Elastic tethers connect telomeres of separating chromosomes in anaphase of animal cells. Immunofluorescence staining of titin in crane-fly spermatocytes, using 4 different antibodies, shows that the giant elastic protein titin seems to be a component of mitotic tethers: titin "strands" extend between separating chromosomes, connecting their telomeres, just as tethers do. Since titin is responsible for elastic forces in myofibrils, we suggest that titin is responsible for the backwards forces exerted on chromosome arms during anaphase.

During normal anaphase in animal cells, elastic tethers connect partner telomeres of segregating chromosomes and exert backward (anti-poleward) forces on those chromosomes. The experiments reported herein test whether microtubules need to be present in order for tethers to produce backwards forces. We disassembled spindle microtubules by treating anaphase crane-fly primary spermatocytes separately with nocodazole, colcemid, or podophyllotoxin. The drug treatments caused anaphase chromosomes to stop moving poleward; almost immediately thereafter they moved backward. The characteristics of the backward movements of the half-bivalents match those of the backwards movements of arm fragments formed by cutting chromosome arms during anaphase – for example the occurrence and lengths of backward movements were a function of tether length. The only difference from movement of arm fragments is that the chromosomes in the treated cells moved backwards slower than arm fragments did. Immunofluorescence of spindle tubulin after the drug treatments indicated that acetylated kinetochore microtubules were not depolymerized by the drugs, though the non-kinetochore spindle microtubules were depolymerized. Our data indicate that tethers move anaphase chromosomes backwards in the absence of functioning spindle microtubules. We suggest that the backward movements that take place when poleward forces are absent are due to tethers, and that the backward movements are slowed by the presence of acetylated kinetochore microtubules.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACT Mitotic tethers connect partner telomeres of all segregating anaphase chromosomes in all animal cells that have been tested, as detected by laser-cutting chromosome arms during anaphase and seeing that the arm fragments move rapidly across the equator to their partner chromosome moving to the opposite pole, telomere moving towards telomere. Tethers exert anti-poleward forces on the poleward separating telomeres, but tether elasticity (that produces the backwards forces) diminishes during anaphase: as determined by the behavior of arm fragments; short tethers (early anaphase) are elastic, long tethers (late anaphase) are not elastic, and medium-length tethers transition between the two states. We developed a procedure in which the tethers still functioned after we partially-lysed anaphase crane-fly spermatocytes. The partial lysis consistently arrested chromosome movements, after which the tethers moved the chromosomes backwards, potentially allowing the elastic tethers to be studied biochemically. To ensure that tether function was not altered by the partial cell-lysis procedure, we compared backward chromosome movements in partially-lysed cells with arm fragment movements in control cells. In the partially-lysed cells the backward chromosomal movements had characteristics identical to those of arm fragments in non-lysed (control) cells. In particular, in both control and partially-lysed cells shorter tethers caused backward movements more often than did longer tethers; shorter tethers caused backward movements over greater fractional distances (of the tether) than did longer tethers; and velocities of the backwards movements were the same for tethers of different lengths. We also compared the effects of Calyculin A (an inhibitor of Protein Phosphatase1) in control versus in partially-lysed cells. Calyculin A (CalA) added to control cells in early anaphase blocks dephosphorylation, thereby maintaining tether elasticity throughout anaphase: after the chromosomes reach the poles they move backwards when the usual poleward forces are reduced. Partial lysis preserves this tether functionality: after partial lysis of CalA-treated cells the chromosomes move backward and reach the partner telomeres at even very long tether lengths. We conclude that partial cell-lysis arrests anaphase chromosome poleward movement but does not affect tether function.

Jeremy Pickett-Heaps was a biologist whose acute observational powers were fed by a deep fascination for how cells work; he had an affinity for the myriad diversity of algae and other protists in general, and for what they could teach us about all cells. An early adopter of the electron microscope, he made fundamental discoveries in plant cell division and green algal phylogeny that developed into studies on cell division in general: first with the highly ordered diatoms and other protists, and later with animal cells. In the latter part of his career, Jeremy mastered time-lapse microcinematography to document the dynamic lives of cells and, through the resultant movies and his enthusiastic teaching, introduced many to the wonders of microscopic life.

