We discriminated cells in early anaphase kinetochores located close to the poles, but no noticeable spindle elongation from cells in late anaphase prominent spindle elongation , and found that all chromosome bridges exhibited kinetochore-microtubule attachments during early anaphase, and nearly all bridges in late anaphase cells had both kinetochores from the same bridge attached to microtubules Fig 4G. Of the very few chromosome bridges we found with no obvious k-fibers at one end, most exhibited a stretched kinetochore appearance with kinetochores positioned at opposite ends, suggesting that microtubule attachment had been present, but either the k-fiber had disassembled immediately before or during fixation, or it was not thick enough for identification based on our fluorescence quantification criteria see materials and methods for details , but still present.
In a few cases 2 out of 86 in vHMECs of bridges with k-fibers only on one side, the unattached kinetochore did not display obvious stretch and was clearly shifted to the opposite pole, suggesting the possibility that the unattached half of the bridged chromosome was simply being dragged towards the opposite pole by the attached half of the bridge as a result of either kinetochore attachment failure or premature k-fiber detachment. These results suggest that only in rare cases, during the segregation of a chromosome bridge, the forces exerted by the intervening chromatin may cause k-fiber detachment.
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. G Frequencies of chromosome bridges with two both , one, or no none k-fibers. A mechanism that would be alternative to k-fiber detachment and that could explain the shifted position of the bridge kinetochores close to the spindle equator is the lengthening of the bridge k-fibers during anaphase.
To test this hypothesis, we used vHMECs because in these cells the total chromosome number is not as large as in HeLa cells important for feasibility of EB1 quantification experiments described below and chromosome size is not as large as in PtK1 cells which may result in a more pronounced bridge-dependent generation of tension at the kinetochore-microtubule interface of vHMECs.
First, we measured the average k-fiber length in metaphase vHMECs, and compared it to the average bridge k-fiber length in anaphase. We found that, although k-fibers from chromosome bridges on average were not longer than metaphase k-fibers, they did not shorten as much as non-bridge k-fibers Fig 5A as cells progressed from metaphase to anaphase.
This suggested that bridge k-fibers either shortened for a limited period of time and then stalled or lengthened slightly, or that they were alternating between periods of shortening and periods of lengthening during anaphase.
To gain further insight, we quantified EB1 a marker of microtubule plus-end polymerization fluorescence Fig 5B at bridge vs. This result indicates that the microtubules bound to bridge kinetochores are more likely than non-bridge kinetochores to be in a polymerization state during anaphase.
The middle and bottom images display overlays of single focal planes corresponding to the focal plane including the bridge KTs. 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.
C Average EB1 background-corrected fluorescence intensity F. D Average EB1 F. The reported n values represent the total number of KTs analyzed from 2 independent experiments. We reasoned that the appearance of bridge kinetochores as stretched or unstretched may depend on the efficiency at which the associated k-fiber polymerized during spindle elongation.
Indeed, unstretched bridge kinetochores were found to exhibit on average higher EB1 fluorescence intensity compared to stretched bridge kinetochores Fig 5D. These data suggest that the stretched appearance of certain bridge kinetochores can be explained by insufficient polymerization of the associated k-fiber during anaphase in response to the tension generated by the bridge.
It could also be possible that the bridge k-fibers alternate between periods of shortening at which times the DNA may become stretched and periods of lengthening at which time no further DNA stretching would occur.
Our data showed that most chromosome bridges that can be visualized in mid-late anaphase persist throughout mitosis and into early G1. Because the DNA is highly condensed in mitosis, it is not surprising that some anaphase chromosome bridges may get stretched without breaking. Indeed, micromanipulation studies showed that isolated newt chromosomes can be extended up to about 80 times their original length without breaking [ 36 ] and breakage occurs when chromosomes are stretched about fold their original length with an applied force of the order of nN [ 37 ].
Similar findings were reported for human chromosomes [ 38 ]. The maximum force that the spindle can exert on individual anaphase chromosomes has been estimated in grasshopper spermatocytes to be approximately pN [ 39 , 40 ], which is substantially lower than the force necessary to stretch a chromosome to the point of breakage [ 37 ].
Thus, whereas spindle elongation may account for some of the stretching observed for anaphase chromosome bridges, it does not produce the type of forces and the amount of stretching necessary to break the chromatin bridge.
Moreover, tension generated at bridge kinetochores may result in kinetochore microtubule polymerization, thus further attenuating the stretching caused by anaphase spindle elongation. What then induces some chromosome bridges to break? The most likely scenario is that chromosome and spindle mechanics act in concert to cause bridge breakage. For example, live-cell studies showed that acentric chromosome fragments in insect spermatocytes are transported poleward during anaphase most likely due to microtubule poleward flux [ 42 , 43 ].
