Scleroderma crest autoantibodies as fluorescent and Immuno-Electron Microscopic probes: Keys to a chromosomal black box

1991 ◽  
Vol 49 ◽  
pp. 12-13
Author(s):  
B. R. Brinkley ◽  
R. P. Zinkowski
Keyword(s):  
Mitotic Spindle ◽  
Attachment Site ◽  
Black Box ◽  
Chromosome Movement ◽  
Electron Microscopic ◽  
Primary Constriction ◽  
Plant Chromosomes ◽  
Spindle Poles ◽  
Spindle Microtubules ◽  
Mitosis And Meiosis

The mammalian kinetochore is a highly differentiated structure found at the centromere (primary constriction) of chromosomes that serves as an attachment site for spindle microtubules. Ultrastructurally, the kinetochore typically appears as a tri-layered plate or disc situated at the sides of the centromere (Fig.1). Recent evidence demonstrates that kinetochores have the ability to capture and stabilize microtubules that grow from the spindle poles. Moreover, the motor(s) for chromosome movement appear to be located in or near the kinetochore which actively participates in the generation of forces necessary for chromosome movement in mitosis and meiosis. To understand how the precise ballet-like movements of chromosomes on the mitotic spindle occur, attention has focused on the “black box” of the chromosome; the centromere-kinetochore complex.The fortuitous discovery that serum from individuals with the CREST variant of scleroderma contain autoantibodies that bind to components of the centromere-kinetochore complex has led to major advancements in the understanding of this chromosomal black box. Indirect immunofluorescence has demonstrated the presence of paired fluorescent structures (Fig.2) at the centromeres of both mammalian and plant chromosomes.

Probing The Mechanisms Underlying Kinetochore Behavior In Vertebate Cells Using Combinations of Advanced Light and 3-D Electron Microscopy

1997 ◽  
Vol 3 (S2) ◽  
pp. 217-218
Author(s):  
B. F. McEwen ◽  
A.B. Heagle ◽  
C.L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Mitotic Spindle ◽  
Attachment Site ◽  
Spindle Pole ◽  
Nuclear Envelope Breakdown ◽  
Daughter Cells ◽  
Primary Constriction ◽  
Sister Kinetochore ◽  
To Receive ◽  
Chromosome Motion

For daughter cells to receive equal copies of the genome during mitosis, the replicated chromosomes must attach to and move bi-directionally on the mitotic spindle. A chromosome becomes attached to the spindle via a pair specialized structures, known as kinetochores, that are positioned on opposite sides of its primary constriction (one on each of the two chromatids). In addition to being the spindle attachment site, kinetochores are also involved in producing and/or transmitting the forces for chromosome motion. In vertebrates the kinetochore closest to a spindle pole at the time of nuclear envelope breakdown usually is the first to attach to the spindle. As a result of this attachment the now “monooriented” chromosome moves toward the closest pole where its only attached kinetochore initiates oscillatory motions toward and away from that pole until the unattached sister kinetochore acquires microtubules (Mts) from the opposite spindle pole.

Electron microscopy of spermatocytes previously studied in life: methods and some observations on micromanipulated chromosomes

1979 ◽  
Vol 35 (1) ◽  
pp. 87-104
Author(s):  
R.B. Nicklas ◽  
B.R. Brinkley ◽  
D.A. Pepper ◽  
D.F. Kubai ◽  
G.K. Rickards
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Target Cell ◽  
Living Cell ◽  
Chromosome Movement ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Direction Of Movement ◽  
Spindle Microtubules ◽  
Future Work

A new method is offered for combined living cell and electron-microscopic studies of spermatocytes (or other cells) which normally do not adhere to glass. The key step is micro-injection of glutaraldehyde near the target cell whenever desired during observation in life. Fixation begins and simultaneously the cell is stuck very firmly to the underlying coverslip. The method is easy and reliable: cells are almost never lost and are well preserved, except for membranes. The application of the method is illustrated by studies of micromanipulated grasshopper spermatocytes. A chromosome was detached from the spindle and placed in the cytoplasm. Before or after the beginning of chromosome movement back toward the spindle, the cell was fixed, sectioned and the manipulated chromosome observed in the electron microscope. If the detached chromosome had not moved by the time of fixation, no or only one or two microtubules were seen at its kinetochore, but if movement had occurred, a few microtubules were always present. The arrangement of these microtubules corresponded to the direction of movement, but they commonly were at an unusual angle relative to the kinetochore. The origin and role in chromosome movement of the microtubules seen near moving chromosomes far from the spindle is not yet established, but a speculation is offered. A goal for future work is the detailed analysis of the microtubules associated with individual moving chromosomes. Such an analysis is feasible because the moving chromosome is far removed from the confusing mass of spindle microtubules, and its value is enhanced because the direction of movement at the time of fixation is known.

