Uteng M et al. (2008), Poleward transport of Eg5 by dynein-dynactin in...

XB-ART-38248

J Cell Biol 2008 Aug 25;1824:715-26. doi: 10.1083/jcb.200801125.

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Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles.

Uteng M , Hentrich C , Miura K , Bieling P , Surrey T .

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Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein-dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.

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Species referenced: Xenopus laevis
Genes referenced: abl1 arhgef7 kif11

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	Figure 1. Microtubule pair sliding driven by purified Eg5 in buffer. (a) Schematic of Eg5 within a pair of antiparallel microtubules. The red microtubule is biotinylated and immobilized to a chemically functionalized glass surface via biotin–neutravidin links. The green microtubule is not biotinylated and mobile. (b) Fluorescence microscopy images of Alexa 568 microtubule pairs formed with Eg5-paGFP. Dimly labeled microtubules immobilized on a biotin-PEG glass surface supporting Eg5-mediated binding of brightly labeled microtubules (see Materials and methods). (c) Time series of confocal fluorescence microscopy images of a Cy5-labeled microtubule (green) moved by Eg5 along an immobilized Alexa 568–labeled microtubule (red). Arrows indicate the initial position of the Cy5-labeled microtubule. The bottom panel shows a kymograph of a line along the microtubule pair during the first 5 min of the time series. (d) Histograms showing distributions for speeds of microtubule sliding driven by Eg5 and Eg5-paGFP.
	Figure 2. Fluorescence intensity profiles from spindles after photobleaching and photoactivation. Time-lapse videos of a confocal section through spindles containing Cy5 microtubules and photoactivatable Eg5-paGFP were recorded simultaneously in the Cy5 and GFP channel of a confocal fluorescence microscope. Between the first and the second image of a time series, one or several rectangular stripes in the confocal section were bleached and simultaneously photoactivated. To convert the images of the two time series into fluorescence intensity profiles, the area of the spindle in the Cy5 microtubule image before the photobleach was determined by applying an intensity threshold to the image (I). This area served then as a mask for the entire time series of the Cy5 microtubule images and the Eg5-paGFP (illustrated here only for the second frame of the time series). The intensity values were projected onto the spindle axis. This created fluorescence intensity profiles (II and V). The turnover of microtubules and of Eg5 was extracted from time series of these intensity profiles (II and V; see Materials and methods). To determine the exact position of the maximum amount of bleached microtubules, the Cy5 intensity profiles were subtracted from the prebleach profile, resulting in an inverted intensity difference profile (III). The positions of the maximum of the intensity difference profiles of Cy5 microtubules and of the intensity profiles of photoactivated Eg5-paGFP were determined from a Gaussian fit (blue) to the profiles (IV and VI) and used to calculate the velocity of the movements of the peaks (see Materials and methods).
	Figure 3. Directed movements of photoactivated Eg5-paGFP and of photobleached Cy5 microtubules along the spindle axis. (a) Spindles in egg extract depleted from endogenous Eg5 and supplemented with Eg5-paGFP and Cy5-tubulin before and after photoactivation of Eg5-paGFP and simultaneous photobleaching of Cy5 microtubules in two different spindle regions. Time series of confocal fluorescence microscopy images of Eg5-paGFP (green) and Cy5 microtubules (MT; red) at the indicated times, and fluorescence intensity profiles (Fig. 2) for Cy5 microtubules and Eg5-paGFP of the same spindle at the indicated times. (b) Intensity difference profiles for bleached Cy5 microtubules and intensity profiles for photoactivated Eg5-paGFP at the indicated times. Only parts of entire profiles are shown for the midzone, halfzone, and pole region. Intensity difference profiles for Cy5 microtubules appear inverted as compared with the intensity profiles (Fig. 2). Note the splitting of the initial single peak of the red microtubule profile in the midzone into two peaks of the blue microtubule profile after 56 s. A corresponding split is not observed in the Eg5 profiles. The intensity profile for the halfzone is derived from the spindle shown in section a, whereas the intensity profiles for the midzone and pole are derived from spindles not shown because of different imaging requirements for the different regions in the spindle. (c) Displacements of the peaks of photoactivated Eg5-paGFP (green) and of photobleached Cy5 microtubules (red) along the spindle axis with time. Linear regression fits (lines) to the experimental values (dots) yielded the velocities as indicated. (d, left) Box plots of the speeds of Eg5 movement (22 measurements in 19 spindles) and of microtubule flux (MT; 16 measurements). (right) Scatter diagram of the speeds of microtubule (MT) flux as a function of the speeds of Eg5 in the halfzone. Each data point represents two simultaneous measurements in the same spindle region.
