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Yang G
,
Cameron LA
,
Maddox PS
,
Salmon ED
,
Danuser G
.
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Continuous poleward movement of tubulin is a hallmark of metaphase spindle dynamics in higher eukaryotic cells and is essential for stable spindle architecture and reliable chromosome segregation. We use quantitative fluorescent speckle microscopy to map with high resolution the spatial organization of microtubule flux in Xenopus laevis egg extract meiotic spindles. We find that the flux velocity decreases near spindle poles by approximately 20%. The regional variation is independent of functional kinetochores and centrosomes and is suppressed by inhibition of dynein/dynactin, kinesin-5, or both. Statistical analysis reveals that tubulin flows in two distinct velocity modes. We propose an association of these modes with two architecturally distinct yet spatially overlapping and dynamically cross-linked arrays of microtubules: focused polar microtubule arrays of a uniform polarity and slower flux velocities are interconnected by a dense barrel-like microtubule array of antiparallel polarities and faster flux velocities.
Figure 1. Regional variation of poleward microtubule flux in control Xenopus egg extract spindles. (A) X-rhodamine tubulin speckles. (B and C) Speckle trajectories separated by direction of poleward movement. Green and red arrows indicate flux towards the left and right poles, respectively. (D and E) Speckle trajectories color coded according to the velocity histogram (E); the flux rate of the spindle shown is mean ± SD = 2.52 ± 0.65 μm/min (N = 9,562 trajectories). (F) Definition of 23 regions along the pole-to-pole axis. (G) Mean and SD (error bars) of speckle velocities within regions in F. (H) Spatial distributions of normalized speckle velocities of 11 control spindles. Bar, 10 μm.
Figure 2. Regional variation of poleward microtubule flux in spindles assembled around plasmid DNA-coated beads. (A) X-rhodamine tubulin speckles and autofluorescent beads. (B and C) Speckle trajectories color coded according to the velocity histogram (C); the flux rate of the spindle shown is mean ± SD = 2.69 ± 0.74 μm/min (N = 2,128 trajectories). (D and E) Mean and SD (error bars) of speckle velocities within regions in D. (F) Spatial distributions of normalized microtubule velocities of nine bead spindles. Bar, 10 μm.
Figure 3. Inhibition of dynein/dynactin, kinesin-5, or both suppresses regional variation of poleward microtubule flux. (A) Spindle treated with excess p50/dynamitin. (B and C) Speckle trajectories color coded according to the velocity histogram (C); the flux rate of the specific spindle shown is mean ± SD = 2.33 ± 0.60 μm/min (N = 2,702 trajectories). (D and E) Mean and SD (error bars) of speckle velocities within regions in D. (F) Normalized flux rate along the pole-to-pole axis of control (average over 11 spindles) versus p50/dynamitin-treated spindles (average over seven spindles). (G) Flux rates at different monastrol concentrations. (H) Spindle treated with 200 μM monastrol. (I and J) Speckle trajectories color coded according to the velocity histogram (J); the flux rate of the spindle shown is mean ± SD = 0.89 ± 0.30 μm/min (N = 3,085 trajectories). (K and L) Mean and SD (error bars) of speckle velocities within regions in K. (M) Normalized flux rate distribution along the pole-to-pole axis of control and monastrol-treated spindles (average over 17 spindles treated with 20â200 μM monastrol). (N) Spindle treated with both 200 μM monastrol and excess p50/dynamitin. (O and P) Speckle trajectories color coded according to the velocity histogram (P); the flux rate of the spindle shown is mean ± SD = 1.59 ± 0.38 μm/min (N = 7,214 trajectories). (Q and R) Mean and SD (error bars) of speckle velocities within regions in Q. (S) Normalized flux rate distribution along the pole-to-pole axis of control versus monastrol + p50/dynamitinâtreated spindles (average over six spindles). Bars, 10 μm.
