Department of Biology

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Open Access
Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes
(Springer Link, 2007-12) Forer, Arthur; Spurck, Tim; Pickett-Heaps, Jeremy
We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphasecrane-fly spermatocytes; then we followed the growth of the “kinetochore stubs”, the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to flux of tubulin in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing flux of tubulin in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward thereby causing stub elongation and generating anaphasechromosome movement in non-irradiated cells.
Open Access
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
(Frontiers, 2018-07-24) Forer, Arthur; Sheykhani, Rozhan; Berns, Michael
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.
Open Access
Chromosomes selectively detach at one pole and quickly move towards the opposite pole when kinetochore microtubules are depolymerized in Mesostoma ehrenbergii spermatocytes.
(Springer Link, 2018-02) Forer, Arthur; Fegaras, Eleni
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.
Open Access
Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations.
(Springer Link, 2013-10) Berns, Michael; Ferraro-Gideon, Jessica; Forer, Arthur
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.
Open Access
Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?
(Springer Link, 2011-01) Johansen, Kristen; Forer, Arthur; Yao, Changfu; Girton, Jack; Johansen, Jørgen
The idea of a spindle matrix has long been proposed in order to account for poorly understood features of mitosis. However, its molecular nature and structural composition have remained elusive. Here we propose that the spindle matrix may be constituted by mainly nuclear-derived proteins that reorganize during the cell cycle to form an elastic gel-like matrix. We discuss this hypothesis in the context of recent observations from phylogenetically diverse organisms that nuclear envelope and intranuclear proteins form a highly dynamic and malleable structure that contributes to mitotic spindle function. We suggest that the visco-elastic properties of such a matrix may constrain spindle length while at the same time facilitating microtubule growth and dynamics as well as chromosome movement. A corollary to this hypothesis is that a key determinant of spindle size may be the amount of nuclear proteins available to form the spindle matrix. Such a matrix could also serve as a spatial regulator of spindle assembly checkpoint proteins during open and semi-open mitosis.
Open Access
Elastic tethers between separating anaphase chromosomes in crane-fly spermatocytes coordinate chromosome movements to the two poles.
(Wiley, 2017-02) Forer, Arthur; Berns, Michael; Sheykhani, Rozhan
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.
Open Access
Elastic ‘tethers’ connect separating anaphase chromosomes in a broad range of animal cells.
(Elsevier, 2017-09) Forer, Arthur; Duquette, Michelle L.; Paliulis, Leocadia V.; Fegaras, E.; Ono, M.; Preece, D.; Berns, Michael
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, produce force on chromosome arms opposite to the direction the anaphase chromosomes move. In crane-fly spermatocytes tethers function to coordinate movements between chromosomes. Their presence in a broad range of cells suggests that they may be important in coordinating movements between chromosomes to ensure normal segregation. Tethers are previously unrecognised force-producing components of general mitotic mechanisms and 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, possibly in coordinating chromosome movements during mitosis.
Open Access
Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane‐fly spermatocytes
(Wiley, 2008-11) Forer, Arthur; Xie, Lele
We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that actin depolymerizing agents such ascytochalasin D or latrunculin B blocked or slowed chromosome movements; we wanted to compare the effects of jasplakinolide, a compound that stabilizes actin filaments against depolymerization. Jasplakinolide had the same effect on movements of each half-bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed movement; slowed pairs did not change their speeds. Confocal microscopy indicated that neither the distributions of spindle actin filaments nor the distributions of spindle microtubules were altered by the jasplakinolide. It is possible that jasplakinolide binds to spindle actin and blocks critical binding sites, but we suggest that jasplakinolide affects anaphase chromosome movement by preventing actin-filament depolymerization that is necessary for anaphase to proceed. Overall, our data indicate that actin is involved in one of the redundant mechanisms cells use to move chromosomes.