	Figure 4. Turnover of Eg5 and microtubules in egg extract spindles measured by time-lapse fluorescence microscopy after photoactivation and photobleaching. (a) Fluorescence decay of photoactivated Eg5-paGFP (green) and fluorescence recovery of Cy5 microtubules (red) in the midzone, the halfzone, and the pole region within the first 2 min after photoactivation and photobleaching. A monoexponential fit (lines) to the experimental values (dots) yielded half-lives as indicated. (b) Box plots of half-lives and residuals of fluorescence decays of photoactivated Eg5-paGFP in the three different spindle regions. 4–12 measurements were made per region.
	Figure 5. Disruption of the dynein–dynactin complex decreases the amount of Eg5 localizing to spindles in X. laevis egg extract. (a) Confocal fluorescence microscopy images of nonfixed spindles assembled either in the absence or additional presence of p50 or cc1 showing the localization of Eg5. The fluorescence of Eg5-paGFP (green) and of Cy5 microtubules (MT; red) was measured immediately after photoactivation of Eg5-paGFP in the entire spindle. (b) Fluorescence intensity profiles for photoactivated Eg5-paGFP and Cy5 microtubules along the spindle axis for the spindles shown in section a. (right) Mean ratios of the total fluorescence intensity of photoactivated Eg5-paGFP divided by the total fluorescence intensity of Cy5 microtubules (MT) for wild-type spindles, p50 spindles, and cc1 spindles are shown. The means were determined from five spindles per condition. The wild-type intensity ratio was set to 100. Error bars indicate standard deviation. (c) Confocal fluorescence microscopy images of fixed spindles assembled either in the absence (top) or presence (bottom) of cc1 showing the localization of dynein. Dynein heavy chain was detected by immunofluorescence (green), tubulin by using incorporated Alexa 568–tubulin (red), and DNA by Hoechst staining (blue). (d) Western blot analysis showing pull-down of p150Glued with Eg5 on anti-Eg5 beads and pull-down of Eg5 with p150Glued on anti-p150Glued beads in X. laevis egg extract, either in the absence (−cc1) or presence (+cc1) of added p150 fragment cc1. Mock represents magnetic beads coated with an irrelevant antibody also incubated in X. laevis egg extract. An anti-p150Glued antibody was used for detection in the top left three lanes and in the bottom right three lanes, whereas an anti-Eg5 antibody was used for detection in the top right three lanes and in the bottom left three lanes. All samples in a horizontal row were run on the same SDS gel. Molecular weight markers to the left of the blots are in kD.
	Figure 6. Dynamics of Eg5 and microtubules in spindles assembled after disruption of the dynein–dynactin complex. (a) Spindle assembled in the presence of p50. Confocal fluorescence microscopy images of photoactivated Eg5-paGFP (green) and Cy5-labeled microtubules (red) before and after photoactivation and simultaneous photobleaching in three different spindle regions. Fluorescence intensity profiles for Eg5-paGFP and Cy5 microtubules (MT) of the same spindle at the indicated times. (b) Sections of intensity difference profiles for bleached Cy5 microtubules and intensity profiles for photoactivated Eg5-paGFP in the halfzone of spindles assembled in the presence of p50 or cc1. (c) Displacements of the peaks of photoactivated Eg5-paGFP (green) and of photobleached Cy5 microtubules (red) along the spindle axis with time. Linear regression fits (lines) to the experimental values (dots) yielded the velocities as indicated. (d) Box plots of the speeds of Eg5 and of microtubule flux (MT) in the halfzone of p50 spindles (11 measurements in 6 spindles) and cc1 spindles (20 measurements in 11 spindles) as compared with wild-type spindles (data from Fig. 3 d). (e) Scatter diagram of speeds of directed movements of photoactivated Eg5-paGFP in the halfzone of p50 spindles (blue), cc1 spindles (green), and in unperturbed spindles (black) as a function of the simultaneously measured speeds of microtubule (MT) flux. (f) Box plots of the half-lives of the fluorescence decays of photoactivated Eg5-paGFP in p50 spindles, cc1 spindles, and unperturbed spindles (data from Fig. 4 b) and box plots of the corresponding residuals. Shown are the same measurements as in section e.

References [+] :

Bieling, Reconstitution of a microtubule plus-end tracking system in vitro. 2007, Pubmed