Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans

Abstract

During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.

Figure 1.

MT ends at the centrosome are either closed or open. Distribution of MT minus ends around centrioles in metaphase (A–C) and anaphase (D–F). (A and D) Selected tomographic slices showing MTs (arrows), the centrosome (c), and the condensed DNA (d, DNA surface outlined in green). (B and E) Corresponding 3-D models showing the centrioles (blue and purple), the MTs (red) and the surface of the chromatin (green). MT minus ends are marked by spheres. (C and F) Models showing the distribution of closed (green) and open (purple) MT minus ends. Bar, 200 nm. Also, see Videos 1–4, available at http://www.jcb.org/cgi/content/full/jcb.200304035/DC1.

Figure 2.

Closed MT minus ends are pointed, open ends are blunt or slightly flared. (A) Capped minus ends. The ends of the MTs are marked by arrowheads. Capped MTs are often pointed and flattened on one side. (B) Open minus ends. Some MT ends show an open, blunt morphology, whereas a few are flared (arrows). Single or double sheet-like extensions can be observed. Bar, 50 nm.

Figure 3.

KMTs do not end directly on the condensed chromatin. (A) Selected 2.7-nm thick tomographic slice showing a region at the metaphase plate. MTs (arrows) and the kinetochore regions (k) are indicated. The surface of the DNA (d) is outlined in green. (B) Corresponding 3-D model showing MTs (red) and the surface of the chromatin (green). MT plus ends are marked by blue spheres. Bar, 500 nm. Also, see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200304035/DC1.

Figure 4.

KMT plus ends have an open, flared, or blunt morphology. (A) Detail of a kinetochore region as obtained by high-voltage electron tomography. KMTs terminate in the ribosome-free zone (outlined in light blue). The chromatin surface is indicated by a green line. (B) 3-D model of the kinetochore region showing MTs (red), the position of their plus ends (yellow spheres), the outer boundary of the ribosome-free zone (blue), and the surface of the DNA (green). The mean width of the ribosome-free zone is 194 nm (±45 nm), and the mean distance of the MT plus end to the chromatin is 115 nm (±52 nm). Also, see Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200304035/DC1. (C) KMT plus ends with an open, flared morphology. Electron tomography revealed double or single extensions (arrows) at the plus ends. (D) Plus ends with an open, blunt morphology. The ends of the open tubes are marked by arrows. Bars: (A) 100 nm; (C) 50 nm.

Figure 5.

Half of the KMT minus ends are open. (A) Partial reconstruction of a metaphase spindle. The surface of the DNA is outlined in green, and MTs that end in the ribosome-free zone are identified in white. Other spindle MTs are shown in red. The centriole pair is shown as blue cylinders. The mean distance from the mother centriole to the open MT minus end (558 ± 139 nm) was not significantly different from the mean distance of closed MT minus ends (530 ± 137) from the mother centriole. (B) KMTs with closed (green) and open (magenta) minus ends. KMTs with minus ends “outside” the volume of the reconstruction are shown in yellow. Bar, 500 nm. Also, see Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200304035/DC1.