Resource for the Visualization of Biological Complexity (RVBC)
Using Electron tomography to assess kinetochore modulation of microtubule dynamics
Chromosome alignment during mitosis and meiosis is essential for genomic stability and accurate gene transmission in eukaryotic organisms. Alignment is achieved through the interaction of a chromosome-associated organelle, known as the kinetochore, with the plus-ends of spindle microtubules (Rieder and Salmon, 1998, Maiato et al., 2004). This interaction is vital and errors in the process lead to several kinds of cancer, birth defects, and miscarriages. Microtubules are dynamic polymers that rapidly transition between distinct phases of growth and shrinkage (Howard and Hyman, 2003). This dynamic behavior is fueled by a GTP-cycle of binding and hydrolysis with both the growing and shrinking states being capable of performing work. Alignment of chromosomes involves a unique form of microtubule-based motility wherein the cargo is attached by the kinetochore to the plus-ends of a bundle of microtubules, rather than to the lateral surface of a single microtubule. Consequently, kinetochore movement towards and away from a spindle pole must be coordinated with kinetochore microtubule dynamics, and drugs that interfere with microtubule dynamics also block chromosome alignment.
A key unresolved question is how the kinetochore modulates microtubule dynamics. We are addressing the question by using electron tomography to determine the plus-end conformations of kinetochore microtubules (Vandenbeldt et al, 2004). In vitro assembly data has revealed that plus ends of growing microtubule take the form of extended sheets, blunt ends, and slightly flared tips, while plus ends of shrinking microtubules fray apart into curled protofilaments (Mandelkow et al., 1991; Chretien et al., 1995, Arnal et al, 2000). There is also a metastable transition state that is neither growing nor shrinking and is thought to adopt a blunt-end conformation (Tran et al., 1997, Janosi et al., 2002). Thus, by determining plus-end conformations we are be able to assess the dynamic status of kinetochore microtubules and thereby obtain critical insight into how microtubule dynamics is modulated by mammalian kinetochores.
Assessing plus-end conformations of kinetochore microtubules. Upper Left: Single 2.0 nm thick slice from the tomographic reconstruction or a high-pressure frozen/freeze-substitution kinetochore. Protein and chromatin are dark in this representation. The indicated plus-end conformations were determined by scanning through all relevant sections of sub-volumes that are extracted with optimal orientation for each microtubule. Upper Right: Examples of manually classified kinetochore microtubule conformations. At total of 405 kinetochore microtubule plus-ends from 103 tomographic reconstructions were manually classified by three researchers independently inspecting the same data. The significance of the difference between capped and open blunt-end conformations is not yet clear so the two groups were combined for subsequent analysis. Classification by correspondence analysis gave similar results (not shown). Lower panel: Summary of manual classifications. During metaphase individual kinetochores can be moving away from or towards their attached spindle pole, or standing still. Therefore, during metaphase kinetochore microtubules are expected to be in a mixture of growing, shrinking, and paused state. Correspondingly, the plus-end conformations are nearly equally distributed between blunt, curled, and forked classes with a slight preponderance of forked ends and slightly lower amount of blunt ends. Treatment with the microtubule stabilizing drug taxol increases the percentage of blunt ends and dramatically decreases the curled ends, while treatment with the microtubule destabilizing drug nocodazole decreases the number of blunt ends and increases the number of curled ends. During anaphase, when most kinetochores are moving poleward, the number of blunt ends is decrease while the number of curled ends is increased. These results are in agreement with the in vitro data indicating that blunt ends are associated with growing microtubules and curled ends with shrinking microtubules. The forked end class is not directly described by previous analyses and we believe that it is a combination of growing, shrinking and paused microtubules. We are currently re-examining this class for possible subclasses.
Application to chromosome motion: correlative DIC- video microscopy with electron tomography. (a), (b): DIC images from two time frames in the video record of a late prometaphase PtK1 cell. Black arrows indicate the positions of two rapidly oscillating chromosomes in each frame. (c): Same cell after chemical fixation. (d): Low magnification electron micrograph of a serial section containing the chromosomes indicated in (a)-(c). (e,), (f): Untitled image from the tomographic tilt series and a single slice from the 3D reconstruction of the kinetochore indicated in (d). (g): Tracking record for sister kinetochores. A plot of the distance between the kinetochore and its attached spindle pole as a function of time for the sister kinetochores located on the lower chromosome in panels a-c. The final time point is after chemical fixation. It appears that fixation might have occurred just at the point the chromosome was switching its direction of motion. (h): Profiles of plus end conformations on the tracked kinetochores. Kinetochore 1 shows a mixed profile resembling the average for metaphase kinetochores. Kinetochore 2 shows a strong preponderance of blunt ends indicating the growing state and motion away for the spindle pole. These results are consistent with the interpretation that kinetochore 2 has just switched its direction of motion while kinetochore 1 is in a transition stage.
Intricate interactions between kinetochores and microtubules are essential for the proper distribution of chromosomes during mitosis. A crucial long-standing question is how vertebrate kinetochores generate chromosome motion while maintaining attachments to the dynamic plus ends of the multiple kinetochore MTs (kMTs) in a kinetochore fibre. Here, we demonstrate that individual kMTs in PtK(1) cells are attached to the kinetochore outer plate by several fibres that either embed the microtubule plus-end tips in a radial mesh, or extend out from the outer plate to bind microtubule walls. The extended fibres also interact with the walls of nearby microtubules that are not part of the kinetochore fibre. These structural data, in combination with other recent reports, support a network model of kMT attachment wherein the fibrous network in the unbound outer plate, including the Hec1-Ndc80 complex, dissociates and rearranges to form kMT attachments. (See: Dong et al, 2007)
References
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Dong, Y, VandenBelt, KJ, Meng, X., Khodjakov, A., and McEwen, B.F. (2007) The outer plate in vertebrate kinetochores is a flexible network with multiple microtubule interactions. Nature Cell Biology 9 (5):516-522. (with cover). Pub Med