Goodman B et al. (2010), Lamin B counteracts the kinesin Eg5 to restrain...

XB-ART-42039

J Biol Chem 2010 Nov 05;28545:35238-44. doi: 10.1074/jbc.M110.140749.

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Lamin B counteracts the kinesin Eg5 to restrain spindle pole separation during spindle assembly.

Goodman B , Channels W , Qiu M , Iglesias P , Yang G , Zheng Y .

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Lamin B is a component of the membranous spindle matrix isolated from Xenopus egg extracts, and it is required for proper spindle morphogenesis. Besides lamin B, the spindle matrix contains spindle assembly factors (SAFs) such as Eg5 and dynein which are known to regulate microtubule organization and SAFs known to promote microtubule assembly such as Maskin and XMAP215. Because lamin B does not bind directly to microtubules, it must affect spindle morphogenesis indirectly by influencing the function of spindle matrix-associated SAFs. Using different assays in Xenopus egg extracts, we found that depleting lamin B caused formation of elongated and multipolar spindles, which could be reversed by partially inhibiting the kinesin Eg5, revealing an antagonistic relationship between Eg5 and lamin B. However, lamin B only very weakly antagonizes Eg5 in mediating poleward microtubule-flux based on fluorescence speckle microscopy. Depleting lamin B led to a very small but statistically significant increase in flux. Furthermore, flux reduction caused by partial Eg5 inhibition is only slightly reversed by removing lamin B. Because lamin B does not bind to Eg5, our studies suggest two nonexclusive mechanisms by which lamin B can indirectly antagonize Eg5. It could function in a network that restricts Eg5-driven microtubule sliding only when microtubules come into transient contact with the network. Lamin B could also function to sequester microtubule polymerization activities within the spindle. Without lamin B, increased microtubule assembly caused by the released SAFs would lead to excessive microtubule sliding that results in formation of elongated and multipolar spindles.

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Species referenced: Xenopus laevis
Genes referenced: aurka ckap5 kif11 lmnb1 lmnb3 tacc3

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	FIGURE 1. Characterization of spindle-like structures stimulated by AurA beads and RanGTP. A, time lapse frames showing formation of bipolar or multipolar spindle-like structures. Clusters of two to three AurA beads initially nucleate MT asters. The separation of the beads within the asters leads to formation of either bipolar or multipolar spindle-like structures (see supplemental Movies S1 and S2). The time (minutes) above the images corresponds to the time following initiation of the reaction that the images were taken. B, MT speckle images of one bipolar spindle-like structure that is undergoing elongation. AurA beads are masked to visualize the speckles better. The yellow dashed line indicates the region used for kymograph analysis in C. C, speckles, labeled green or red, moving toward each of the AurA beads, are demonstrating speckle movement in each direction. Below, a kymograph shows the speckle movements with respect to the movement of AurA beads. D, speckles color coded according to their average instantaneous speckle velocities shown on the histogram in E (flux rate, 2.229; S.D., 0.532; speckles tracked, 632). The same bipolar spindle-like structure shown in C was used. F, reduction of the rate of AurA bead separation in bipolar spindle-like structures by inhibiting Eg5 with 15 μm Monastrol. The distances between two AurA beads during spindle assembly in the presence or absence of Monastrol were measured and plotted against time. The plot shows average distance for each time point across all four experiments for each condition. Scale bars, 10 μm.
	FIGURE 2. LB3 and Eg5 oppose each other in maintaining the separation of AurA beads in spindle-like structures. A, sample images of MT structures formed in the presence or absence of Monastrol. Images were acquired randomly using an automated stage control. The small white dots and white lines in these and other images in this figure represent AurA beads and the linear distance between nearest neighboring beads connected by MTs, respectively, as characterized by the image recognition software developed in Matlab (see supplemental Fig. S1B). B, dose-dependent decrease of the distances between neighboring AurA beads in MT structures caused by inhibition of Eg5 by Monastrol. The distances are normalized to 0 μm Monastrol (set to 1) to allow comparisons among different egg extracts. C, sample images of MT structures formed in the presence or absence of LB3 and Monastrol. D, increase in the distance between AurA beads caused by LB3 depletion. Quantification of distances between neighboring AurA beads in the presence or absence of LB3 without Monastrol is shown. E, depletion of LB3 relief of the effect of Eg5 inhibition at each concentration of Monastrol tested. Quantification of distances between neighboring AurA beads in the presence or absence of LB3 and an increasing concentration of Monastrol is shown. Error bars, S.E. from at least three experiments performed in different egg extracts on different days with ∼200 measurements of distances in each condition and each experiment. Scale bars, 50 μm.
	FIGURE 3. LB3 limits whereas Eg5 promotes the separation of clustered AurA beads during formation of spindle-like structures. A, images of MT structures formed in the presence or absence of various concentrations of Monastrol at 15 min into the reaction. B, increase of AurA bead clustering caused by Eg5 inhibition. Quantification of the bead-clustering index at different concentrations of Monastrol is shown. Values are normalized to 0 μm Monastrol (dimethyl sulfoxide carrier) to allow comparisons among different egg extracts. Monastrol caused a dose-dependent increase in AurA-bead-clustering. C, images of AurA beads in MT structures assembled in the presence or absence of LB3 and Monastrol at 15 min into the reaction. D, decrease in AurA bead clustering caused by LB3 inhibition. Quantification of the bead-clustering index in the presence or absence of LB3 without inhibiting Eg5 is shown. E, depletion of LB3 relief of the clustering effect caused by Monastrol. Quantification of clustering index in the presence or absence of LB3 and an increasing concentration of Monastrol is shown. Arrows and arrowheads in all images indicate clustered and separated AurA beads, respectively. Error bars are S.E. from at least three experiments performed in different egg extracts on different days with 700 beads counted per condition per experiment. Scale bars, 10 μm.
	FIGURE 4. Effects of LB3 and Eg5 on the length, poles, and MT flux of spindles assembled from sperm chromatin. A, images of spindles formed in mock-depleted and LB3-depleted egg extracts. LB3 depletion resulted in larger spindles and an increase in multipolar spindles. B, quantifications of spindle length, excluding half-spindles and monopolar spindles, in both mock-depleted and LB3-depleted extracts in the presence or absence of 15 μm Monastrol. Monastrol rescued the spindle length change caused by LB3 depletion. C, quantification of the percentage of bipolar spindles, excluding half-spindles and monopolar spindles, in mock-depleted extracts and LB3-depleted extracts in the presence or absence of 15 μm Monastrol. LB3 depletion caused a decrease in the percentage of bipolar spindles, which was corrected with the addition of 15 μm Monastrol. Approximately 35 spindles were analyzed per condition per experiment, and three experiments performed on different days were used for quantifications. Error bars, S.E. D, speckle images of sperm spindles. The gray image shows a frame of the speckled spindle. Speckles moving toward opposite spindle poles are labeled as green and red dots. E, speckle trajectories color-coded according to their average instantaneous speckle velocities shown on the histogram to the right (flux rate, 2.031; S.D., 0.488; speckles tracked, 4488). Scale bars, 10 μm.

References [+] :

Adam, Regulation of nuclear lamin polymerization by importin alpha. 2008, Pubmed, Xenbase