Fig 1.
Many chromosome bridges do not break during anaphase.
(A, C, E) Examples of chromosome bridges in vHMEC (A), PtK1 (C), and HeLa (E) cells at different mitotic stages. DAPI staining is shown for all cells. Scale bars, 5 μm. (B, D, F) Frequencies (mean ± s.e.) of bridges in vHMEC (B), PtK1 (D), and HeLa (F) cells. The reported n values represent the total number of cells analyzed from 2 (vHMECs) or 3 (PtK1 and HeLa) independent experiments.
Fig 2.
Chromosome bridges persist beyond completion of mitosis.
(A) Example of early G1 vHMEC with a chromosome bridge whose ends are completely detached from the bulk of the chromatin in the daughter nuclei. Kinetochores (KTs, immunostained using CREST antibodies) are shown in green, microtubules (MTs) in red, and DNA in blue. Two KTs are visible at one end of the bridge and one KT at the other end (arrows). Scale bar, 10 μm. (B) Diagram illustrating how the bridges were classified. (C-E) Frequencies (mean ± s.e.) of bridges with KTs visible at both ends (Both), at one end (One), or not visible (None) because embedded in the bulk of the chromatin. The reported n values represent the total number of chromosome bridges analyzed from 2 independent experiments.
Fig 3.
Bridge kinetochores are shifted close to the spindle equator compared to non-bridge kinetochores.
(A) Example of anaphase vHMEC with chromosome bridge with staining for kinetochores (KTs, green, immunostained using CREST antibodies), microtubules (MTs, red), and DNA (blue). The symbols represent the location at which various elements of the mitotic apparatus were mapped. Blue triangle = position of spindle pole; green square = average position of 5 non-bridge KTs; red diamond = position of bridge KT. Scale bar, 10 μm. (B-D) Graphs displaying the relative distances of the various mapped elements from the spindle equator in vHMEC (B), PtK1 (C), and HeLa (D) cells. The position of the spindle equator was determined as the middle point between the two spindle poles. Each line in the graph represents an individual cell. For each cell, the position of the non-bridge KTs is reported as an average of five randomly selected KTs, whereas the bridge KT positions are reported individually. Thus, if a cell had multiple chromatin bridges, multiple pairs of red diamonds appear on the corresponding line. The reported n values represent the total number of cells analyzed from 2 independent experiments.
Fig 4.
Nearly all bridge kinetochores are bound to k-fibers during anaphase.
(A-C) Example of anaphase vHMEC stained for DNA (grey/blue), kinetochores (KTs, green, immunostained using CREST antibodies), and microtubules (MTs, red) and possessing a chromosome bridge. The overlay of KTs and MTs is shown in B and the overlay of all three colors is shown in C. The KT and MT images in (B) and (C) are maximum intensity projections of Z-stacks. The insets in B show enlarged (300%) views of the boxed regions. Scale bar, 10 μm. (D-E) Single focal planes of the left (D) and right (E) portions of the mitotic spindle that provide better visualization of the bridge KTs and their associated k-fibers. Enlarged (300%) views of the boxed regions are displayed at the bottom. (F) Diagram illustrating how KT-MT attachment (presence/absence of k-fibers) was classified in anaphase cells. To ensure unbiased evaluation, the presence of a k-fiber was determined by background-corrected fluorescence intensity quantification of α-tubulin immunostaining (see materials and methods section for details on fluorescence intensity quantification). (G) Frequencies of chromosome bridges with two (both), one, or no (none) k-fibers. The data represent the average from 2 independent experiments in which a total of 86 (vHMEC), 92 (PtK1), or 22 (HeLa), chromosome bridges were analyzed.
Fig 5.
Bridge k-fibers do not display typical anaphase behavior.
(A) Average metaphase k-fiber length and anaphase k-fiber length for bridge and non-bridge kinetochores (KTs) in vHMECs. Note that whereas the non-bridge k-fibers shorten by nearly 50% in anaphase, the bridge k-fibers exhibit a very modest length change. (B) Example of chromosome bridge in an anaphase vHMEC immunostained for DNA (blue), kinetochores (KTs, red, immunostained using CREST antibodies), and EB1 (green). The top image shows an overlay of maximum intensity projections of Z-stacks of immunostained KTs and EB1 with a single focal plane image of DAPI-stained DNA. The middle and bottom images display overlays of single focal planes corresponding to the focal plane including the bridge KTs. The insets in the bottom image display enlarged (300%) views of the boxed regions and include the bridge KTs as well as a non-bridge KT. White arrowheads point at the EB1 signal associated with the bridge KTs, whereas the yellow arrowhead points at the MT face of the non-bridge KT and illustrates the low level of EB1 labeling. Scale bar, 10 μm. (C) Average EB1 background-corrected fluorescence intensity (F.I.) at bridge and non-bridge KTs. (D) Average EB1 F.I. at non-bridge KTs vs. stretched and unstretched bridge KTs. For both (C) and (D), the asterisk denotes statistically significant difference (t-test, p<0.01) when data were compared to data from non-bridge KTs. The reported n values represent the total number of KTs analyzed from 2 independent experiments.
Fig 6.
Differential shortening/lengthening of k-fibers during anaphase can lead to segregation of the bridged chromosomes to the same daughter cell.
(A-C) Diagram illustrating how differential shortening/lengthening of k-fibers during anaphase would lead to segregation of the bridged chromosomes to the same daughter cell. Depending on the extent of the length differential between the two k-fibers bound to the bridge kinetochores, the bridged chromosome may end up in the main nucleus (B) or in a micronucleus (C). (D-E) Example of vHMEC in which the bridged chromosomes are segregating to the same daughter cell. DNA is shown in blue, kinetochores (KTs, immunostained using CREST antibodies) in green, and microtubules (MTs) in red. Open arrowheads point to the two ends/KTs of the chromosome bridge. Note that the cleavage furrow (arrows) is ingressing on one side of the chromosome bridge, thus pushing the whole bridge into one of the daughter cells. Scale bar, 5 μm.