Augmin-dependent microtubule nucleation at microtubule walls in the spindle - PubMed (original) (raw)
Augmin-dependent microtubule nucleation at microtubule walls in the spindle
Tomoko Kamasaki et al. J Cell Biol. 2013.
Abstract
The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end-wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.
Figures
Figure 1.
MT organization and end distribution in the mitotic spindle of human U2OS cells. (A–D) Partial reconstructions of metaphase spindles in control and augmin RNAi cells. MTs are colored green. Chromosome-facing ends (all open) and pole-facing closed and open ends are marked with white, magenta, and yellow spheres, respectively. Surfaces of chromosomes are outlined in blue. The centriole pair and associated MTs are in gray. In three out of four augmin knockdown cells, abnormal centriolar MTs were observed (
Fig. S2, D and E
). The connection sites between mother and daughter MTs are red in D. Bar, 1 µm. (E) Distribution of putative minus ends near or away from the centriole. Combined data from three control or four augmin knockdown cells are displayed. Data on each cell are presented in
Tables S1
and
S2
.
Figure 2.
Ultrastructure of the augmin-dependent end-links that connect two adjacent MTs. Two types of connection between an MT closed (minus) end and the lattice of an adjacent MT. (A) Selected tomographic slices (1.4 nm/slice; three out of seven serial slices) of two end-lattice connections, at which rod-shaped end-links were detected (arrows). Note that the links are most clearly visible in the fourth plane, which includes the daughter MT end. Manual tracings of the mother MT wall (green; a part of the wall lying in this slice was marked) and the capped daughter MT ends (magenta) are displayed on the right. See
Videos 1
,
2
, and
3
for more serially sliced or rotated images. (B) Two examples of the direct end-link. See
Fig. S3
for more images. Bars, 30 nm.
Figure 3.
Quantitative description of the end-links. (A) Comparison of the probability of end-link appearance between control and augmin RNAi–treated cells in the centriole-proximal area or the body of the spindle. Combined data from three control or four augmin knockdown cells are displayed. Data on each cell are presented in
Tables S1
and S2. (B) Frequency of the end-link appearance on the closed ends near or away from the centriole. End-links were observed more frequently in the body of the spindle than around the centrioles. Combined data from three control cells are displayed in this graph. Data on each cell are presented in Table S1. (C) Length distributions of the rod-shaped end-links. (D–G) Angles between mother and daughter MTs (α; E), a rod-shaped end-link and a mother MT (β; F), and a rod-shaped end-link and a daughter MT (γ; G). Mother and daughter MTs were predominantly at small angles (E). The data presented in C and E–G are derived from tomographic volumes of four control cells. The sample numbers for analysis are presented for each cell in Tables 1 and S1.
Figure 4.
MT branching from the kMTs and non-kMTs. (A–C) Models showing examples of the “branching” sites on mother MTs. Mother, daughter, and granddaughter MTs are colored green, orange, and aqua, respectively. Closed ends, chromosome-facing open ends, and the sites of end-links are shown with magenta, white, and red spheres, respectively. The surface of the chromosomes is outlined in blue. The centriole pair is colored gray. Note that both kMTs and non-kMTs served as mother MTs (A), and in one case, a mother MT was connected by the end-links to two daughter MTs (B), and a daughter MT was further end-linked to a granddaughter MT (C). Arrows indicate the branching sites. Bars: (A and B) 1 µm; (C) 0.5 µm.
References
- Byers B., Shriver K., Goetsch L. 1978. The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae. J. Cell Sci. 30:331–352 - PubMed
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