Structural evidence for elastic tethers connecting separating chromosomes in crane-fly spermatocytes.

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.

Blocking Protein Phosphatase 1 [PP1] Prevents Loss of Tether Elasticity in Anaphase Crane-Fly Spermatocytes.

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.

Evidence of Non-microtubule Spindle Forces in Mesostoma ehrenbergii 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 Between Separating Anaphase Chromosomes Regulate the Poleward Speeds of the Attached Chromosomes in Crane-Fly Spermatocytes.

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.

The role of phosphorylation in the elasticity of the tethers that connect telomeres of separating anaphase chromosomes.

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.

Anaphase Chromosomes in Crane-Fly Spermatocytes Treated With Taxol (Paclitaxel) Accelerate When Their Kinetochore Microtubules Are Cut: Evidence for Spindle Matrix Involvement With Spindle Forces.

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.

Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in Mesostoma ehrenbergii spermatocytes.

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.

Chromosomes selectively detach at one pole and quickly move towards the opposite pole when kinetochore microtubules are depolymerized in Mesostoma ehrenbergii spermatocytes.

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.

A review of "tethers": elastic connections between separating partner chromosomes in anaphase.

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.

Mitotic tethers connect sister chromosomes and transmit "cross-polar" force during anaphase A of mitosis in PtK2 cells.

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.

Elastic 'tethers' connect separating anaphase chromosomes in a broad range of animal cells.

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.

Elastic tethers between separating anaphase chromosomes in crane-fly spermatocytes coordinate chromosome movements to the two poles.

Separating anaphase chromosomes in crane-fly spermatocytes are connected by elastic tethers, as originally described by LaFountain et al. (2002): telomere-containing arm fragments severed from the arms move backwards to the partner telomeres. We have tested whether the tethers coordinate the movements of separating partner chromosomes. In other cell types anaphase chromosomes move faster, temporarily, when their kinetochore microtubules are severed. However, in crane-fly spermatocytes the chromosomes move at their usual speed when their kinetochore microtubules are severed. To test whether the absence of increased velocity is because tethers link the separating chromosomes and coordinate their movements, we cut tethers with a laser microbeam and then cut the kinetochore microtubules. After this procedure, the associated chromosome sped up, as in other cells. These results indicate that the movements of partner anaphase chromosomes in crane-fly spermatocytes are coordinated by elastic tethers connecting the two chromosomes and confirm that chromosomes speed up in anaphase when their kinetochore microtubules are severed. © 2016 Wiley Periodicals, Inc.

Movement of chromosomes with severed kinetochore microtubules.

Experiments dating from 1966 and thereafter showed that anaphase chromosomes continued to move poleward after their kinetochore microtubules were severed by ultraviolet microbeam irradiation. These observations were initially met with scepticism as they contradicted the prevailing view that kinetochore fibre microtubules pulled chromosomes to the pole. However, recent experiments using visible light laser microbeam irradiations have corroborated these earlier experiments as anaphase chromosomes again were shown to move poleward after their kinetochore microtubules were severed. Thus, multiple independent studies using different techniques have shown that chromosomes can indeed move poleward without direct microtubule connections to the pole, with only a kinetochore 'stub' of microtubules. An issue not yet settled is: what propels the disconnected chromosome? There are two not necessarily mutually exclusive proposals in the literature: (1) chromosome movement is propelled by the kinetochore stub interacting with non-kinetochore microtubules and (2) chromosome movement is propelled by a spindle matrix acting on the stub. In this review, we summarise the data indicating that chromosomes can move with severed kinetochore microtubules and we discuss proposed mechanisms for chromosome movement with severed kinetochore microtubules.

Meiosis-I in Mesostoma ehrenbergii spermatocytes includes distance segregation and inter-polar movements of univalents, and vigorous oscillations of bivalents.

In this article, we describe meiosis-I in spermatocytes of the free-living freshwater flatworm Mesostoma ehrenbergii. The original observations of Oakley (1983, 1985) and Fuge (Eur J Cell Biol 44:294-298, 1987, Cell Motil Cytoskeleton 13:212-220, 1989, Protoplasma 160:39-48, 1991), the first to describe these cells, challenge our understanding of cell division, and we have expanded on these descriptions with the aim of laying the framework for further experimental work. These cells contain three bivalents and four univalent chromosomes (two pairs). Bivalent kinetochores oscillate vigorously and regularly throughout prometaphase, for up to several hours, until anaphase. Anaphase onset usually begins in the middle of the kinetochore oscillation cycle. Precocious cleavage furrows form at the start of prometaphase, ingress and then remain arrested until the end of anaphase. The four univalents do not pair, yet by anaphase there is one of each kind at each pole, an example of "distance segregation" (Hughes-Schrader in Chromosoma 27:109-129, 1969). Until proper segregation is achieved, univalents move between spindle poles up to seven times in an individual cell; they move with velocities averaging 9 µm/min, which is faster than the oscillatory motions of the bivalent kinetochores (5-6 µm/min), and much faster than the anaphase movements of the segregating half-bivalents (1 µm/min). Bipolar bivalents periodically reorient, most often resulting in the partner kinetochores exchanging poles. We suggest that the large numbers of inter-polar movements of univalents, and the reorientations of bivalents that lead to partners exchanging poles, might be because there is non-random segregation of chromosomes, as in some other cell types.

Methods for rearing Mesostoma ehrenbergii in the laboratory for cell biology experiments, including identification of factors that influence production of different egg types.

