Chromosome fragments possessing only one kinetochore can congress to the spindle equator

Abstract

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.

Figure 1

(A–C) Diagram of how two different size CF1Ks can be created from a bioriented chromosome. (D–I) Video micrographs of a prometaphase cell in which the laser was used to sever the region between two kinetochores on a congressed chromosome (E, black arrow, arrowhead) to produce two CF1Ks (F, black arrow, arrowhead) that moved towards their respective polar areas (G). In PtK₁, as in most animals, large metaphase chromosomes are usually folded at their primary constriction so that their arms lie on top of one another (A and D). The centromere region on these chromosomes is positioned on the surface of the spindle, and the axis between its associated and opposing sister kinetochores is parallel to the spindle long axis (A). When both kinetochore regions stretch poleward, the area between them can be cut with the laser without damage to either kinetochore (B and E). Then, as the kinetochore regions continue to move towards their respective poles, a small section that contains a single kinetochore can be loped from the bulk of the chromosome (C and F). The cell followed in D–G was fixed shortly after G and processed for the fluorescent localization of DNA (H) and kinetochores (I). A comparison of G, H, and I clearly reveals that both CF1Ks produced by this operation (H, white arrow, arrowhead) contain a single kinetochore (I, white arrow, arrowhead). Time in seconds is noted in the bottom right corner of A–G. Bars, 5 μm.

Figure 1

(A–C) Diagram of how two different size CF1Ks can be created from a bioriented chromosome. (D–I) Video micrographs of a prometaphase cell in which the laser was used to sever the region between two kinetochores on a congressed chromosome (E, black arrow, arrowhead) to produce two CF1Ks (F, black arrow, arrowhead) that moved towards their respective polar areas (G). In PtK₁, as in most animals, large metaphase chromosomes are usually folded at their primary constriction so that their arms lie on top of one another (A and D). The centromere region on these chromosomes is positioned on the surface of the spindle, and the axis between its associated and opposing sister kinetochores is parallel to the spindle long axis (A). When both kinetochore regions stretch poleward, the area between them can be cut with the laser without damage to either kinetochore (B and E). Then, as the kinetochore regions continue to move towards their respective poles, a small section that contains a single kinetochore can be loped from the bulk of the chromosome (C and F). The cell followed in D–G was fixed shortly after G and processed for the fluorescent localization of DNA (H) and kinetochores (I). A comparison of G, H, and I clearly reveals that both CF1Ks produced by this operation (H, white arrow, arrowhead) contain a single kinetochore (I, white arrow, arrowhead). Time in seconds is noted in the bottom right corner of A–G. Bars, 5 μm.

Figure 2

(A–I) A bioriented chromosome (A, black arrow) is cut between its sister kinetochores (B, black arrow) to produce two CF1Ks (C, black arrow, arrowhead) that then moved into their respective polar areas (D–E). The small CF1K (C–E, black arrow) initiated congression in F, and was fully congressed by the time of anaphase onset in H. This CF1K then segregated to one of the poles during anaphase. The larger CF1K (C–I, black arrowhead) remained monooriented until anaphase onset, at which time it disjoined into a kinetochore-containing chromatid that moved into the pole and two smaller acentric fragments (I, white arrowheads). The white arrow in A–D notes a nonirradiated monooriented chromosome, the congression behavior of which is plotted in Fig. 3 (curve 2). Time in seconds is at lower right corner of each frame. Bar, 10 μm.

Figure 3

Time-versus-distance plots depicting the behavior of the two CF1Ks noted by the black arrow and arrowhead in Fig. 2, as well as the naturally monooriented chromosome noted by the white arrow in this figure. Plot 1 (top, solid circles) represents changes in distance between the right-hand pole and the kinetochore region on the CF1K (Fig. 2, C–H, black arrow), while plot 2 (top, open squares) depicts changes in distance between the right-hand pole and the nonirradiated control chromosome (Fig. 2, A–D, white arrow). Note that both the CF1K and the control chromosome exhibited low amplitude oscillatory motions until they initiated congression (open arrows) and that each underwent a single oscillation at about the same point during the congression period. The bottom part of this figure depicts the behavior of the larger CF1K (Fig. 2, black arrowhead) relative to its (i.e., the left-hand) pole. This CF1K remained monooriented until anaphase onset. The black bar at about 100 s represents time of laser surgery (corresponding to Fig. 2 B).

Figure 4

(A–H) Highly magnified selected images of the small CF1K noted by the black arrow in Fig. 2 as it congresses. Note that once congression is initiated (between B and C), the kinetochore region (black arrow) leads in motion towards the spindle equator. Bar, 5 μm.