scholarly journals Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle

2014 ◽  
Vol 25 (25) ◽  
pp. 4034-4048 ◽  
Author(s):  
Natalie J. Nannas ◽  
Eileen T. O’Toole ◽  
Mark Winey ◽  
Andrew W. Murray
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Simple Model ◽  
Mitotic Spindle ◽  
Cell Types ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Poles ◽  
Length Regulation ◽  
Spindle Microtubules ◽  
Different Cell Types

The length of the mitotic spindle varies among different cell types. A simple model for spindle length regulation requires balancing two forces: pulling, due to micro­tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles. In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore–microtubule interactions generate an inward force to balance forces that elongate the spindle. Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number.

scholarly journals Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum.

1984 ◽  
Vol 99 (1) ◽  
pp. 155-165 ◽  
Author(s):  
T Schedl ◽  
T G Burland ◽  
K Gull ◽  
W F Dove
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Microscopic Analysis ◽  
Nucleic Acid Hybridization ◽  
Electron Microscopic ◽  
Two Dimensional Gel Electrophoresis ◽  
Spindle Microtubules ◽  
Beta 2

The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.

scholarly journals Odd chromosome movement and inaccurate chromosome distribution in mitosis and meiosis after treatment with protein kinase inhibitors

1993 ◽  
Vol 104 (4) ◽  
pp. 961-973 ◽  
Author(s):  
R.B. Nicklas ◽  
L.E. Krawitz ◽  
S.C. Ward
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Chromosome Movement ◽  
Chromosome Distribution ◽  
Protein Kinase Inhibition ◽  
Spindle Microtubules ◽  
Mitosis And Meiosis ◽  
Normal Mitosis

Errors in chromosome orientation in mitosis and meiosis are inevitable, but normally they are quickly corrected. We find that such errors usually are not corrected in cells treated with protein kinase inhibitors. Highly inaccurate chromosome distribution is the result. When grasshopper spermatocytes were treated with the kinase inhibitor 6-dimethylaminopurine (DMAP), 84% of maloriented chromosomes failed to reorient; in anaphase, both partner chromosomes were distributed to the same daughter cell. These chromosomes were observed for a total of over 60 h, and not a single reorientation was seen. In contrast, in untreated cells, maloriented chromosomes invariably reoriented, and quickly: in 10 min, on average. A second protein kinase inhibitor, genistein, had exactly the same effect as DMAP. DMAP affected PtK1 cells in mitosis as it did spermatocytes in meiosis: improper chromosome orientations persisted, leading to frequent errors in distribution. We micromanipulated chromosomes in spermatocytes treated with DMAP to learn why maloriented chromosomes often fail to reorient. Reorientation requires the loss of improper microtubule attachments and the acquisition of new, properly directed kinetochore microtubules. Micromanipulation experiments disclose that neither the loss of old nor the acquisition of new microtubules is sufficiently affected by DMAP to account for the indefinite persistence of malorientations. Drug treatment causes a novel form of chromosome movement in which one kinetochore moves toward another kinetochore. Two kinetochores in the same chromosome or in different chromosomes can participate, producing varied, dance-like movements executed by one or two chromosomes. These kinetochore-kinetochore interactions evidently are at the expense of kinetochore-spindle interactions. We propose that malorientations persist in treated cells because the kinetochores have numerous, short microtubules with a free end that can be captured by a second kinetochore. Kinetochores capture each other's kinetochore microtubules, leaving too few sites available for the efficient capture of spindle microtubules. Since the efficient capture of spindle microtubules is essential for the correction of errors, failure of capture allows malorientations to persist. Whether the effects of DMAP actually are due to protein kinase inhibition remains to be seen. In any case, DMAP reveals interactions of one kinetochore with another, which, though ordinarily suppressed, have implications for normal mitosis.

scholarly journals Mechanical and genetic separation of aster- and midzone-positioned cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 381-383 ◽  
Author(s):  
Henrik Bringmann
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mitotic Spindle ◽  
Heterotrimeric G Proteins ◽  
Contractile Ring ◽  
Spindle Poles ◽  
Spindle Midzone ◽  
Spindle Microtubules ◽  
Two Populations ◽  
Provided Examples

The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.

