Department of Biology

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Open Access
Crane Fly Spermatocytes and Spermatids: A System for Studying Cytoskeletal Components
(Academic Press, Inc., 1982) Forer, Arthur
This chapter studies cytoskeletal components of the crane fly spermatocytes and spermatids. Normal living crane fly spermatocytes have been studied using phase-contrast and polarization microscopy. Information has been obtained about chromosomal orientations during various stages of meiosis and about velocities of movements during the different stages of meiosis. The IV-instar larvae are distinguishable from III-instar larvae by the size of the spiracles at the rear end of the larvae. Male larvae cannot be distinguished from female larvae using external markers, however, so it is impossible to choose male larvae with 100% accuracy. It is found that 80% of the males had become IV-instar when only 62% of the total had become IV-instar. It is observed that even if there were no way to distinguish male from female crane fly larvae, one increases one's chances of choosing males by restricting the choice to the first 50–60% of the group to become IV-instar. It is suggested that better synchrony might be obtained by keeping the animals at constant density and at constant humidity in a constant-temperature incubator.
Open Access
Chromosome movements ln the meiosis of insects, especially crane-fly spermatocytes
(Blackwell Scientific Publications, 1980) Forer, Arthur
I write as a cell biologist interested in the mechanisms of chromosome movements during mitosis and meiosis: studies on insect cells have contributed greatly to our understanding of these processes (for example, see review by Schrader 1953). Much of my work has utilized primary spermatocytes from crane-flies (Diptera: Tipulidae); I will try to explain here some of the characteristics of these cells which make them useful as objects of study. To see how studies on crane-fly spermatocytes fit into the general picture it is necessary to first describe the general state of our understanding of chromosome movements. Chromosome movements during various stages of mitosis and meiosis have been well described from studies of fixed and of living cells under the light microscope. Such studies give good estimates of the timing of stages, chromosome velocities, distances travelled, and so forth. From these data one can calculate the force and energy requirements for moving chromosomes (reviews in Forer 1969; Nicklas 1971); very small forces and very little energy are required. The cytological component responsible for transmitting the force to the chromosome is identified at the light-microscopical level as the chromosomal spindle fibre, which extends between the chromosome and the spindle pole (see Cornman 1944; Schrader 1953; Mazia 1961; Forer 1969, 1974; Nicklas 1971, 1975). Studies with the electron microscope show that microtubules, 25nm-diameter, hollow-looking cylinders, are attached to the chromosome's centromere (i.e. the spindle fibre attachment region of the chromosome), and thus would seem to have some important role or roles in mitosis and meiosis. But it is not known what microtubules do. One of the important problems at present is to identify chemically and ultrastructurally those chromosomal spindle fibre components that produce the force for chromosome movement. Eventually one wants to understand how force production is regulated, but it is difficult to deal with this question until one knows what the 'motor' is. Hence, a prime concern at present is to identify the force producers, and to see how they work, chemically.
Open Access
Chromosome Movements During Cell-Division: Possible involvement of actin filaments
(Academic Press, 1978) Forer, Arthur
In this chapter I concentrate on the question of whether or not actin is involved in producing force for chromosome movements during cell-division. the data to date concern whether or not actin is a genuine spindle component: these data will be considered in detail. By way of introduction, I first review some of the basic and generally agreed upon aspects of mitosis, such as the forces involved, the role of chromosomal spindle fibres, and so on. Then I briefly summarize the main hypotheses in which microtubules are considered to be the force producing agents, and I briefly summarize my view of the status of these hypotheses. Finally, I discuss critically and in detail the electron microscopic and light microscopic data which suggest that actin is a component of chromosomal spindle fibres. I discuss also the negative evidences, and I discuss the counter-arguments that are used to argue that actin is not a component of spindles. I conclude that actin is probably a spindle component, and is probably involved in chromosome movement. However, none of the evidences for actin being a spindle component are unequivocal, and I disucss the kinds of data that need to be obtained to substantiate that actin is a spindle fibre component and that actin is involved in producing the force for chromosome movement.
Open Access
Chromosome movements during cell-division
(1969) Forer, Arthur
This chapter is concerned with chromosome movements during unuphur. of cell-division. It deals with such problems as the morphological identification of the material(s) which transmit force to the chromosomes, and the composition and function of this material. At the present time very little is known about such problems, but I hope that this survey will point out some of the questions which need be, and can now be, asked. The material will be discussed in the following order: (a) the time course of cell-division as seen in living and fixed cells, defining the terms to be used, and defining the problem: chromosome movements during anaphase; (b) the morphological and chemical nature of the materials controlling chromosome movement as deduced from light microscopic observations and experiments; (c) electron microscopic observations and their relation to the light microscopic observations; and finally, (d) biochemical analyses of the spindle as studied by isolation techniques. In all four sections I touch on many problems which are discussed in more detail in the following general reviews: Wilson; Cornman; Hughes; Schrader; and Mazia (1961).
Open Access
Measurements of forces produced by the mitotic spindle using optical tweezers
(Scientific Society Publisher Alliance, 2013-03-13) Ferraro-Gideon, Jessica; Sheykhani, Rozhan; Zhu, Qingyuan; Duquette, Michelle L.; Berns, Michael; Forer, Arthur
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.
Open Access
Open Access photos of University Professor Dawn Bazely, recipient of the Royal Canadian Institute for Science's 2022 Fleming Medal for Excellence in Science Communication
(2022) Bazely, Dawn
Collection of open access images of University Professor Dawn Bazely spanning the 1960s to 2019. Bazely is the recipient of the 2022 Sandford Fleming Medal for Excellence in Science Communication awarded annually by the Royal Canadian Institute for Science since 1982. Bazely joined the Department of Biology as an Assistant Professor in March 1990. Image 1: Dawn Bazely has contributed her knowledge of arctic botany to Adventure Canada's team of experts travelling on ecotourism expedition cruises since 2016. Bazely has sailed the northwest passage from west to east, including being part of a Parks Canada test visit at the site of the HMS Erebus shipwreck in 2019. Image 2. Professors Dawn Bazely (Science) and Steve Alsop (Education) at Wapusk National Park Headquarters in Churchill MB in October 2019. Image 3. Dawn Bazely in 2018. Bazely was a visiting professor in the Environmental Studies Faculty of Visva Bharati University, Santiniketan, India, marking her first time back in her country of birth since 1962. Her sabbatical activities included teaching graduate student workshops on open access and science communication. Image 4. Dawn Bazely and colleagues at the Nature Canada, Women for Nature Ball in Ottawa, 2016. Image 5. Headshot of Dawn Bazely in 2016. Image 6. Fieldwork in a Southwestern Ontario forest in 2008. Image 7. Fieldwork on the island of Hirta, St Kilda in Scotland,1991. Image 8. Dawn Bazely at La Pérouse Bay, Hudson Bay in 1982. For more images from the long-term research see YorkSpace's Churchill Communities of Knowledge, and https://www.cbc.ca/news/canada/manitoba/research-photos-manitoba-tundra-open-public-1.5330742 Image 9. Dawn Bazely with her mother (at left) and younger sister, Sue Bazely (at right) and a family friend on a visit to Tring Natural History Museum, UK, c.1964
Open Access
Data files for "Molecular Modeling the Proteins from the exo-xis Region of Lambda and Shigatoxigenic Bacteriophages"
(MDPI, 2021-10-20) Donaldson, Logan
Open Access
Ploidy and reduction divisions in cancer and mosquito hind-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
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’ 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
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
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
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
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
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
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
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
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.
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
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.