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1

Electronic Resource

Effects of griseofulvin on mitosis in PtK1 cells (1979)

Mullins, J. Michael ; Snyder, Judith A.

Springer

Chromosoma 72 (1979), S. 105-113

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ISSN: 1432-0886

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract The concentration dependent effects of griseofulvin (GF) on mitosis in PtK1 cells were studied using a combination of time lapse cinematography and polarization and electron microscopy. Low concentrations of GF (4×10−5 M) allowed a substantial number of cells to enter and complete an apparently normal mitosis. At higher concentrations of GF (1×10−4 M and 2.5×10−4 M) all cells entering mitosis were arrested. Typical c-mitotic chromosome arrays were observed at 1×10−4 M GF with microtubules present but no spindle formed. At 2.5×10−4 M GF chromosomes did not orient toward a common center to form a c-mitotic figure, but instead remained in a loosely clustered grouping at the center of the cell. Electron microscopy showed microtubules to be absent but revealed an irregularly shaped electron dense cloud around the centrioles. Quantitative polarization microscopy of metaphase cells perfused with GF showed rapid loss of spindle birefringence after exposure to the drug. Coinciding with loss of birefringence the spindle shrank rapidly with a pronounced shortening of pole to pole distance.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00286432

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2

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Anaphase progression and furrow establishment in nocodazole-arrested PtK1 cells (1981)

Mullins, J. Michael ; Snyder, Judith A.

Springer

Chromosoma 83 (1981), S. 493-505

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ISSN: 1432-0886

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract The relationship between progression through anaphase and furrow establishment was investigated in PtK1 cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 μm or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK1 cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00328275

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3

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Selective reduction of anaphase B in quinacrine-treated PtK1 cells (1989)

Armstrong, L. ; Snyder, Judith A.

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 14 (1989), S. 220-229

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ISSN: 0886-1544

Keywords: microtubules ; mitosis ; kinetochore ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non-kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10-19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 μM to determine energy requirements for chromosome motion. The rate and extent of chromosome-to-pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore-kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore-kinetochore separation by 20%, and addition of 12 μM quinacrine reduced the kinetochore-kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongationquinacrine-treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373-379, 1985). Metaphase cells treated with 2-10 μM concentrations of quinacrine for 2-5 min reduced spindle lengths by 10-50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose-induced elongation was concomitantly diminished. These data suggest that quinacrine-sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose-induced metaphase spindle elongation.

Additional Material: 11 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/cm.970140208

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4

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Cytochalasin J affects chromosome congression and spindle microtubule organization in PtK1 cells (1995)

Snyder, Judith A. ; Cohen, Laura

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 32 (1995), S. 245-257

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ISSN: 0886-1544

Keywords: actin ; cytochalasin ; microfilaments ; microtubules ; mitosis ; mitotic apparatus ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: PtK1 cells were treated with 10 μg/ml cytochalasin J (CJ) for 15 min at various stages of mitosis. When applied at nuclear envelope breakdown (NEB) chromosome congression was blocked or substantially slowed, and chromosomes failed to show organization patterns typical of prometaphase. Spindle microtubule (MT) numbers appeared unaffected as judged by the pattern of birefringent retardation. However, ultrastructural analysis showed MTs to be reorganized within the spindle domain with some exhibiting fragmentation and others failing to interact with poorly defined kinetochore laminae. The spindle domain took on a curved, almost banana-like shape, as related to the position of the centrosomes and lack of orientation of chromosomes. Serial section analysis of kinetochore regions showed reduced contour length and maturation of the kinetochore plate with few MTs associated with this structure. Cells similarly treated with 10 μg/ml CJ at NEB for 15 min and then released into conditioned medium for 15 min showed that most chromosomes resumed congression to the metaphase plate. Ultrastructural analysis revelaed a more normal organization of spindle MTs, but kinetochore structure remained affected. CJ treatment of cells in prometaphase slightly affected chromosome congression with most chromosomes aligning at the metaphase plate after 10-15 min of treatment. Ultrastructural analysis showed that astral MTs were disrupted and spindle MTs were fragmented; few MTs coursed from kinetochore to pole. Kinetochore structure was also affected with only small numbers of short MTs seen associated with kinetochores. Application of CJ at anaphase onset had little effect on anaphase A and B, but cytokinesis failed to occur. Anti-tubulin staining of a monolayer of cells treated with 10 μg/ml CJ for 15 min showed that over 60% of mitotic figures exhibited changes in MT organization. Cells showing the greatest effect of treatment had several foci of bundles of MTs, as if the spindle were multipolar. Chromosomes were arranged near the periphery of the spindle which could be a result of abnormalities of kinetochore structure. Improper association of spindle MTs with kinetochores and, thus, changes in kinetochore position could account for these changes in spindle architecture. © 1995 Wiley-Liss, Inc.