Similarly, in plant endosperm cells acentric chromosome fragments are pulled poleward at the time of phragmoplast formation [ 44 — 46 ] via a kinetochore-independent mechanism [ 45 ]. It is possible that microtubule poleward flux or other microtubule-dependent forces may similarly act on chromosome bridges, thus causing them to stretch poleward and eventually break.
This phenomenon could explain the prevalence of bridge breakage in plant cells [ 26 ] as opposed to mammalian tissue culture cells this study.
Indeed, poleward movement of acentric fragments in vertebrate somatic cells has not been reported, and acentric chromosome fragments are instead believed to lag behind at the spindle equator during anaphase [ 47 , 48 ]. What other forces could account for bridge breakage in vertebrate somatic cells? A recent study reported that maximum chromosome compaction in mammalian tissue culture cells is achieved in late anaphase [ 49 ].
Such chromosome condensation may cause some regions of the chromosome bridge to become stretched and break as other regions attempt to undergo anaphase compaction. Alternatively, some regions of the chromosome may be more easily stretched and broken due to intrinsic structural features see for example [ 52 — 54 ].
Our observations also suggest that if any bridge breakage occurs due to mechanical stress in mammalian cells, this must happen mostly in anaphase, given that when chromosome bridges persist long enough they are most likely to result in cleavage furrow regression or delayed abscission [ 31 ].
Several studies have previously identified some degree of aneuploidy under conditions that would be expected to induce chromosome structural aberrations, and hence chromosome bridges as a main defect. For example, chemical inhibition of topoisomerase II to a level that induces high frequencies of anaphase chromosome bridges, also results in both chromosome breakage and aneuploidy [ 7 ].
Moreover, certain cancer cell types exhibit high frequencies of both anaphase chromosome bridges and aneuploidy [ 28 , 57 ]. Finally, in vHMECs the chromosomes with the shortest telomeres are frequently found in aneuploid numbers within the cell population [ 27 ].
It was previously hypothesized that this could be a consequence of bridge kinetochore detachment from spindle microtubules [ 27 , 28 , 30 ]. However, this hypothesis was never tested before. We now show that bridge kinetochores very rarely, if ever, lose their attachment to spindle microtubules Fig 4 , and the ends of the chromosome bridge only become free to move upon spindle disassembly Fig 2. Instead, we present evidence Fig 5 for a mechanism in which the k-fibers bound to bridge kinetochores do not significantly shorten, and possibly elongate, during anaphase.
If the two k-fibers change length differentially e. Whether the bridged chromosome ends up in the main nucleus Fig 6B or in a micronucleus Fig 6C may simply depend on the extent of the length differential between the two k-fibers bound to the bridge kinetochores. In a few cells see Fig 3 and example in Fig 6D and 6E we found evidence of differential k-fiber lengthening. However, it is possible that such segregation of bridged chromosomes to the same daughter cell may be hard to visualize if it occurs early in anaphase, when the arms of many chromosomes still span the spindle midzone, thus making the identification of this type of segregation very challenging.
Moreover, segregation of a chromosome bridge into a micronucleus may result in both aneuploidy there may be a chromosome loss in the cell without the micronucleus and accumulation of DNA damage within the micronucleus itself [ 58 , 59 ].
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. 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. Indeed, it appears that in animal somatic cells a chromosome bridge may lead to a number of different outcomes, including chromosome breakage, polyploidy by cleavage furrow regression , aneuploidy, and possibly cell cycle arrest by abscission checkpoint activation.
Given the myriad of possible outcomes anaphase chromosome bridges can produce, future studies should be aimed at elucidating what determines a bridge to break, mis-segregate, or inhibit cytokinesis.
One could argue that the multiple fates of chromosome bridges may explain the complex karyotypes of cancer cells, in which high rates of both aneuploidy and chromosome rearrangements are observed. However, elevated frequencies of chromosome bridges have only been reported for certain specific cancer types [ 60 — 63 ], whereas in most other cancer cells anaphase lagging chromosomes appear to be a far more common chromosome segregation defect [ 64 — 68 ].
Thus, it is possible that chromosome bridges contribute to tumor initiation, when chromosome rearrangements and aneuploidy may initially arise, whereas once telomeres become stabilized by re-activation of telomere maintenance mechanisms, the major contributors to chromosomal instability may be spindle multipolarity and anaphase lagging chromosomes [ 69 ]. For experiments, cells were plated on sterilized acid-washed coverslips inside sterile 35 mm Petri dishes.
