scholarly journals The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

2001 ◽  
Vol 154 (6) ◽  
pp. 1135-1146 ◽  
Author(s):  
Aime A. Levesque ◽  
Duane A. Compton
Keyword(s):  
Cultured Cells ◽  
Time Lapse ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Poles ◽  
Ejection Force ◽  
Chromosome Congression ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.

scholarly journals Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push

1996 ◽  
Vol 109 (12) ◽  
pp. 2823-2831 ◽  
Author(s):  
J.C. Waters ◽  
R.V. Skibbens ◽  
E.D. Salmon
Keyword(s):  
Cell Cycle Progression ◽  
Immunofluorescence Microscopy ◽  
Differential Interference Contrast Microscopy ◽  
Cycle Progression ◽  
Spindle Poles ◽  
Constant Tension ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Regulate Cell Cycle Progression ◽  
Rest Length

Experimentally introduced tension on kinetochores and their centromeres has been shown to stabilize kinetochore attachment to microtubules, modify kinetochore directional instability, and regulate cell-cycle progression into anaphase. In mitosis, kinetochore tension and the stretch of centromere chromatin are produced by the movement of sister kinetochores toward opposite poles and astral ejection forces on the chromosome arms. However, newt lung cell kinetochores oscillate between poleward and away from the pole motility states throughout mitosis, indicating kinetochores are not under constant tension. To test whether kinetochores are under net tension while they are oscillating, and how often they are under compression and pushing into the chromosome, we measured the distance between sister kinetochores in newt lung cells using both video-enhanced differential interference contrast microscopy (VE-DIC) and immunofluorescence microscopy. We found that for chromosomes in which sister kinetochores are attached to opposite spindle poles, centromeres are, on average, stretched (2.2 microns in living cells and 1.8 microns in fixed cells) with respect to the inter-kinetochore ‘rest’ length (1.1 microns in living and fixed cells). For chromosomes in which only one kinetochore is attached to the spindle, the centromere chromatin associated with the tethered kinetochore is, on average, stretched to approximately half of the average inter-kinetochore distance measured for chromosomes in which both kinetochores are attached. We conclude that while newt lung cell kinetochores oscillate between states of P and AP movement, they are under tension approximately 90% of the time and under compression less than 6% of the time.

scholarly journals The structure of cytoplasm in directly frozen cultured cells. II. Cytoplasmic domains associated with organelle movements.

1986 ◽  
Vol 102 (4) ◽  
pp. 1510-1521 ◽  
Author(s):  
P C Bridgman ◽  
B Kachar ◽  
T S Reese
Keyword(s):  
Cultured Cells ◽  
Cytoplasmic Domains ◽  
Differential Interference Contrast Microscopy ◽  
Cultured Fibroblasts ◽  
Central Type ◽  
Contrast Microscopy ◽  
Central Regions ◽  
Distinct Individual ◽  
Interference Contrast Microscopy ◽  
The Relationship

The relationship between organelle movement and cytoplasmic structure in cultured fibroblasts or epithelial cells was studied using video-enhanced differential interference contrast microscopy and electron microscopy of directly frozen whole mounts. Two functional cytoplasmic domains are characterized by these techniques. A central domain rich in microtubules is associated with directed as well as Brownian movements of organelles, while a surrounding domain rich in f-actin supports directed but often intermittent organelle movements more distally along small but distinct individual microtubule tracks. Differences in the organization of the cytoplasm near microtubules may explain why organelle movements are typically continuous in central regions but usually intermittent along the small tracks through the periphery. The central type of cytoplasm has a looser cytoskeletal meshwork than the peripheral cytoplasm which might, therefore, interfere less frequently with organelles moving along microtubules there.

scholarly journals Optical sectioning in differential interference contrast microscopy

2009 ◽  
Vol 282 (16) ◽  
pp. 3223-3230 ◽  
Author(s):  
Atsushi Noguchi ◽  
Hiroshi Ishiwata ◽  
Masahide Itoh ◽  
Toyohiko Yatagai
Keyword(s):  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Optical Sectioning ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy

scholarly journals Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

2009 ◽  
Vol 20 (21) ◽  
pp. 4575-4585 ◽  
Author(s):  
Paul Chang ◽  
Margaret Coughlin ◽  
Timothy J. Mitchison
Keyword(s):  
Binding Proteins ◽  
Magnetic Beads ◽  
Spindle Pole ◽  
Covalent Modification ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Tankyrase 1 ◽  
Mitotic Spindle Pole

Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

scholarly journals Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

2003 ◽  
Vol 160 (5) ◽  
pp. 671-683 ◽  
Author(s):  
Alexey Khodjakov ◽  
Lily Copenagle ◽  
Michael B. Gordon ◽  
Duane A. Compton ◽  
Tarun M. Kapoor
Keyword(s):  
Three Dimensional ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Formation ◽  
Capture Mechanism ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5 ◽  
Dependent Mechanism

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living α-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

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