Additional Material: 10 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/cm.970320402

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5

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Nocodazole pretreatment in anaphase selectively reduces anaphase B in PtK1 cells (1983)

Snyder, Judith A. ; Vogt, Sandra I. ; McLelland, Sandra L.

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 3 (1983), S. 79-91

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ISSN: 0886-1544

Keywords: mitosis ; anaphase ; microtubules ; nocodazole ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: During early anaphase PtK1 cells were briefly treated with the rapidly reversible microtubule (MT) poison nocodazole. This treatment abruptly stopped chromosome motion and effected a large decrease in spindle birefringence. On removal of the drug, chromosome to pole motion (anaphase A) returned, though at a lesser rate but not extent than untreated cells. In most cases elongation of the pole-pole distance (anaphase B) also occured, at both a rate and to an extent less than in untreated cells. During the recovery period following drug arrest spindle birefringence did not return to pretreatment levels. Electron microscopic analysis of nocodazole arrested, or arrested and released, cells revealed extensive disassembly of the nonkinetochore class of MTs (nkMTs), particularly evident in the astral region. Microtubules seen in the interzone region were largely fragments of midbody precursors. Kinetochore MTs (kMTs) appeared to be unaffected by the brief drug treatment chosen for these experiments. Analysis of MT profiles seen in transverse sections of the interzone region indicated in treated and released cells approximately 60% fewer MTs. This may suggest that chromosome motion during anaphase is not dependent on interactions between kMTs and nkMTs and separation of the spindle poles can occur in the presence of disrupted interzonal MTs.

Additional Material: 6 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/cm.970030107

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6

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UV microbeam irradiations of the mitotic spindle I. The UV-microbeam apparatus (1989)

Stonington, O. G. ; Spurck, T. P. ; Snyder, Judith A. ; [et al.]

Springer

Protoplasma 153 (1989), S. 62-70

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ISSN: 1615-6102

Keywords: Mitosis ; Ultraviolet microbeam

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Summary We describe the assembly of a UV microbeam microscope based on a Zeiss IM35 inverted microscope. The important UV transmitting elements are standard UV epifluorescence attachments available from Zeiss; the main modification involves fitting an adjustable slit in place of the field diaphragm. We describe how to align and focus the UV source for optimal irradiations. Our current version of this machine is also fitted with a monochromator and using monochromatic UV light, we can reproduceably create Areas of Reduced Birefringence in spindle fibres with ca. 2–3 s irradiations, while continually observing the fibres. The microscope is stable and easy to set up, allowing many consecutive experiments to be done, including multiple irradiations on the one cell. In conjunction with video image processing techniques, the cells can be observed continuously using polarising, Nomarski or other optical systems. Some preliminary observations demonstrating the versatility of the machine are described.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01322466

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7

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Localization of kinetochore fragments isolated from single chromatids in mitotic CHO cells (1995)

Christy, Cathleen S. ; Deden, Michelle ; Snyder, Judith A.

