The Experts below are selected from a list of 105 Experts worldwide ranked by ideXlab platform
Ronald D Vale - One of the best experts on this subject based on the ideXlab platform.
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length control of the metaphase spindle
Current Biology, 2005Co-Authors: Gohta Goshima, Ronald D Vale, Roy Wollman, Nico Stuurman, Jonathan M ScholeyAbstract:BACKGROUND: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given Cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. RESULTS: Using the Drosophila S2 Cell Line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. CONCLUSIONS: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.
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the roles of microtubule based motor proteins in mitosis comprehensive rnai analysis in the drosophila S2 Cell Line
Journal of Cell Biology, 2003Co-Authors: Gohta Goshima, Ronald D ValeAbstract:Kinesins and dyneins play important roles during Cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 Cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but Cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan Cell type.
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drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
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drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
Stephen L Rogers - One of the best experts on this subject based on the ideXlab platform.
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drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
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drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
David J Sharp - One of the best experts on this subject based on the ideXlab platform.
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drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
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drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
Gregory C Rogers - One of the best experts on this subject based on the ideXlab platform.
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drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
-
drosophila eb1 is important for proper assembly dynamics and positioning of the mitotic spindle
Journal of Cell Biology, 2002Co-Authors: Stephen L Rogers, Gregory C Rogers, David J Sharp, Ronald D ValeAbstract:EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 Cell Line that promotes Cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase Cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic Cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the Cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live Cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the Cell cortex.
Gohta Goshima - One of the best experts on this subject based on the ideXlab platform.
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length control of the metaphase spindle
Current Biology, 2005Co-Authors: Gohta Goshima, Ronald D Vale, Roy Wollman, Nico Stuurman, Jonathan M ScholeyAbstract:BACKGROUND: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given Cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. RESULTS: Using the Drosophila S2 Cell Line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. CONCLUSIONS: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.
-
the roles of microtubule based motor proteins in mitosis comprehensive rnai analysis in the drosophila S2 Cell Line
Journal of Cell Biology, 2003Co-Authors: Gohta Goshima, Ronald D ValeAbstract:Kinesins and dyneins play important roles during Cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 Cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but Cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan Cell type.
