Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans.

Specimen preparation for electron microscopy Wild-type C. elegans hermaphrodites were cryoimmobilized using a BAL-TEC HPM 010 high-pressure freezer (BAL-TEC). Fixation was performed by freeze substitution over 3 d at −90°C in anhydrous acetone containing 1% OsO 4 and 0.1% uranyl acetate (EM AFS; Leica) ( McDonald and Müller-Reichert, 2002 ). Epon/Araldite infiltrated samples were flat embedded in a thin layer of resin and polymerized for 3 d at 60°C ( Rappleye et al., 1999 ). Worms containing >5 early embryos were selected by light microscopy before remounting. Serial semithick sections (300–400 nm) were cut using a Leica Ultracut UCT Microtome. Sections were collected on Formvar-coated copper slot grids and poststained with 2% uranyl acetate in 70% methanol and Reynold's lead citrate.

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 × 1K CCD camera at a pixel size of 1.4 nm. In some instances, montages of 2 × 1 or 3 × 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.

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.

Supplemental Material [Supplemental Material Index]