After washing out the drug, cells were re-incubated in fresh media for 24 hours before fixation. The subsequent steps were the same for all cell types. The confocal head harbored a filter set for illumination at nm, nm and nm wavelengths through a mW argon laser and a mW krypton laser.
Images were acquired using a x 1. During prometaphase , chromosomes move back and forth. Kinesins anchor the chromosomes to the kinetochore microtubules beyond the tip where Kinesin is depolymerizing the microtubules, aided by a shortage of available tubulin dimers. A combination of motor proteins, microtubule interacting proteins and treadmilling serves to move the chromosomes.
Meanwhile, dynein and dynactin — motor proteins which walk towards the - end — work on the astral microtubules, pulling the MTOCs toward the cell periphery. During this process the nuclear envelope dissolves and so nuclear import becomes irrelevant. Cells have some mechanism for detecting the tension in the microtubules that indicates their attachment chromatids before mitosis can proceed. Making sure that every chromatid is properly anchored is crucial for avoiding aneuploidy.
By the way, other cytoskeletal elements besides microtubules also play a key role in the cell cycle. In cytokinesis , actin forms a contractile ring and, with the help of myosin II motor proteins, cinches the cell into two. The discovery of cell cycle regulatory processes relied heavily on some neat features of popular model organisms. Saccharomyces cerevisiae budding yeast and Schizosaccharomyces pombe fission yeast can exist as haploids or diploids.
This makes it possible to study the knockout phenotype at the non-permissive temperature while still having the convenience of being able to easily propagate the organisms at the permissive temperature. The entire S. That is how many of the genes that regulate the cell cycle were discovered. Temperature-sensitive mutants at the nonpermissive temperature get stuck unable to bud and enter the S phase.
We are now in a position to speculate with some authority about the specific molecular pathways that underlie spindle assembly, chromosome motility, and segregation. Here, we focus on our emerging understanding of the molecular basis of perhaps the most dramatic of all mitotic events, the disjunction and poleward movement of sister chromatids during anaphase A. Anaphase must be understood within the broader context of the entire process of mitosis.
The earliest phase of mitosis, known as prophase, is marked by the condensation of duplicated chromosomes which occurs prior to nuclear envelope breakdown NEB. NEB stimulates the onset of prometaphase, when chromosomes are captured by spindle microtubules, many of which are nucleated from duplicated centrosomes, and maneuvered to the spindle equator or metaphase plate.
Metaphase begins once all chromosomes have achieved an equatorial position. During this time, chromosomes often continue to oscillate back and forth across the metaphase plate Skibbens et al. The metaphase-to-anaphase transition is then signaled by a dramatic shift in cell chemistry resulting in the disjunction and poleward separation of sister chromatids. This occurs in two subphases: anaphase A chromatid-to-pole motion and anaphase B spindle elongation. The separated chromosomal masses finally decondense into daughter nuclei during telophase.
How does the spindle move chromosomes? The primary functional interface between chromosomes and spindle microtubules is the kinetochore, a multiprotein complex that assembles onto the centromere of each sister chromatid. Early in prometaphase, kinetochores can attach to spindle microtubules laterally leading to their poleward transport by the minus-end directed motor cytoplasmic dynein Pfarr et al. However, as mitosis progresses, kinetochores capture and make stable associations with numerous microtubule plus-ends Cheeseman and Desai , and it is in this configuration that the majority of chromosome movements, including anaphase, occur.
Over the years, two main models have emerged to describe the role of kinetochores and their associated microtubules in the poleward translocation of chromosomes during anaphase. The first posited that kinetochore microtubules serve as traction fibers which pull kinetochores and chromatids poleward Schrader ; Pickett-Heaps et al. Linkage of kinetochores to fluxing tubulins would provide a means of reeling chromatids into the poles. While there has been some controversy as to whether one or the other mechanism is the most important factor in anaphase A, it is now known that both play a role and often occur simultaneously but to various extents depending on cell type.
A model for Pacman-Flux mechanism-based chromosome segregation during anaphase. C Microtubule plus-ends embedded in the kinetochore are uncapped by microtubule-severing enzyme, Katanin. The uncapped microtubule end is linked to the kinetochore via multiple interactions of Ndc Dynein attached to the coronal fibers and the microtubule also keeps the plus-ends tethered to the kinetochore.
Dynein attached to the coronal fibers walks toward the minus ends of the kinetochore microtubules, thereby pushing the microtubule into the depolymerizing machinery at the kinetochores. The first published description of Fluxing spindle fibers microtubules can be attributed to Arthur Forer in the s Forer Using a UV microbeam to irradiate the spindle fibers of crane fly spermatocytes, Forer noticed that the resulting region of reduced birefringence moved toward the poles and then disappeared.