Open Access
Mesostoma ehrenbergii spermatocytes - a unique and advantageous cell for studying meiosis
(Wiley, 2013-05) Forer, Arthur; Hoang, Carina; Ferraro-Gideon, Jessica
Mesostoma ehrenbergii have a unique male meiosis: their spermatocytes have three large bivalents that oscillate for 1-2 hours 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, i.e., 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’. In the present article we review the literature on meiosis in Mesostoma spermatocytes and describe the current research that we are doing using Mesostoma spermatocytes, rearing the animals in the laboratory using methods that we describe in our companion article (Hoang et al., 2013).
Open Access
Methods for rearing Mesostoma ehrenbergii in the laboratory for cell biology experiments, including identification of factors that influence production of different egg types
(Wiley, 2013-10) Forer, Arthur; Gauthier, Kimberley; Ferraro-Gideon, Jessica; Hoang, Carina
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 3 bivalent and 4 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. ehrenbergiiare 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 diapausing eggs hatch, which is irregular and creates unpredictable wait times. As a result, to maintain M. ehrenbergii stocks in order to study their spermatocytes, we studied various factors that might influence egg type production. We have found that feeding them daily and keeping them at 25°C favours S egg production. Currently, our cultures have reached the 45th generation. In this article we describe our rearing and dissection methods and describe experiments which led to our present rearing methods.
Open Access
Mitosis: spindle evolution and the matrix model
(Springer Link, 2009-03) Forer, Arthur; Pickett-Heaps, Jeremy
Current spindle models explain “anaphase A” (movement of chromosomes to the poles) in terms of a motility system based solely on microtubules (MTs) and that functions in a manner unique to mitosis. We find both these propositions unlikely. An evolutionary perspective suggests that when the spindle evolved, it should have come to share not only components (e.g., microtubules) of the interphase cell but also the primitive motility systems available, including those using actin and myosin. Other systems also came to be involved in the additional types of motility that now accompany mitosis in extant spindles. The resultant functional redundancy built reliability into this critical and complex process. Such multiple mechanisms are also confusing to those who seek to understand how chromosomes move. Narrowing this commentary down to just anaphase A, we argue that the spindle matrix participates with MTs in anaphase A and that this matrix may contain actin and myosin. The diatom spindle illustrates how such a system could function. This matrix may be motile and work in association with the MT cytoskeleton, as it does with the actin cytoskeleton during cell ruffling and amoeboid movement. Instead of pulling the chromosome polewards, the kinetochore fibre’s role might be to slow polewards movement to allow correct chromosome attachment to the spindle. Perhaps the earliest eukaryotic cell was a cytoplast organized around a radial MT cytoskeleton. For cell division, it separated into two cytoplasts via a spindle of overlapping MTs. Cytokinesis was actin-based cleavage. As chromosomes evolved into individual entities, their interaction with the dividing cytoplast developed into attachment of the kinetochore to radial (cytoplast) MTs. We think it most likely that cytoplasmic motility systems participated in these events.
Open Access
Movement of chromosomes with severed kinetochore microtubules
(Springer Link, 2015-01) Forer, Arthur; Johansen, Kristen M.; Johansen, Jørgen
Experiments from as early as 1966 and thereafter showed that anaphase chromosomes continued to move poleward after their kinetochore microtubules were severed by ultraviolet microbeam irradiation; these conclusions were initially met with skepticism as this 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 summarize the data indicating that chromosomes can move with severed kinetochore microtubules and we discuss proposed mechanisms of chromosome movement with severed kinetochore microtubules.
Open Access
Polyploidy and reduction divisions in cancer and mosquito gut cells
(Wiley, 2013-01) Forer, Arthur
Several articles in a recent issue of this journal have called attention to a possible way by which cancer cells can evade death and become resistant to treatments (discussed in Erenpreisa et al., 2008; Wheatley, 2008). Some cancer cells duplicate chromosomes inside their nucleus without undergoing mitosis. The resultant large polyploid cells remain quiescent, but eventually a small percentage undergoes reduction divisions to form diploid or pseudo-diploid cells which then proliferate via normal mitosis, and which sometimes are more resistant to treatment than were the original cells (e.g., Puig et al., 2008). However, this is not a specific trait of cancer cells because somatic reduction divisions regularly occur in non-cancerous cells, the best-studied example being cells of the mosquito gut.
