Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles

Abstract

The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through 'flux', where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed.

Keywords: anaphase A; biased diffusion; chromosome-to-pole motion; conformational wave; kinetochore; microtubule poleward flux.

Figure 1

Schematic diagram of a spindle in metaphase (a) and anaphase (b). Only the chromosome-to-pole, “anaphase A” motion is depicted here; it is the focus of this chapter. Separation of the two spindle poles from one another via elongation of the spindle, “anaphase B”, is discussed in the subsequent chapter by Scholey et al. [3].

Figure 2

Light micrographs of metaphase (a) and late anaphase (b) plant endosperm (Haemanthus) spindles. During metaphase in these plant cells the chromosome arms are bent in the direction of the spindle poles. This behavior differs from what is seen in animal somatic cells, where chromosome arms are pushed continually away from spindle poles [11]. These Haemanthus images are reprinted from [9], and are displayed under the terms of a Creative Commons License (Attribution-Noncommerical-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). Scale bar, 10 μm.

Figure 3

Example of kinetochore directional instability during anaphase A in a newt lung cell. Anaphase A chromosome-to-pole movement of the kinetochore is interrupted by transient reversals in directionality. This graph is reprinted from [12], and is displayed under the terms of a Creative Commons License (Attribution-Noncommerical-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Figure 4

Chromosome-to-pole motion during anaphase A is coupled to microtubule disassembly. (a) Simple mechanism with disassembly occurring only at microtubule plus ends, as seen in yeasts, where minus end attachments to the poles are static and no flux occurs [26,27,28,29]. (b) Dual mechanism, as in cultured mitotic human cells, where chromosome-to-pole motion is a superposition of a kinetochore’s movement relative to the microtubules, which is coupled to plus end disassembly, and the microtubules’ flux relative to the poles, which is coupled to minus end disassembly [30]. (c) Mechanism observed for autosomal half-bivalents in meiotic crane-fly spermatocytes, with disassembly at minus ends and assembly at plus ends [10,31]. Switching between mechanism (b) and mechanism (c) has been directly observed in Xenopus egg extract spindles [32].

Figure 5

Kinetochores can adopt two distinct states, an active state that generates pole-directed pulling force, and a ‘neutral’ state that remains stationary or passively slips anti-poleward in response to external forces. (a) Motions of sister kinetochore regions in a metaphase PtK1 cell before, during (horizontal bar) and after micro-surgically separating the sisters. (b) Motion of a trailing kinetochore before, during (horizontal bar), and after selectively destroying its poleward moving sister kinetochore. In both cases the trailing kinetochore abruptly stops once it is micro-surgically freed from its sister. Then, after a ~20 s delay, it reverses its original directionality and begins to move poleward. These graphs are reprinted from [57], and are displayed under the terms of a Creative Commons License (Attribution-Noncommerical-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Figure 6

Model for kinetochore-microtubule tip-coupling based on conventional motor proteins and microtubule-regulators. Conventional ATP-powered, minus end-directed motor enzymes anchored at the kinetochore could reach around the tip of the microtubule, moving along the sides of the filament and thereby dragging the chromosome poleward (leftward in the diagram). The activities of additional microtubule depolymerases or severing enzymes, somehow coordinated with the conventional motor activity, could explain how poleward chromosome movement is coupled to plus end-disassembly.

Figure 7

Laser trap assay for studying tip-coupling by purified kinetochore subcomplexes and native kinetochore particles. (a) Time-lapse images showing a bead decorated sparsely with native yeast kinetochore particles tracking with microtubule growth (0–700 s) and shortening (700–800 s). The laser trap (yellow crosshair) is moved automatically to keep a constant level of tension (here, ~1 pN) on the kinetochore as it moves with the microtubule tip. Scale bar, 4 μm. (b) Cartoon showing force clamp operation. The laser trap is servo-controlled to keep a fixed offset, Δx, between the trap and the bead, thereby maintaining a constant tensile force. (c) Upper plot: Record of position versus time for a native kinetochore isolated from yeast cells depleted of the TOG-family protein, Stu2. Arrows indicate switching of the microtubule tip from growth to shortening (↓, ‘catastrophes’) and from shortening back to growth (↑, ‘rescues’). Lower plot: Mean attachment lifetime as a function of force for wild-type (WT, black) and Stu2-depleted (stu2-AID, red) kinetochore particles. Plots in (c) are adapted from [135], and are displayed with permission from Elsevier Publishing (http://www.sciencedirect.com/science/journal/00928674).

Figure 8

Models for tip-coupling without conventional motor activity. (a) Two versions of the conformational wave mechanism are shown, one (ring-based) in which elements of the kinetochore assemble into a microtubule encircling ring that is hooked by curling protofilaments, and another (fibril-based) where fibrillar kinetochore elements bind independently to the curling protofilaments. In either case, the curling action of the protofilaments exerts pulling force (directed leftward in the diagrams) on the chromosome. (b) In the biased diffusion mechanism, an array of kinetochore fibrils rapidly binds and unbinds the microtubule lattice at or near the tip. Thermal fluctuations of the chromosome that allow more fibrils to bind (leftward movements of the chromosome in the diagram) are favored by the energy of binding those elements. This biased thermal movement produces a thermodynamic pulling force. (c) A hybrid model is also shown, where force is produced by a combination of protofilament curling and biased thermal fluctuations. These diagrams are adapted from [159], and are displayed with permission from Elsevier Publishing (http://www.sciencedirect.com/science/journal/09628924).

Figure 9

Change in distance from chromatids to poles before and after ablation of their kinetochore-associated microtubule fibers (k-fibers) during anaphase. Chromatids attached to ablated k-fibers (blue traces) are pulled toward poles faster than anaphase movement of their unmanipulated sisters (green traces) before resuming normal anaphase movement (at ~70 s). This graph is reprinted from [60], and is displayed under the terms of a Creative Commons License (Attribution-Noncommerical-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Figure 10

Spindle self-repair mechanism observed after micro-surgical ablation of kinetochore-associated microtubule fibers (k-fibers) in mammalian cells expressing fluorescent tubulin [60,61]. (a) Ablation of a k-fiber (yellow lightning bolt) during anaphase. (b) NuMA (cyan) and dynein/dynactin (green) rapidly localize to new microtubule minus ends on the k-fiber stub after ablation. (c) When the new minus end-localized dynein contacts neighboring microtubules, it walks processively along them, pulling the k-fiber stub as cargo and moving the attached chromosome. These diagrams are redrawn based on similar cartoons from [60], and are included here under the terms of a Creative Commons License (Attribution-Noncommerical-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).