Figure 5

(A–P) Selected frames from a video series showing the formation of two CF1Ks and their reorientation. In this cell, the arms were first separated from the centromere region of a large chromosome (compare arrows in A and B). The resultant fragment was then split along its long axis (C) to create two CF1Ks similar in size (D–O, arrow, arrowhead). The CF1K noted by the arrowhead in C moved towards its pole (D and E), and then towards the spindle equator (F and G). This fragment then ultimately crossed the equator (H) and became permanently associated with the other spindle pole (I–O). After moving into its pole (C–H, arrow) the other CF1K also reoriented (H–J, arrow) and moved through the spindle equator (K–L, arrow) until it reached the other pole (M–O, arrow). Bar, 10 μm.

Figure 6

(A–H) Same conditions as in Fig. 2 except that after congression, the large CF1K produced by laser microsurgery in this cell (B–H, black arrow) remained stably bioriented on the spindle equator. In this example, the small CF1K (B–D, black arrowhead) moved out of the focal plane about 3.5 min after it was generated. However, the larger CF1K, after moving towards its associated pole (B–D, black arrow), moved back to the spindle equator (E and F, black arrow), where it remained until mid anaphase (G–H, black arrow), at which time the cell was fixed for a subsequent three-dimensional EM analysis. The white arrow in A–F notes a small oscillating bioriented chromosome positioned near the top surface of the spindle, while the white arrowhead notes a larger bioriented chromosome positioned opposite that of the congressed CF1K (see Fig. 7). Bar, 10 μm.

Figure 7

Time versus distance from the pole plots of the large CF1K shown in Fig. 6 (black arrow), as well as the two bioriented metaphase chromosomes noted in this figure (Fig. 6, white arrow and arrowhead). The top curve (1, solid circles) depicts the behavior of the kinetochore region on the large CF1K (Fig. 6, black arrow), which was tracked relative to the top pole. Once created (solid bar at time 0), this CF1K exhibited oscillatory motions that favored a net displacement towards its associated pole (0–220 s). It then initiated congression (near the 220-s timepoint), and during this process, it exhibited one oscillation before reaching the spindle equator (near the 550-s timepoint; compare with Fig. 3). Once positioned on the spindle equator, the amplitude of its oscillations became dampened (compare with plot 1). The middle curve (2, open squares) depicts the behavior of a large nonirradiated metaphase chromosome (Fig. 6, white arrowhead), while the bottom curve (3, open squares) follows the small bioriented nonirradiated metaphase chromosome (Fig. 6, white arrow). The kinetochore region on both of these chromosomes was tracked relative to the bottom pole. Note that the amplitudes of the oscillations exhibited by these two “control” chromosomes varies widely.

Figure 8

Three-dimensional structure of the kinetochore region on the congressed CF1K noted by the black arrow in Fig. 6 H. (A–C) Selected 3-nm-thick slices from the tomographic volume generated from a thick (0.25-μm) section through this region. Note that a number of microtubules impact and terminate on both sides of the kinetochore/chromatin complex. White arrows note structural differentiations that resemble pieces of the kinetochore outer plate, which is highly distorted. (D) Color-coded stereo volume generated from stacking all of the pertinent information found in sequential slices of the tomogram shown in A–C. Recognizable portions of the kinetochore outer plate are red and associated microtubules are blue. In this example, several Mts derived from the upper pole in Fig. 6 terminate in the upper part of the kinetochore plate, while those from the bottom pole terminate in the bottom half. Mts from both poles also appear to be connected to another region of the plate that is stretched between the poles. Bar, 0.25 μm.

Figure 8

Three-dimensional structure of the kinetochore region on the congressed CF1K noted by the black arrow in Fig. 6 H. (A–C) Selected 3-nm-thick slices from the tomographic volume generated from a thick (0.25-μm) section through this region. Note that a number of microtubules impact and terminate on both sides of the kinetochore/chromatin complex. White arrows note structural differentiations that resemble pieces of the kinetochore outer plate, which is highly distorted. (D) Color-coded stereo volume generated from stacking all of the pertinent information found in sequential slices of the tomogram shown in A–C. Recognizable portions of the kinetochore outer plate are red and associated microtubules are blue. In this example, several Mts derived from the upper pole in Fig. 6 terminate in the upper part of the kinetochore plate, while those from the bottom pole terminate in the bottom half. Mts from both poles also appear to be connected to another region of the plate that is stretched between the poles. Bar, 0.25 μm.