Open Access
Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes
(Springer Link, 2007-10) Forer, Arthur; Fabian, Lacramioara
We tested whether the mechanisms of chromosome movement during anaphase in locust [Locusta migratoria (L.)] spermatocytes might be similar to those described in crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes as indicated by effects of inhibitors and by localisations of actin and myosin in spindles. In this study we tested whether locust spermatocytes spindles also utilize actin and myosin and whether actin is involved in microtubule flux. Living locust spermatocytes were treated with inhibitors of actin (Latrunculin B and Cytochalasin D), an inhibitor of myosin (BDM), or inhibitors of myosin phosphorylation (Y-27632 and ML-7). We added drugs (individually) during anaphase. Actin inhibitors alter anaphase: chromosomes either completely stop moving, slow, or sometimes accelerate. The myosin inhibitor, BDM, also alters anaphase: in most cases, the chromosomes drastically slow or stop. ML-7, an inhibitor of MLCK, causes chromosomes to stop, slow, or sometimes accelerate, similar to actin inhibitors. Y27632, an inhibitor of Rho-kinase, drastically slows or stops anaphase chromosome movements. The effects of the drugs on anaphase movement are reversible: most of the half-bivalents resume movement at normal speed after these drugs are washed out. Actin and myosin were present in the spindles in locations consistent with their possible involvement in force production. Microtubule flux along kinetochore fibres is an actin-dependent process, since LatB removes completely or drastically reduces the gap in microtubule acetylation at the kinetochore. These results suggest that actin and myosin are involved in anaphase chromosome movements in locust spermatocytes.
Open Access
Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in Mesostoma ehrenbergii spermatocytes.
(Springer Link, 2018-03) Forer, Arthur; Fegaras, Eleni
A “precocious” cleavage furrow develops and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes (Forer and Pickett-Heaps, 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 (FerraroGideon et al. 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 MTs are implicated in various furrow positioning models and our experiments studied the responses of the precocious furrows to the absence of spindle microtubules (MTs). We depolymerized spindle MTs during prometaphase using various concentrations of nocodazole (NOC) and colcemid. The expected result is the furrow should regress and chromosomes remain in the midzone of the cell (Cassimeris et al. 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.
Open Access
The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle
(Wiley, 2013-05) Forer, Arthur; Berns, Michael; Shah, Jagesh; Liaw, Lih-Huei; Gomez, Veronica; Baker, Norman; Sheykhani, Rozhan
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 un-irradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and mono-phosphorylated myosin are associated with spindle fibres and showed that some actin and mono-phosphorylated 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 un-irradiated pole to move toward the equator until a balanced length is obtained.
Open Access
The role of myosin phosphorylation in anaphase chromosome movement
(Elsevier, 2013-04) Sheykhani, Rozhan; Shirodkar, Purnata V.; Forer, Arthur
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 biphosphorylated 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 2Pmyosin. 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.
Open Access
Tethers: elastic connections between separating partner chromosomes in anaphase
(Springer Link, 2018-05) Forer, Arthur; Paliulis, Leocadia
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 19th 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.
Open Access
What generates flux of tubulin in kinetochore microtubules?
(Springer Link, 2008-04) Forer, Arthur; Pickett-Heaps, Jeremy; Spurck, Tim
We discuss models for production of tubulin flux in kinetochore microtubules. Current models concentrate solely on microtubules and their associated motors and enzymes. For example, in some models the driving force for flux is enzymes at the poles and the kinetochores; in others the driving force is motor molecules that are associated with a stationary spindle matrix. We present a different viewpoint, that microtubules are propelled poleward by forces arising from the spindle matrix, that the forces on the microtubules "activate" polymerising and depolymerising enzymes at kinetochores and poles, that matrix forces utilise actin, myosin, and microtubule motors, and that the matrix itself may not necessarily be static.

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