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Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

Goshima G, Nédélec F, Vale RD - J. Cell Biol. (2005)

Bottom Line: Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes.Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs.From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

View Article: PubMed Central - PubMed

Affiliation: The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

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FRAP studies of K fiber-associated Ncd-GFP. (A) Comparison of expression levels of Ncd-GFP with endogenous Ncd. A control wild-type cell (1) and a Ncd-GFP–expressing cell in which endogenous Ncd was selectively knocked down by RNAi against UTR region of endogenous Ncd gene (2) were stained with an anti-Ncd antibody. The immunofluorescence intensity of Ncd-GFP in 2 was similar to endogenous Ncd (1). Bar, 5 μm. (B) FRAP experiment reveals dynamic interaction of Ncd with K fiber. (Left) Ncd-GFP fluorescence in the subregion of K fiber (white square) was bleached. (Right) Fluorescence recovery after photobleaching of Ncd-GFP on K fiber. Mean value of relative GFP intensity is shown by square dots with standard deviation (n = 9). Relative GFP intensity was plotted at each time point after normalization using NBA as reference. GFP intensity before bleaching was adjusted to 1.00 (Materials and methods). Immobile fraction was <10% and half time of equilibrium was 2.5 ± 1.0 s, indicating a fast turnover of Ncd-GFP. A FRAP result of GFP-tubulin (subregion of K fiber) is also plotted (open square) and significant fluorescence recovery is not seen in this time range.
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fig3: FRAP studies of K fiber-associated Ncd-GFP. (A) Comparison of expression levels of Ncd-GFP with endogenous Ncd. A control wild-type cell (1) and a Ncd-GFP–expressing cell in which endogenous Ncd was selectively knocked down by RNAi against UTR region of endogenous Ncd gene (2) were stained with an anti-Ncd antibody. The immunofluorescence intensity of Ncd-GFP in 2 was similar to endogenous Ncd (1). Bar, 5 μm. (B) FRAP experiment reveals dynamic interaction of Ncd with K fiber. (Left) Ncd-GFP fluorescence in the subregion of K fiber (white square) was bleached. (Right) Fluorescence recovery after photobleaching of Ncd-GFP on K fiber. Mean value of relative GFP intensity is shown by square dots with standard deviation (n = 9). Relative GFP intensity was plotted at each time point after normalization using NBA as reference. GFP intensity before bleaching was adjusted to 1.00 (Materials and methods). Immobile fraction was <10% and half time of equilibrium was 2.5 ± 1.0 s, indicating a fast turnover of Ncd-GFP. A FRAP result of GFP-tubulin (subregion of K fiber) is also plotted (open square) and significant fluorescence recovery is not seen in this time range.

Mentions: Localization of Ncd was also investigated by stably expressing GFP-tagged Ncd. Functionality of Ncd-GFP for K fiber focusing was confirmed using rescue experiment, in which UTR-based RNAi knockdown of endogenous Ncd was combined with ectopic expression of Ncd-GFP fusion protein (Goshima and Vale, 2005). We also quantitatively measured the K fiber distance in this rescue condition, and found that Ncd-GFP expression rescued focusing of the fibers to control level (Ncd-GFP expression restored K fiber distance after Ncd RNAi from 5.2 ± 2.3 μm [n = 21] to 2.0 ± 0.6 μm [n = 21], a value close to that of control cells [2.2 ± 1.2 μm]). Unlike dynein, Ncd-GFP was clearly localized to microtubules in the mitotic spindle. To verify that GFP expression levels used for imaging in this study were in a similar range to the endogenous protein level, UTR-based RNAi was performed to deplete endogenous Ncd; Ncd-GFP was then expressed from an inducible promoter and the cells were stained with an anti-Ncd antibody (Fig. 3 A). We then examined low expressing cells that were typically used for live imaging (e.g., Fig. 3 A, 2) and compared the level of anti-Ncd antibody staining with that of endogenous Ncd in control wild-type cells (e.g., Fig. 3 A, 1). The intensity of anti-Ncd staining in the mitotic spindles in the Ncd-GFP–expressing cells was similar to wild-type cells (Fig. 3 A, compare 1 and 2). This level of expression also rescues the Ncd pole unfocusing phenotype (Fig. 3 A, 2). Thus, we believe that imaging of GFP-tagged Ncd in this study was performed under expression conditions that produced comparable levels to the endogenous protein.


Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

Goshima G, Nédélec F, Vale RD - J. Cell Biol. (2005)

FRAP studies of K fiber-associated Ncd-GFP. (A) Comparison of expression levels of Ncd-GFP with endogenous Ncd. A control wild-type cell (1) and a Ncd-GFP–expressing cell in which endogenous Ncd was selectively knocked down by RNAi against UTR region of endogenous Ncd gene (2) were stained with an anti-Ncd antibody. The immunofluorescence intensity of Ncd-GFP in 2 was similar to endogenous Ncd (1). Bar, 5 μm. (B) FRAP experiment reveals dynamic interaction of Ncd with K fiber. (Left) Ncd-GFP fluorescence in the subregion of K fiber (white square) was bleached. (Right) Fluorescence recovery after photobleaching of Ncd-GFP on K fiber. Mean value of relative GFP intensity is shown by square dots with standard deviation (n = 9). Relative GFP intensity was plotted at each time point after normalization using NBA as reference. GFP intensity before bleaching was adjusted to 1.00 (Materials and methods). Immobile fraction was <10% and half time of equilibrium was 2.5 ± 1.0 s, indicating a fast turnover of Ncd-GFP. A FRAP result of GFP-tubulin (subregion of K fiber) is also plotted (open square) and significant fluorescence recovery is not seen in this time range.
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Related In: Results  -  Collection

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fig3: FRAP studies of K fiber-associated Ncd-GFP. (A) Comparison of expression levels of Ncd-GFP with endogenous Ncd. A control wild-type cell (1) and a Ncd-GFP–expressing cell in which endogenous Ncd was selectively knocked down by RNAi against UTR region of endogenous Ncd gene (2) were stained with an anti-Ncd antibody. The immunofluorescence intensity of Ncd-GFP in 2 was similar to endogenous Ncd (1). Bar, 5 μm. (B) FRAP experiment reveals dynamic interaction of Ncd with K fiber. (Left) Ncd-GFP fluorescence in the subregion of K fiber (white square) was bleached. (Right) Fluorescence recovery after photobleaching of Ncd-GFP on K fiber. Mean value of relative GFP intensity is shown by square dots with standard deviation (n = 9). Relative GFP intensity was plotted at each time point after normalization using NBA as reference. GFP intensity before bleaching was adjusted to 1.00 (Materials and methods). Immobile fraction was <10% and half time of equilibrium was 2.5 ± 1.0 s, indicating a fast turnover of Ncd-GFP. A FRAP result of GFP-tubulin (subregion of K fiber) is also plotted (open square) and significant fluorescence recovery is not seen in this time range.
Mentions: Localization of Ncd was also investigated by stably expressing GFP-tagged Ncd. Functionality of Ncd-GFP for K fiber focusing was confirmed using rescue experiment, in which UTR-based RNAi knockdown of endogenous Ncd was combined with ectopic expression of Ncd-GFP fusion protein (Goshima and Vale, 2005). We also quantitatively measured the K fiber distance in this rescue condition, and found that Ncd-GFP expression rescued focusing of the fibers to control level (Ncd-GFP expression restored K fiber distance after Ncd RNAi from 5.2 ± 2.3 μm [n = 21] to 2.0 ± 0.6 μm [n = 21], a value close to that of control cells [2.2 ± 1.2 μm]). Unlike dynein, Ncd-GFP was clearly localized to microtubules in the mitotic spindle. To verify that GFP expression levels used for imaging in this study were in a similar range to the endogenous protein level, UTR-based RNAi was performed to deplete endogenous Ncd; Ncd-GFP was then expressed from an inducible promoter and the cells were stained with an anti-Ncd antibody (Fig. 3 A). We then examined low expressing cells that were typically used for live imaging (e.g., Fig. 3 A, 2) and compared the level of anti-Ncd antibody staining with that of endogenous Ncd in control wild-type cells (e.g., Fig. 3 A, 1). The intensity of anti-Ncd staining in the mitotic spindles in the Ncd-GFP–expressing cells was similar to wild-type cells (Fig. 3 A, compare 1 and 2). This level of expression also rescues the Ncd pole unfocusing phenotype (Fig. 3 A, 2). Thus, we believe that imaging of GFP-tagged Ncd in this study was performed under expression conditions that produced comparable levels to the endogenous protein.

Bottom Line: Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes.Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs.From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

View Article: PubMed Central - PubMed

Affiliation: The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

Show MeSH