Using Electron Tomography to Determine How Kinetochores Bind and Alter the Conformations of Microtubule Plus Ends

2001 ◽  
Vol 7 (S2) ◽  
pp. 86-87
Author(s):  
B.F. McEwen ◽  
R.M. Barnard ◽  
C-E. Hsieh ◽  
J. Frank
Keyword(s):  
Electron Tomography ◽  
Unique Form ◽  
Primary Constriction ◽  
Polar Polymers ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
Chromosome Motion ◽  
Mitosis And Meiosis ◽  
Delay Progression ◽  
Cellular Organelles

Chromosome alignment during mitosis and meiosis is mediated through the interaction between kinetochores and spindle microtubules (Mts). Kinetochores are fibrous mat-like structures, located at the primary constriction of chromosomes, that bind spindle Mts, generate poleward chromosome motion, and delay progression through mitosis until chromosome alignment is complete. Mts are polar polymers that function as tracks for the movement of a variety of cellular organelles and vesicles. Chromosome alignment requires a unique form of Mt-based motion because kinetochores capture and bind the plus ends of a bundle of Mts, rather than moving along the lateral surface of a single Mt . As a result, the direction of chromosome motion must be coordinated with the growth and shrinkage of Mts. Furthermore, the kinetochore maintains continuous attachment to growing and shrinking Mts and simultaneously permits the addition and dissociation of tubulin subunits within its confines

THE MITOTIC SPINDLE APPARATUS OF NEUROSPORA CRASSA

1971 ◽  
Vol 13 (4) ◽  
pp. 873-887 ◽  
Author(s):  
W. Barry Van Winkle ◽  
John J. Biesele ◽  
R. P. Wagner
Keyword(s):  
Neurospora Crassa ◽  
Mitotic Spindle ◽  
Nuclear Division ◽  
External Surface ◽  
Fungal Species ◽  
Electron Microscopic ◽  
Spindle Apparatus ◽  
Microscopic Studies ◽  
Microtubule Growth ◽  
Spindle Microtubules

Electron microscopic studies of the sl-327 mutant of Neurospora crassa reveal that somatic nuclear division in hyphlet somatic cells involves a definite spindle apparatus consisting of spindle microtubules terminating at specific loci on the external surface of the nuclear envelope. These loci, termed spindle plaques, have been observed in a variety of other fungal species and seem to play a role in the initiation of spindle microtubule growth during mitosis. The development of the various spindle components and their possible functions in nuclear division in this strain of Neurospora are discussed.

scholarly journals Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Kimberly K. Fong ◽  
Trisha N. Davis ◽  
Charles L. Asbury
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Force Generation ◽  
Initial Contact ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules ◽  
Pole Component

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.

scholarly journals An Asp–CaM complex is required for centrosome–pole cohesion and centrosome inheritance in neural stem cells

2015 ◽  
Vol 211 (5) ◽  
pp. 987-998 ◽  
Author(s):  
Todd Schoborg ◽  
Allison L. Zajac ◽  
Carey J. Fagerstrom ◽  
Rodrigo X. Guillen ◽  
Nasser M. Rusan
Keyword(s):  
Stem Cells ◽  
Cell Polarity ◽  
Mitotic Spindle ◽  
Function Analysis ◽  
Tissue Homeostasis ◽  
Cell Cortex ◽  
Cross Link ◽  
Spindle Poles ◽  
Spindle Microtubules ◽  
Spindle Function

The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, our understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. Here we report that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Our binding and structure–function analysis support this hypothesis. Disruption of the Asp–CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. We further show that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, we provide evidence that CaM is required for Asp’s spindle function; however, it is completely dispensable for Asp’s role in microcephaly suppression.

Sign in / Sign up

Export Citation Format

Share Document