Mesostoma ehrenbergii spermatocytes are uniquely useful to study various aspects of cell division. Their chromosomes are large in size and few in number, with only three bivalent and four univalent chromosomes. During prometaphase, bipolar bivalents oscillate regularly to and from the poles for 1-2 hours. The univalents remain at the poles but occasionally move from one pole to the other. In addition, a precocious cleavage furrow forms during prometaphase and remains partially constricted until anaphase. Attempts to rear these animals indefinitely in laboratory conditions, however, have been mostly unsuccessful because of their reproductive strategy. M. ehrenbergii are hermaphroditic flatworms that can produce viviparous offspring (termed S eggs) and/or diapausing eggs (termed D eggs) and they follow either one of two reproductive patterns: (1) they first form S eggs and following the delivery of these eggs produce D eggs, or (2) they only produce D eggs. When only D eggs are formed, which is common under laboratory conditions, the stocks die out until the D eggs hatch, which is irregular and creates unpredictable wait times. Consequently, in order to maintain M. ehrenbergii stocks to study their spermatocytes, we examined various factors that might influence egg-type production. Feeding them daily and keeping them at 25°C favours S egg production. Currently, our cultures have reached the 53rd generation. We herein describe our rearing and dissection methods, and some experiments which led to our present rearing methods.

Mesostoma ehrenbergii spermatocytes--a unique and advantageous cell for studying meiosis.

Mesostoma ehrenbergii have a unique male meiosis: their spermatocytes have three large bivalents that oscillate for 1-2 h before entering into anaphase without having formed a metaphase plate, have a precocious ('pre-anaphase') cleavage furrow, and have four univalents that segregate between spindle poles without physical interaction between them, that is via 'distance segregation'. These unique and unconventional features make Mesostoma spermatocytes an ideal organism for studying the force produced by the spindle to move chromosomes, and to study cleavage furrow control and 'distance segregation'. We review the literature on meiosis in Mesostoma spermatocytes and describe our current research with Mesostoma spermatocytes, rearing the animals in the laboratory using methods that described in our companion article [Hoang et al. (2013); Cell Biol Int].

The role of myosin phosphorylation in anaphase chromosome movement.

This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production. Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin.

The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle.

This study investigates spindle biomechanical properties to better understand how spindles function. In this report, laser microbeam cutting across mitotic spindles resulted in movement of spindle poles toward the spindle equator. The pole on the cut side moved first, the other pole moved later, resulting in a shorter but symmetric spindle. Intervening spindle microtubules bent and buckled during the equatorial movement of the poles. Because of this and because there were no detectable microtubules within the ablation zone, other cytoskeletal elements would seem to be involved in the equatorial movement of the poles. One possibility is actin and myosin since pharmacological poisoning of the actin-myosin system altered the equatorial movements of both irradiated and unirradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and monophosphorylated myosin are associated with spindle fibers and showed that some actin and monophosphorylated myosin remained in the irradiated regions. Overall, our experiments suggest that actin, myosin and microtubules interact to control spindle length. We suggest that actin and myosin, possibly in conjunction with the spindle matrix, cause the irradiated pole to move toward the equator and that cross-talk between the two half spindles causes the unirradiated pole to move toward the equator until a balanced length is obtained.

Measurements of forces produced by the mitotic spindle using optical tweezers.

We used a trapping laser to stop chromosome movements in Mesostoma and crane-fly spermatocytes and inward movements of spindle poles after laser cuts across Potorous tridactylus (rat kangaroo) kidney (PtK2) cell half-spindles. Mesostoma spermatocyte kinetochores execute oscillatory movements to and away from the spindle pole for 1-2 h, so we could trap kinetochores multiple times in the same spermatocyte. The trap was focused to a single point using a 63× oil immersion objective. Trap powers of 15-23 mW caused kinetochore oscillations to stop or decrease. Kinetochore oscillations resumed when the trap was released. In crane-fly spermatocytes trap powers of 56-85 mW stopped or slowed poleward chromosome movement. In PtK2 cells 8-mW trap power stopped the spindle pole from moving toward the equator. Forces in the traps were calculated using the equation F = Q'P/c, where P is the laser power and c is the speed of light. Use of appropriate Q' coefficients gave the forces for stopping pole movements as 0.3-2.3 pN and for stopping chromosome movements in Mesostoma spermatocytes and crane-fly spermatocytes as 2-3 and 6-10 pN, respectively. These forces are close to theoretical calculations of forces causing chromosome movements but 100 times lower than the 700 pN measured previously in grasshopper spermatocytes.

Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations.

Univalent sex chromosomes in crane-fly spermatocytes have kinetochore spindle fibres to each spindle pole (amphitelic orientation) from metaphase throughout anaphase. The univalents segregate in anaphase only after the autosomes approach the poles. As each univalent moves in anaphase, one spindle fibre shortens and the other spindle fibre elongates. To test whether the directionality of force production is fixed at anaphase, that is, whether one spindle fibre can only elongate and the other only shorten, we cut univalents in half with a laser microbeam, to create two chromatids. In both sex-chromosome metaphase and sex-chromosome anaphase, the two chromatids that were formed moved to opposite poles (to the poles to which their fibre was attached) at speeds about the same as autosomes, much faster than the usual speeds of univalent movements. Since the chromatids moved to the pole to which they were attached, independent of the direction to which the univalent as a whole was moving, the spindle fibre that normally elongates in anaphase still is able to shorten and produce force towards the pole when allowed (or caused) to do so.