scholarly journals Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to-pole motion.

1992 ◽  
Vol 116 (6) ◽  
pp. 1409-1420 ◽  
Author(s):  
E Shelden ◽  
P Wadsworth
Keyword(s):  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Pole Motion ◽  
Anaphase Onset ◽  
Tubulin Subunit ◽  
Confocal Fluorescence ◽  
Anaphase B ◽  
The Relationship ◽  
Chromosome Motion ◽  
In Cells

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.

scholarly journals Two S. pombe septation phases differ in ingression rate, septum structure, and response to F-actin loss

2019 ◽  
Vol 218 (12) ◽  
pp. 4171-4194 ◽  
Author(s):  
Mariona Ramos ◽  
Juan Carlos G. Cortés ◽  
Mamiko Sato ◽  
Sergio A. Rincón ◽  
M. Belén Moreno ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Dual Function ◽  
Second Phase ◽  
Glucan Synthase ◽  
Two Phases ◽  
Anaphase B ◽  
The Relationship ◽  
Contractile Structure ◽  
In Cells

In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.

scholarly journals Metabolic inhibitors block anaphase A in vivo.

1986 ◽  
Vol 102 (6) ◽  
pp. 1995-2005 ◽  
Author(s):  
P K Hepler ◽  
B A Palevitz
Keyword(s):  
Metabolic Inhibitors ◽  
Pole Motion ◽  
Carbonyl Cyanide ◽  
Transport Inhibitors ◽  
Spindle Microtubules ◽  
Guard Mother Cells ◽  
Mother Cells ◽  
Anaphase B ◽  
Chromosome Motion

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells of Tradescantia virginiana consists almost entirely of chromosome-to-pole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome motion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to "freeze" in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol treatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and/or electrical gradients generated by cellular membranes.

Artifact-free electron microscopic immunocytochemistry on rat brain tissue

1991 ◽  
Vol 49 ◽  
pp. 272-273
Author(s):  
Veronika Burmeister ◽  
N. Ludvig ◽  
P.C. Jobe
Keyword(s):  
Microscopic Examination ◽  
Free Electron ◽  
Electron Microscopic Examination ◽  
Ultrastructural Localization ◽  
Rabbit Serum ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Phosphate Buffered Saline ◽  
Rat Brain Tissue ◽  
Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

Ultrastructural Immunocytochemistry of Five Rat Pituitary Adenomas

1980 ◽  
Vol 38 ◽  
pp. 724-725
Author(s):  
D. J. McComb ◽  
N. Ryan ◽  
E. Horvath ◽  
K. Kovacs ◽  
E. Nagy ◽  
...  
Keyword(s):  
Pituitary Adenomas ◽  
Pituitary Tumors ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Transmission Electron ◽  
Rat Pituitary ◽  
Radiation Induced ◽  
Group 5 ◽  
Group 2 ◽  
Microscopic Techniques

Conventional light and electron microscopic techniques failed to clarify the cellular composition and derivation of spontaneous and induced, intrasellar and transplanted pituitary adenomas in rats (1). In the present work, electron microscopic immunocytochemistry was applied to evaluate five adenohypo-physial tumors using a technique described by Moriarty and Garner (2). Spontaneously occurring pituitary adenomas (group 1) were harvested from aging female Long-Evans rats. R-Amsterdam rats were treated with 2 x 1.0 mg estrone acetate (HogivaI) s.c. weekly for 6 months. Pituitary adenomas in excess of 30 mg were removed from these animals to make up the tumors of group 2. Groups 3 and 4 consisted of estrogen-induced autonomous transplan¬ted pituitary tumors MtT.WlO and MtT.F4. Group 5 was a radiation-induced transplanted autonomous pituitary tumor MtT.W5. The tumors of groups 3,4 and 5 were allowed to proliferate in host rats 6-8 weeks prior to removal for processing. Tissue was processed for transmission electron microscopy (glutaraldehyde fixation, OsO4 postfixation and epoxy resin embedding), and electron microscopic immunocytochemistry (3% paraformaldehyde fixation and Araldite embedding).

scholarly journals Microtubule Flux and Sliding in Mitotic Spindles ofDrosophila Embryos

2002 ◽  
Vol 13 (11) ◽  
pp. 3967-3975 ◽  
Author(s):  
Ingrid Brust-Mascher ◽  
Jonathan M. Scholey
Keyword(s):  
Mitotic Spindles ◽  
Pole Motion ◽  
Spindle Poles ◽  
Balance Of Forces ◽  
Anaphase B

We proposed that spindle morphogenesis in Drosophilaembryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 μm/s, too slow to drive chromatid-to-pole motion at 0.11 μm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.

Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactive neurons in the rat hypothalamus

1984 ◽  
Vol 302 (2) ◽  
pp. 221-230 ◽  
Author(s):  
Y. Ibata ◽  
F. Kawakami ◽  
K. Fukui ◽  
H.L. Obata-Tsuto ◽  
M. Tanaka ◽  
...  
Keyword(s):  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Rat Hypothalamus
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