Yang G et al. (2008), Regional variation of microtubule flux reveals ...

XB-ART-38249

J Cell Biol 2008 Aug 25;1824:631-9. doi: 10.1083/jcb.200801105.

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Regional variation of microtubule flux reveals microtubule organization in the metaphase meiotic spindle.

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.

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Species referenced: Xenopus laevis
Genes referenced: arhgef7 dctn2

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	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 20–200 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/dynamitin–treated 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/dynamitin–treated spindles (J; N = 6). (K–O) 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 1–2 and 22–23 define left and right poles in our analysis, respectively.

References [+] :

Burbank, A new method reveals microtubule minus ends throughout the meiotic spindle. 2006, Pubmed, Xenbase