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Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states.

Cheerambathur DK, Brust-Mascher I, Civelekoglu-Scholey G, Scholey JM - J. Cell Biol. (2008)

Bottom Line: The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely.KLP61F appears to transiently bind MTs, moving short distances along them before detaching.Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California at Davis, Davis, CA 95616, USA.

ABSTRACT
The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable "spindle matrix" to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.

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Related in: MedlinePlus

Localization and function of KLP61F-GFP in D. melanogaster embryo mitosis. (A) Micrographs from a time-lapse video of a representative spindle showing KLP61F-GFP (left), rhodamine-tubulin (center), and double-label fluorescence (right) at various stages of mitosis. The plots (far right) are line scans extending pole to pole along an ipMT (10 pixels wide; ∼0.129 μm/pixel) for KLP61F (green) and tubulin (red). The y axis shows normalized fluorescence intensity. Bar, 5 μm. (B) Spindle pole dynamics in wild-type embryos, GFP-KLP61F rescued mutant embryos, and anti-KLP61F microinjected wild-type embryos showing how bipolar spindles collapse into monoasters after the loss of KLP61F function. Pole–pole separation dynamics are very similar in wild-type and rescued mutant embryos.
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fig1: Localization and function of KLP61F-GFP in D. melanogaster embryo mitosis. (A) Micrographs from a time-lapse video of a representative spindle showing KLP61F-GFP (left), rhodamine-tubulin (center), and double-label fluorescence (right) at various stages of mitosis. The plots (far right) are line scans extending pole to pole along an ipMT (10 pixels wide; ∼0.129 μm/pixel) for KLP61F (green) and tubulin (red). The y axis shows normalized fluorescence intensity. Bar, 5 μm. (B) Spindle pole dynamics in wild-type embryos, GFP-KLP61F rescued mutant embryos, and anti-KLP61F microinjected wild-type embryos showing how bipolar spindles collapse into monoasters after the loss of KLP61F function. Pole–pole separation dynamics are very similar in wild-type and rescued mutant embryos.

Mentions: To study the dynamics of KLP61F in vivo, standard fly genetics and transformation techniques (Roberts, 1986; Ashburner, 1989) were used to generate stable transgenic fly lines expressing KLP61F-GFP under the control of a poly-ubiquitin promoter in a form that localizes to spindles and supports mitosis (Fig. 1). This promoter drives near normal levels of KLP61F because quantitative immunoblotting revealed that wild-type embryos harboring two copies of the transgene contained KLP61F-GFP/endogenous KLP61F in the ratio 0.4–0.7:1.0. Also, KLP61F-GFP displayed similar spindle localization and dynamics in either the presence (unpublished data) or absence (see below) of wild-type KLP61F, which suggests that it can compete effectively with endogenous untagged KLP61F for binding sites in spindles. One or two copies of the KLP61F-GFP transgene were able to rescue several or severe loss-of-function mutant alleles (namely klp61f3, klp61f4, klp61f6836, and klp61f7415) that, in the absence of the rescuing transgene, only survive as homozygotes or transheterozygotes to second or third instar larval stages, when they die due to mitotic failures when the maternal load of KLP61F is depleted (Heck et al., 1993). For example, klp61f3/klp61f3 homozygotes and klp61f3/klp61f6836 transheterozygotes display larval lethality but were rescued by either one or two copies of the KLP61F-GFP transgene and produced viable, fertile adult flies that could be propagated as stable transgenic lines, which suggests that the KLP61F-GFP is functional (studies described in the following sections refer to the latter mutant rescued with two copies of the transgene). As with transgenic lines expressing other fluorescent mitotic proteins (e.g., GFP-Ncd; Endow and Komma, 1997), these KLP61F-GFP–expressing lines should be useful for studying mitosis in various D. melanogaster cell types.


Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states.

Cheerambathur DK, Brust-Mascher I, Civelekoglu-Scholey G, Scholey JM - J. Cell Biol. (2008)

Localization and function of KLP61F-GFP in D. melanogaster embryo mitosis. (A) Micrographs from a time-lapse video of a representative spindle showing KLP61F-GFP (left), rhodamine-tubulin (center), and double-label fluorescence (right) at various stages of mitosis. The plots (far right) are line scans extending pole to pole along an ipMT (10 pixels wide; ∼0.129 μm/pixel) for KLP61F (green) and tubulin (red). The y axis shows normalized fluorescence intensity. Bar, 5 μm. (B) Spindle pole dynamics in wild-type embryos, GFP-KLP61F rescued mutant embryos, and anti-KLP61F microinjected wild-type embryos showing how bipolar spindles collapse into monoasters after the loss of KLP61F function. Pole–pole separation dynamics are very similar in wild-type and rescued mutant embryos.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2500124&req=5

fig1: Localization and function of KLP61F-GFP in D. melanogaster embryo mitosis. (A) Micrographs from a time-lapse video of a representative spindle showing KLP61F-GFP (left), rhodamine-tubulin (center), and double-label fluorescence (right) at various stages of mitosis. The plots (far right) are line scans extending pole to pole along an ipMT (10 pixels wide; ∼0.129 μm/pixel) for KLP61F (green) and tubulin (red). The y axis shows normalized fluorescence intensity. Bar, 5 μm. (B) Spindle pole dynamics in wild-type embryos, GFP-KLP61F rescued mutant embryos, and anti-KLP61F microinjected wild-type embryos showing how bipolar spindles collapse into monoasters after the loss of KLP61F function. Pole–pole separation dynamics are very similar in wild-type and rescued mutant embryos.
Mentions: To study the dynamics of KLP61F in vivo, standard fly genetics and transformation techniques (Roberts, 1986; Ashburner, 1989) were used to generate stable transgenic fly lines expressing KLP61F-GFP under the control of a poly-ubiquitin promoter in a form that localizes to spindles and supports mitosis (Fig. 1). This promoter drives near normal levels of KLP61F because quantitative immunoblotting revealed that wild-type embryos harboring two copies of the transgene contained KLP61F-GFP/endogenous KLP61F in the ratio 0.4–0.7:1.0. Also, KLP61F-GFP displayed similar spindle localization and dynamics in either the presence (unpublished data) or absence (see below) of wild-type KLP61F, which suggests that it can compete effectively with endogenous untagged KLP61F for binding sites in spindles. One or two copies of the KLP61F-GFP transgene were able to rescue several or severe loss-of-function mutant alleles (namely klp61f3, klp61f4, klp61f6836, and klp61f7415) that, in the absence of the rescuing transgene, only survive as homozygotes or transheterozygotes to second or third instar larval stages, when they die due to mitotic failures when the maternal load of KLP61F is depleted (Heck et al., 1993). For example, klp61f3/klp61f3 homozygotes and klp61f3/klp61f6836 transheterozygotes display larval lethality but were rescued by either one or two copies of the KLP61F-GFP transgene and produced viable, fertile adult flies that could be propagated as stable transgenic lines, which suggests that the KLP61F-GFP is functional (studies described in the following sections refer to the latter mutant rescued with two copies of the transgene). As with transgenic lines expressing other fluorescent mitotic proteins (e.g., GFP-Ncd; Endow and Komma, 1997), these KLP61F-GFP–expressing lines should be useful for studying mitosis in various D. melanogaster cell types.

Bottom Line: The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely.KLP61F appears to transiently bind MTs, moving short distances along them before detaching.Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California at Davis, Davis, CA 95616, USA.

ABSTRACT
The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable "spindle matrix" to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.

Show MeSH
Related in: MedlinePlus