Springer

Protoplasma 186 (1995), S. 193-200

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ISSN: 1615-6102

Keywords: Mitosis ; Microtubules ; Kinetochores ; Sucrose ; MUGs (mitotic cells with an unreplicated genome)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Summary Chinese hamster ovary (CHO) cells are treated with hydroxurea followed by a caffeine treatment to form detached kinetochore fragments in the absence of sister chromatids. Detached kinetochores in mitotic CHO cells display a functional association with MTs initiated from one or both centrosomes such that these association(s) have a significant influence on the location and orientation of detached kinetochores and/or their fragments. Kinetochore fragments which are amphitelically oriented are positioned approximately midway between the two centrosomes. Thus, a kinetochore isolated from a single chromatid can capture MTs from both poles. Monotelic orientation of these fragments is more frequently observed with kinetochore fragments located an average distance of 2.5 μm from the nearest centrosome, compared to an average distance of 4.4 μm in amphitelically oriented fragments. In cells treated with the potent MT poison, nocodazole, kinetochore isolation also occurs and therefore is not dependent on the presence of MTs. CHO cells treated to produce isolated kinetochores or kinetochore fragments then subsequently hyperosmotically shocked show no MTs directly inserted into kinetochore lamina, similar to the response of sucrose-treated metapbase PtK1 cells. This treatment shows circular kinetochores tangentially associated with bundles of MTs that are located an average of 1.5 μm from the centrosome. Our results suggest that a single kinetochore fragment can attach to MTs initiated from one or both centrosomes and that their specific association to MT fibers defines orientation of detached kinetochores within the spindle domain.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01281329

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8

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Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells (1993)

Armstrong, Lydia ; Snyder, Judith A.

Springer

Protoplasma 173 (1993), S. 133-143

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ISSN: 1615-6102

Keywords: Centrosomes ; Microtubule ; Mitosis ; Prometaphase ; Quinacrine

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Summary Quinacrine, an acridine derivative, has previously been shown to disrupt lateral associations between non-kinetochore microtubules (nkMTs) of opposite polarity in PtK1 metaphase spindles such that the balance of spindle forces is significantly altered. We extended the analysis of the spatial relationship of spindle microtubules (MTs) in this study by using quinacrine to compare ATP-dependent requirements for early prometaphase centrosome separation and spindle formation. The route used for centrosome migration can take a variety of pathways in PtK1 cells, depending on the location of the centrosomes at the time of nuclear envelope breakdown. Following quinacrine treatment centrosome separation decresased by 1.9 to 14.0 μm depending on the pathway utilized. However, birefringence of the centrosomal region increased approximately 50% after quinacrine treatment. Quinacrine-treated mid-prometaphase cells, where chromosome attachment to MTs had occurred, showed a decrease in spindle length of approximately 6.0 μm with only a slight increase in astral birefringence. Computer-generated reconstructions of quinacrine-treated prometaphase cells were used to confirm changes in MT reorganization. Early-prometaphase cells showed more astral MTs (aMTs) of varied length while mid-prometaphase cells showed only a few short aMTs. Late prometaphase cells again showed a large number of aMTs. Our results suggest that: (1) quinacrine treatment affects centrosome separation, (2) recruitment of nkMTs by kinetochores is quinacrine-sensitive, and (3) development of the prometaphase spindle is dependent on quinacrine-sensitive lateral interactions between nkMTs of opposite polarity. These data also suggest that lateral interactions between MTs formed during prometaphase are necessary for centrosome separation and normal spindle formation but not necessarily chromosome motion.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01379002

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Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells (1993)

Geck, D. C. ; Snyder, Judith A.

Springer

Protoplasma 174 (1993), S. 83-90

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ISSN: 1615-6102

Keywords: Mitosis ; Microtubules ; Kinetochore ; Metaphase ; Anaphase

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Summary Treatment of metaphase PtK1 cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK1 metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK1 cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 μm in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK1 cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 μm in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01379040

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