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Address correspondence to T. Müller-Reichert, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, D-01307 Dresden, Germany. Tel.: 49-351-210-1763. Fax: 49-351-210-2000. email: mueller-reichert{at}mpi-cbg.de
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Abstract |
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Key Words: Caenorhabditis elegans; centrosome; electron tomography; mitosis; 3-D reconstruction
Abbreviations used in this paper: 3-D, three-dimensional; KMT, kinetochore MT; MT, microtubule; PCM, pericentriolar material.
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Introduction |
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Results and discussion |
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Fine structure of kinetochore MT (KMT) plus ends
We have used electron tomography to ask whether different minus end structures are associated with specific subpopulations of spindle MTs. First, we identified chromatin-associated ends of spindle MTs. In C. elegans this interaction occurs in a ribosome-free zone that surrounds each chromosome (Howe et al., 2001). When chromatin is aligned on the metaphase plate, the two pole-facing regions are peppered with MT ends (Fig. 3 A). In the corresponding three-dimensional (3-D) model, 118 MTs were identified (Fig. 3 B). 85 of these MTs terminated in the ribosome-free zone at the face of the condensed chromatin and were considered to be kinetochore MTs (KMTs). At 610 nm resolution we have found no distinct electron-dense protein complex physically linking chromatin to the MTs. Instead, there was a gap between the MT plus ends and the apparent surface of the chromatin. This gap contained a loose mat of fine filaments. KMT ends lay at a mean (±SD) distance from the chromatin surface of 115 (±52) nm. The mean width of the ribosome-free zone was 194 (±45) nm. Interestingly, the kinetochores of PtK1 cells prepared by high-pressure freezing/freeze-substitution have been reported to contain a "ribosome-excluded" zone 100150-nm wide (McEwen et al., 1998), which is similar in appearance and dimension to the zone described here.
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What is the functional consequence of these different minus-end structures? It seems likely that the capped ends are stable minus ends, nucleated from the centrosome. -tubulin appears to be the kinetically dominant nucleator of mitotic centrosomes in C. elegans (Hannak et al., 2002), suggesting that these minus ends are capped by
-tubulin during nucleation. Interestingly, the capped ends make up the majority (
80%) of the minus ends. Open ends, on the other side, are likely to be dynamic, perhaps accounting for the dynamic properties of minus ends at spindle poles. The open minus-end structure could be a structural consequence of MT severing where release of the
-tubulin cap could then lead to more dynamic behavior of the MT minus ends. MT severing by katanin-like proteins has been documented in meiotic spindles of C. elegans, to serve a possible role in limiting the size of MTs in these smaller spindles (Srayko et al., 2000), as well as in mitotic spindles from other systems (McNally et al., 1996; McNally and Thomas, 1998). Possibly, open MTs do not associate with nucleating sites but rather with structures capable of force generation and MT disassembly activity in the PCM. Such ends could be generated by release and anchoring of centrosome-nucleated MTs (for reviews see Bornens, 2002; McIntosh et al., 2002). Dynamic MT minus ends could also participate in poleward MT flux of mitotic spindles (for review see Wittmann et al., 2001). Future experiments combining RNA-mediated interference (RNAi) and tomographic reconstruction of different spindles should distinguish these different possibilities.
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Materials and methods |
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High-voltage electron tomography
Electron tomography was performed essentially as described in O'Toole et al. (1999). Briefly, 15-nm colloidal gold particles (Sigma-Aldrich) were attached to both surfaces of the semi-thick sections to serve as fiducial markers for subsequent image alignment. The specimens were placed in a tilt-rotate specimen holder (Model 650; Gatan, Pleasanton, CA) and tomographic datasets recorded using a JEM-1000 high-voltage electron microscope (JEOL, USA) operated at 750 kV. Images were captured every 1.5° over a ± 60° range using a Gatan 1K x 1K CCD camera at a pixel size of 1.4 nm. In some instances, montages of 2 x 1 or 3 x 1 frames were collected and used to image larger areas (Marsh et al., 2001). For dual axis tomography, the grids were imaged in one tilt series then rotated 90°, and a similar tilt series was acquired. For image processing, images were transferred to a Silicon Graphics workstation, and the tilted views were aligned using the positions of the colloidal gold particles as fiducial points. Tomograms were computed for each tilt axis using the R-weighted back-projection algorithm (Gilbert, 1972). We used the ratio of the section thickness, as defined by the microtome's setting, to the section's thickness measured after microscopy to calculate a "thinning factor," which was then used to correct the tomogram's dimension along the beam axis (O'Toole et al., 1999). For double tilt data sets, the two tomograms were aligned to each other and combined (Mastronarde, 1997). In addition, tomograms computed from adjacent serial sections were aligned and joined to increase the reconstructed volume (Ladinsky et al., 1999; Marsh et al., 2001). We recorded 11 double tilt series of mitotic spindles. In total, we analyzed 5 mitotic centrosomes.
Modeling and analysis of tomographic data
Tomograms were displayed and analyzed using the IMOD software package (Kremer et al., 1996). Features of interest were modeled in the serial slices extracted from the tomogram. An "image slicer" window in IMOD was used to display a slice extracted from the 3-D volume in any position or orientation; this feature was useful for unambiguous tracking of MTs in 3-D (O'Toole et al., 1999). With the slicer window, we analyzed the morphology of MT ends near the centrosomes and kinetochores by extracting a slice of image data 1-voxel thick and adjusting its orientation to contain the axis of the MT in a single view (O'Toole et al., 1999). A projection of the 3-D model was displayed and rotated to study its 3-D geometry. For this display in 3-D, MTs were shown as tubular graphic objects.
A program was written to compute the distance between the points on a selected object and a chosen reference location. The centroid of each centriole was located and used as the reference for positions within the spindle. A single model point was located at the pole-proximal end of each MT, and the 3-D distance of those points from the reference was calculated. A neighbor density analysis was performed to determine if there were preferred inter-fiber distances. These have been seen as indicative of interactions between different classes of MTs in two (McDonald et al., 1992) and three dimensions (Mastronarde et al., 1993, Marsh et al., 2001). MT ends were classified as described (Müller-Reichert et al., 1998).
Online supplemental material
Supplemental videos are available at http://www.jcb.org/cgi/content/full/jcb.200304035/DC1. Videos 1 and 2 show the complete tomographic volume of centrosomes in metaphase and anaphase corresponding to Fig. 1, A and D, respectively. Videos 3 and 4 are the projected 3-D models corresponding to Fig. 1, B and C, and E and F, respectively. Video 5 corresponds to Fig. 3 A and shows the tomographic reconstruction of a kinetochore region in metaphase, and Video 6 shows the projected 3-D model displayed in Fig. 3 B. The video sequence associated with Fig. 4 B (Video 7) shows a movie through a tomographic reconstruction of a kinetochore region in greater detail. Video 8, corresponding to Fig. 5, A and B, illustrates the tracing of KMTs in a partially reconstructed metaphase spindle.
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Acknowledgments |
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This work was supported in part by grant RR00592 from the National Center for Research Resources of the National Institutes of Health to J.R. McIntosh, who is a Research Professor of the American Cancer Society.
Submitted: 7 April 2003
Accepted: 17 September 2003
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