Forer also noted that the rate of this Flux approximated that of anaphase A. This idea was developed further by Margolis and Wilson who posited that spindle microtubules function as molecular treadmills based on observations of microtubule treadmilling in vitro Margolis and Wilson In particular, Margolis and Wilson speculated that tightly controlled modifications of such behaviors within the spindle could be used to control the position of chromosomes.
During metaphase, plus-end polymerization at kinetochores balanced by minus-end depolymerization at the poles would create a steady state in which kinetochore microtubules maintain a constant length, albeit with the tubulin subunits within them flowing poleward, allowing chromosomes to persist at the spindle equator Margolis and Wilson ; Margolis and Wilson The cessation of plus-end polymerization at the onset of anaphase would cause kinetochore-associated microtubules to shorten, through unbalanced minus-end depolymerization, thus reeling chromatids into the pole.
Direct observation of the Flux of tubulin subunits within spindle microtubules had to wait until the mids. This was first demonstrated by Mitchison and colleagues who injected biotin-labeled tubulins into live monkey fibroblast BSC1 and followed their incorporation into kinetochore microtubules using electron microscopy.
New tubulin subunits were observed to incorporate primarily at the plus ends and then spread poleward over time Mitchison et al. Similar results were obtained when the spindles of LL-CPK1 and PtK2 cells were labeled with photoactivatable caged fluorophore-tagged tubulins.
Live observation of photoactivated bands within the half spindles of these cells revealed the continual poleward flow of the tagged tubulin subunits Mitchison et al. And as additional components continue to be found, it appears that Flux within anaphase spindles generally involves at least three distinct enzymatic activities: 1 a pole-associated minus-end depolymerase, 2 centrosome-associated microtubule-severing enzymes, and 3 microtubule-sliding motors within the central spindle Fig.
Probably the best known and characterized of the protein factors involved in the stimulation of Flux are the kinesins. Members of this kinesin subfamily are immotile but instead bind to microtubule ends and catalyze their depolymerization, in vitro Desai et al. Work in a variety of systems, ranging from fruitflies to humans, has indicated that kinesins, positioned on spindle poles, promote Flux by depolymerizing pole-focused minus-ends.
Although Drosophila contains two additional kinesin family members both of which are discussed below , KLP10A is the only one to associate with spindle poles note that the spindle pole is a clearly distinct entity from the centrosome in Drosophila cells; Rogers et al.
Specifically, injection of embryos with anti-KLP10A antibodies was found to induce the near complete cessation of poleward Flux in both metaphase and anaphase spindles and a commensurate decrease in the rate of anaphase A Rogers et al. Inhibition of pole-associated kinesins in vertebrate spindles has been found to reduce Flux rate and anaphase A indicating that this portion of the Flux machinery is highly conserved Ganem et al. The mechanisms used to target kinesins to spindle poles have also received attention.
Studies in vertebrates and fruitflies have suggested that these proteins are transported poleward by cytoplasmic dynein. Inhibition of dynein in Xenopus egg extract spindles or Drosophila tissue culture cells causes kinesins that are normally associated with poles to become spread across the spindle Gaetz and Kapoor ; Morales-Mulia and Scholey Interestingly, KLP59D appears to work by shuttling KLP10A through centrosomes KLP59D localizes to centrosomes but not poles and we have proposed that this generates a localized region of active depolymerase activity in the vicinity of the pole Rath et al.
Within the cytoplasm, the majority of KLP10A proteins are known to be maintained in a low activity state, through the phosphorylation of their motor domains Mennella et al. Passage through the centrosome, which houses numerous phosphatases as well as kinases , could provide a means of locally activating a subpopulation of the protein. The vertebrate protein NuMA has also been proposed to form such a pole matrix Dionne et al.
Of course, this awaits further investigation. Kinesins are not the only proteins involved in promoting the depolymerization of microtubule minus-ends at the poles. Using time-lapse photography, it is possible to watch the chromosome halves move in opposite directions at speeds which can reach 0. This movement is directed and under the control of the spindle fibers and the microtubules. Although it is by no means certain, the most likely mechanism to account for this movement is the continuous polymerization and depolymerization of the microtubules themselves.
If this concept is correct, the spindle microtubules attached to the kinetochores of the sister chromatids, shorten by depolymerization removal of protein subunits at their polar ends. This would shorten the microtubule and "pull" on it, tugging the chromosome half towards that pole.
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