Chromosome movement speeds during anaphase are regulated by depolymerization of microtubules. Several models describe chromosome movement during cell division but none of them consider post-translational modifications of tubulin, even though such modifications help specify microtubules for unique cellular activities. Among these modifications, acetylation of Lysine 40 is one of the common post-translational modifications. Acetylation of microtubules greatly improves their stability, especially when subjected to cooling or drug treatment. Since kinetochore microtubules are acetylated in a variety of eukaryote cells, we wondered whether deacetylation of kinetochore microtubules was necessary in order for microtubules to be able to depolymerize during anaphase. HDAC6 (Histone Deacetylase 6) deacetylates acetylated tubulin. To study whether tubulin must be deacetylated during anaphase, we added to living cells two different HDAC6 inhibitors (Tubacin and Trichostatin A), separately, as chromosomes moved poleward in anaphase. Both HDAC6 inhibitors altered chromosome movement: chromosomes either completely stopped moving, or moved more slowly, or sometimes continued movement without speed changes. The effects of the inhibitors on chromosome movement are reversible: half-bivalents either restarted anaphase movement by themselves before washing out the inhibitor or resumed their poleward movement after the inhibitor was washed out. We suggest that kinetochore microtubules need to be deacetylated in order for normal anaphase movements to occur.

Different types of anaphase bridges are reported to form between segregating chromosomes during cell division. Previous studies using laser microsurgery suggested that elastic tethers connect the telomeres of separating anaphase chromosomes in many animal meiotic and mitotic cells. However, structural evidence is lacking for their existence. In this study, by correlating live imaging with electron tomography, we examined whether visible structures connect separating telomeres in meiosis I of crane-fly primary spermatocytes. We found structures extending between separating telomeres in all stages of anaphase. The structures consist of two components: one is darkly stained, looking somewhat like chromatin, whereas the other is more lightly stained, appearing filamentous. Although in early anaphase both structures extend between telomeres, in later anaphase, the darker structure extends shorter distances from the telomeres but the lighter structure still extends between the separating telomeres. From these observations, we deduced that these structures represent the "tethers" inferred from the laser-cutting experiments. Because elastic tethers have been detected in a variety of animal cells, they probably are present during anaphase in all animal cells.

In normal anaphase cells, telomeres of each separating chromosome pair are connected to each other by tethers. Tethers are elastic at the start of anaphase: arm fragments cut from anaphase chromosomes in early anaphase move across the equator to the oppositely-moving chromosome, telomere moving toward telomere. Tethers become inelastic later in anaphase as the tethers become longer: arm fragments no longer move to their partners. When early anaphase cells are treated with Calyculin A (CalA), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), at the end of anaphase chromosomes move backward from the poles, with telomeres moving toward partner telomeres. Experiments described herein show that in cells treated with CalA, backwards movements are stopped in a variety of ways, by cutting the tethers of backwards moving chromosomes, by severing arms of backwards moving chromosomes, by severing arms before the chromosomes reach the poles, and by cutting the telomere toward which a chromosome is moving backwards. Measurements of arm-fragment velocities show that CalA prevents tethers from becoming inelastic as they lengthen. Since treatment with CalA causes tethers to remain elastic throughout anaphase and since inhibitors of PP2A do not cause the backwards movements, PP1 activity during anaphase causes the tethers to become inelastic.

Elastic “tethers” connect separating anaphase chromosomes in most (or all) animal cells. We tested whether tethers are involved in coordinating movements of separating anaphase chromosomes in crane-fly spermatocytes. In these cells the coupled movements of separating chromosomes become uncoupled after the tethers are severed by laser microbeam irradiation of the interzone region between the chromosomes (Sheykhani et al., 2017). While this strongly suggests that tethers are involved with coordinating the poleward chromosome movements, the experiments are open to another interpretation: laser irradiations that cut the tethers also might damage something else in the interzone, and those non-tether components might regulate chromosome movements. In the experiments reported herein we distinguish between those two possibilities by disabling the tethers without cutting the interzone. We cut the arms from individual chromosomes, thereby severing the mechanical connection between separating chromosomes, disconnecting them, without damaging components in the interzone. Disabling tethers in this way uncoupled the movements of the separating chromosomes. We thus conclude that tethers are involved in regulating the speeds of separating anaphase chromosomes in crane-fly spermatocytes.

