliding the nuclear microtubules, using the microtubules to push out on the spindle poles (Hoyt et al., 1992) though other mechanisms are also possible. The bipolar spindle now has two types of forces acting in opposition on the spindle poles, represented by open arrows. Once anaphase begins, the antagonistic relationship between Cin8p and Kar3p changes. Kar3p-driven spindle collapse no longer occurs in cin8 kip1 mutants (Saunders et al., 1992); Kar3p is no longer found at the spindle poles, and there is a net polymerization of the spindle microtubules (and to a lesser extent the cytoplasmic microtubules) resulting in a great increase in spindle length. Thus the outwardly directed force of Cin8p and Kip1p are retained, but the inward pull of Kar3p is lost. It is proposed that this transition is a major contributor to the onset of anaphase B and can occur in the absence of chromosomes (Zhang and Nicklas, 1996). Spindle pole bodies are shown as discs embedded in a nuclear envelope, small open circles represent microtubule depolymerization, and dark arrows the presumed direction of Kar3p movement.
Mentions: The results reported here support the following model (Fig. 9): Kar3p is proposed to use its minus end–directed motor capacity to move to the spindle pole region of the cells before or at the START point of the cell cycle. At this time Kar3p functions to stimulate microtubule depolymerization at the minus ends associated with the spindle pole. As the bipolar spindle forms, the two poles become physically linked by intrapolar microtubules via kinetochore attachments and polar microtubule crosslinking. Microtubule depolymerization now would have the consequence of pulling the linked spindle poles together. (This model assumes a net polymerization at the kinetochore or plus end of the spindle microtubules and a net depolymerization at the minus end. This type of spindle flux has been observed in spindles assembled in Xenopus oocyte extracts [Sawin and Mitchison, 1991] but has not yet been documented in the smaller yeast spindles.) In the normal spindle the assembly motors Cin8p and Kip1p resist this collapsing action producing a balanced level of motor function in the spindle. As anaphase begins, the microtubule dynamics in the cell change. Now there is a net polymerization of the nuclear microtubules (and to a lesser extent of the cytoplasmic microtubules, as shown here) to produce the long anaphase spindle necessary for proper chromosome segregation. During this time of net microtubule polymerization, the depolymerizing activity of Kar3p is no longer useful, and Kar3p is lost from the spindle poles. The activity of Cin8p and Kip1p is retained, and this shift in the balance of motor activity is proposed to make a major contribution to the timing of the onset of anaphase B. Further experimentation will be required to confirm and expand upon this model. For example, it is currently unknown whether Kar3p acts directly to depolymerize microtubules in vivo and whether it acts on all microtubules as shown, or on a more limited set.

Bottom Line: We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner.Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization.These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA. wsaund@vms.cis.pitt.edu

ABSTRACT
The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

Show MeSH
Related in: MedlinePlus