Figure 4. Spatial organization of spindle microtubules. (A, C, E, G, and I) Mode analysis of flux velocity distributions for representative individual spindles: the control spindle in Fig. 1 A (A), the DNA bead spindle in Fig. 2 A (C), the spindle treated with p50/dynamitin in Fig. 3 A (E), the spindle treated with 200 μM monastrol in Fig. 3 C (G), and the spindle treated with 200 μM monastrol and p50/dynamitin in Fig. 3 N (I). Red, fast mode; green, slow mode; cyan, mixture of both modes. (B, D, F, H, and J) Regional contribution of velocity modes. Averages of control spindles (B; N = 11), DNA bead spindles (D; N = 9), p50/dynamitin-treated spindles (F; N = 7), monastrol-treated spindles (H; N = 7), and monastrol and p50/dynamitinâtreated spindles (J; N = 6). (KâO) Spatial distribution of speckle appearances of spindles analyzed in A, C, E, G, and I. Orange, speckles moving toward the left pole; blue, speckles moving toward the right pole. (P) Normalized distribution of average speckle intensity (average of 11 control spindles). (Q) Normalized speckle number along the pole-to-pole axis of nine control spindles. Gray, normalized speckle number of a spindle treated with 200 μM monastrol. (R) Model: two slow-fluxing polar microtubule arrays (green) are dynamically coupled via motors to a barrel array (red) with antiparallel microtubule polarity and faster flux velocities. Gray bands representing regions 1â2 and 22â23 define left and right poles in our analysis, respectively.
Burbank,
Slide-and-cluster models for spindle assembly.
2007, Pubmed,
Xenbase
Burbank,
Slide-and-cluster models for spindle assembly.
2007,
Pubmed
,
Xenbase
Burbank,
A new method reveals microtubule minus ends throughout the meiotic spindle.
2006,
Pubmed
,
Xenbase
Buster,
Poleward tubulin flux in spindles: regulation and function in mitotic cells.
2007,
Pubmed
Cameron,
Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells.
2006,
Pubmed
Desai,
The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.
1999,
Pubmed
,
Xenbase
Gaetz,
Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles.
2004,
Pubmed
,
Xenbase
Ganem,
The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK.
2004,
Pubmed
Ganem,
Efficient mitosis in human cells lacking poleward microtubule flux.
2005,
Pubmed
Heald,
Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.
1996,
Pubmed
,
Xenbase
Hyman,
Preparation of modified tubulins.
1991,
Pubmed
Kapitein,
The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks.
2005,
Pubmed
,
Xenbase
Kwok,
Microtubule flux: drivers wanted.
2007,
Pubmed
Maddox,
Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles: implications for spindle mechanics.
2003,
Pubmed
,
Xenbase
Maddox,
Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells.
2003,
Pubmed
Maiato,
Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres.
2005,
Pubmed
Mayer,
Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.
1999,
Pubmed
,
Xenbase
Mitchison,
Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence.
1989,
Pubmed
Mitchison,
Mechanism and function of poleward flux in Xenopus extract meiotic spindles.
2005,
Pubmed
,
Xenbase
Mitchison,
Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles.
2005,
Pubmed
,
Xenbase
Miyamoto,
The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles.
2004,
Pubmed
,
Xenbase
Murray,
Cell cycle extracts.
1991,
Pubmed
Ohi,
Nonredundant functions of Kinesin-13s during meiotic spindle assembly.
2007,
Pubmed
,
Xenbase
Ponti,
Computational analysis of F-actin turnover in cortical actin meshworks using fluorescent speckle microscopy.
2003,
Pubmed
Rogers,
Spindle microtubules in flux.
2005,
Pubmed
Rogers,
Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase.
2004,
Pubmed
Shirasu-Hiza,
Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked.
2004,
Pubmed
,
Xenbase
Vallotton,
Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study.
2003,
Pubmed
,
Xenbase
Waterman-Storer,
Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.
1998,
Pubmed
,
Xenbase
Wittmann,
Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes.
1999,
Pubmed
,
Xenbase
Yang,
Architectural dynamics of the meiotic spindle revealed by single-fluorophore imaging.
2007,
Pubmed
,
Xenbase
Zhang,
Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes.
2007,
Pubmed