We tested conclusions reached in previous experiments in which Mesostoma spermatocyte chromosomes moved rapidly to a pole in the absence of microtubules: after 10 μM nocodazole (NOC) depolymerized metaphase spindle microtubules, kinetochores from each of the 3 bivalents detached from the same pole and rapidly moved to the other pole, at speeds averaging 37.7 μm/min. with some as high as 100 μm/min. We concluded that these very fast movements were due to non-microtubule forces arising from a spindle matrix. However, since the chromosomes stretch out before detaching, there is tension in the chromosomes from the stretch. Thus the movements of detached kinetochores conceivably might be due to recoil from the tension, though we argued against this possibility (Fegaras and Forer, 2018a). In this article we test whether recoil causes the movements. We cut bivalents into 2 pieces, using a femtosecond laser, before addition of NOC. When 1 bivalent was severed, all kinetochores moved to one pole in 12/15 cells; when 2 bivalents were severed, all kinetochores moved to one pole in 4/6 cells; and when all 3 bivalents were severed all kinetochores moved to one pole in 3/9 cells. The bivalent “halves” moved rapidly, with average speeds of 47 μm/min, velocities that are not significantly different from those in cells without any laser-cut bivalents (p > 0.05). Since kinetochores move at the same speeds whether they are part of bivalents or not, NOC-induced chromosome movements are not due to recoil from tension along the full-length bivalent, strongly supporting the idea that non-microtubule forces move chromosomes in Mesostoma spermatocytes.

Elastic tethers, connecting telomeres of all separating anaphase chromosome pairs, lose elasticity when they lengthen during anaphase. Treatment with phosphatase inhibitor CalyculinA causes anaphase chromosomes to move backwards after they reach the poles, suggesting that dephosphorylation causes loss of tether elasticity. We added 50nM CalyculinA to living anaphase crane-fly spermatocytes with different length tethers. When tethers were short, almost all partner chromosomes moved backwards after nearing the poles. When tethers were longer, fewer chromosomes moved backwards. With yet longer tethers none moved backward. This is consistent with tether elasticity being lost by dephosphorylation. 50nM CalyculinA blocks both PP1 and PP2A. To distinguish between PP1 and PP2A we treated cells with short tethers with 50nM okadaic acid which blocks solely PP2A, or with 1µM okadaic acid which blocks both PP1 and PP2A. Only 1µM okadaic acid caused chromosomes to move backward. Thus, tether elasticity is lost because of dephosphorylation by PP1.

A “precocious” cleavage furrow develops and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes (Forer and Pickett-Heaps Eur J Cell Biol 89:607-618, 2010). In response to chromosome movements which regularly occur during prometaphase and that alter the balance of chromosomes in the two half-spindles, the precocious furrow shifts its position along the cell, moving 2–3 μm towards the half cell with fewer chromosomes (Ferraro-Gideon et al. Cell Biol Int 37:892-898, 2013). This process continues until proper segregation is achieved and the cell enters anaphase with the cleavage furrow again in the middle of the cell. At anaphase, the furrow recommences ingression. Spindle microtubules (MTs) are implicated in various furrow positioning models, and our experiments studied the responses of the precocious furrows to the absence of spindle MTs. We depolymerized spindle MTs during prometaphase using various concentrations of nocodazole (NOC) and colcemid. The expected result is that the furrow should regress and chromosomes remain in the midzone of the cell (Cassimeris et al. J Cell Sci 96:9-15, 1990). Instead, the furrows commenced ingression and all three bivalent chromosomes moved to one pole while the univalent chromosomes, that usually reside at the two poles, either remained at their poles or moved to the opposite pole along with the bivalents, as described elsewhere (Fegaras and Forer 2018). The microtubules were completely depolymerized by the drugs, as indicated by immunofluorescence staining of treated cells (Fegaras and Forer 2018), and in the absence of microtubules, the furrows often ingressed (in 33/61 cells) at a rate similar to normal anaphase ingression (~ 1 μm/min), while often simultaneously moving toward one pole. Thus, these results indicate that in the absence of anaphase and of spindle microtubules, cleavage furrows resume ingression.

In a typical cell division, chromosomes align at the metaphase plate before anaphase commences. This is not the case in Mesostoma spermatocytes. Throughout prometaphase, the three bivalents persistently oscillate towards and away from either pole, at average speeds of 5–6 μm/min, without ever aligning at a metaphase plate. In our experiments, nocodazole (NOC) was added to prometaphase spermatocytes to depolymerize the microtubules. Traditional theories state that microtubules are the producers of force in the spindle, either by tubulin depolymerizing at the kinetochore (PacMan) or at the pole (Flux). Accordingly, if microtubules are quickly depolymerized, the chromosomes should arrest at the metaphase plate and not move. However, in 57/59 cells, at least one chromosome moved to a pole after NOC treatment, and in 52 of these cells, all three bivalents moved to the same pole. Thus, the movements are not random to one pole or other. After treatment with NOC, chromosome movement followed a consistent pattern. Bivalents stretched out towards both poles, paused, detached at one pole, and then the detached kinetochores quickly moved towards the other pole, reaching initial speeds up to more than 200 μm/min, much greater than anything previously recorded in this cell. As the NOC concentration increased, the average speeds increased and the microtubules disappeared faster. As the kinetochores approached the pole, they slowed down and eventually stopped. Similar results were obtained with colcemid treatment. Confocal immunofluorescence microscopy confirms that microtubules are not associated with moving chromosomes. Thus, these rapid chromosome movements may be due to non-microtubule spindle components such as actin-myosin or the spindle matrix.

Recent work has demonstrated the existence of elastic connections, or tethers, between the telomeres of separating partner chromosomes in anaphase. These tethers oppose the poleward spindle forces in anaphase. Functional evidence for tethers has been found in a wide range of animal taxa, suggesting that they might be present in all dividing cells. An examination of the literature on cell division from the nineteenth century to the present reveals that connections between separating partner chromosomes in anaphase have been described in some of the earliest observations of cell division. Here, we review what is currently known about connections between separating partner chromosomes in anaphase, and we speculate on possible functions of tethers, and on what they are made of and how one might determine their composition.

Various experiments have indicated that anaphase chromosomes continue to move after their kinetochore microtubules are severed. The chromosomes move poleward at an accelerated rate after the microtubules are cut but they slow down 1-3 min later and move poleward at near the original speed. There are two published interpretations of chromosome movements with severed kinetochore microtubules. One interpretation is that dynein relocates to the severed microtubule ends and propels them poleward by pushing against non-kinetochore microtubules. The other interpretation is that components of a putative "spindle matrix" normally push kinetochore microtubules poleward and continue to do so after the microtubules are severed from the pole. In this study we distinguish between these interpretations by treating cells with taxol. Taxol eliminates microtubule dynamics, alters spindle microtubule arrangements, and inhibits dynein motor activity in vivo. If the dynein interpretation is correct, taxol should interfere with chromosome movements after kinetochore microtubules are severed because it alters the arrangements of spindle microtubules and because it blocks dynein activity. If the "spindle matrix" interpretation is correct, on the other hand, taxol should not interfere with the accelerated movements. Our results support the spindle matrix interpretation: anaphase chromosomes in taxol-treated crane-fly spermatocytes accelerated after their kinetochore microtubules were severed.

Originally described in crane-fly spermatocytes, tethers physically link and transmit force between the ends of separating chromosomes. Optical tweezers and laser scissors were used to sever the tether between chromosomes, create chromosome fragments attached to the tether which move toward the opposite pole, and to trap the tethered fragments. Laser microsurgery in the intracellular space between separating telomeres reduced chromosome strain in half of tested chromosome pairs. When the telomere-containing region was severed from the rest of the chromosome body, the resultant fragment either traveled towards the proper pole (poleward), towards the sister pole (cross-polar), or movement ceased. Fragment travel towards the sister pole varied in distance and always ceased following a cut between telomeres, indicating the tether is responsible for transferring a cross-polar force to the fragment. Optical trapping of cross-polar traveling fragments places an upper boundary on the tethering force of ~1.5 pN.

During the anaphase stage of mitosis, a motility force transports genetic material in the form of chromosomes to the poles of the cell. Chromosome deformations during anaphase transport have largely been attributed to viscous drag force, however LaFountain et. al. found that a physical tether connects separating chromosome ends in crane-fly spermatocytes such that a backwards tethering force elongates the separating chromosomes. In the presented study laser microsurgery was used to deduce the mechanistic basis of chromosome elongation in rat-kangaroo cells. In half of tested chromosome pairs, laser microsurgery between separating chromosome ends reduced elongation by 7 +/- 3% suggesting a source of chromosome strain independent of viscous drag. When microsurgery was used to sever chromosomes during transport, kinetochore attached fragments continued pole ward travel while half of end fragments traveled towards the opposite pole and the remaining fragments either did not move or segregated to the proper pole. Microsurgery directed between chromosome ends always ceased cross-polar fragment travel suggesting the laser severed a physical tether transferring force to the fragment. Optical trapping of fragments moving towards the opposite pole estimates an upper boundary on the tethering force of 1.5 pN.

We describe the general occurrence in animal cells of elastic components ("tethers") that connect individual chromosomes moving to opposite poles during anaphase. Tethers, originally described in crane-fly spermatocytes, exert force on chromosome arms opposite to the direction the anaphase chromosomes move. We show that they exist in a broad range of animal cells. Thus tethers are previously unrecognised components of general mitotic mechanisms that exert force on chromosomes and they need to be accounted for in general models of mitosis in terms of forces on chromosomes and in terms of what their roles might be.