scholarly journals Electron tomography reveals aspects of spindle structure important for mechanical stability at metaphase

2020 ◽  
Vol 31 (3) ◽  
pp. 184-195 ◽  
Author(s):  
Eileen O’Toole ◽  
Mary Morphew ◽  
J. Richard McIntosh
Keyword(s):  
Mechanical Stability ◽  
Electron Tomography ◽  
Mitotic Spindles ◽  
Metaphase Chromosomes ◽  
Spindle Structure

Electron tomography of mitotic spindles reveals connections that link kinetochore-associated microtubules with other microtubules that cross the spindle midplane, providing support for the tension that acts on metaphase chromosomes.

3-D reconstruction and analysis of mammalian mitotic spindle structure

1992 ◽  
Vol 50 (1) ◽  
pp. 578-579
Author(s):  
Kent McDonald ◽  
David Mastronarde ◽  
Rubai Ding ◽  
Eileen O'Toole ◽  
J. Richard McIntosh
Keyword(s):  
Cross Sections ◽  
Mitotic Spindle ◽  
Experimental Studies ◽  
Video Camera ◽  
Three Dimensions ◽  
Mitotic Spindles ◽  
Black And White ◽  
Reconstruction Methods ◽  
Spindle Structure ◽  
Tissue Culture Line

Mammalian spindles are generally large and may contain over a thousand microtubules (MTs). For this reason they are difficult to reconstruct in three dimensions and many researchers have chosen to study the smaller and simpler spindles of lower eukaryotes. Nevertheless, the mammalian spindle is used for many experimental studies and it would be useful to know its detailed structure.We have been using serial cross sections and computer reconstruction methods to analyze MT distributions in mitotic spindles of PtK cells, a mammalian tissue culture line. Images from EM negatives are digtized on a light box by a Dage MTI video camera containing a black and white Saticon tube. The signal is digitized by a Parallax 1280 graphics device in a MicroVax III computer. Microtubules are digitized at a magnification such that each is 10-12 pixels in diameter.

scholarly journals Mutations at the asp locus of Drosophila lead to multiple free centrosomes in syncytial embryos, but restrict centrosome duplication in larval neuroblasts

1990 ◽  
Vol 96 (4) ◽  
pp. 605-616 ◽  
Author(s):  
C. Gonzalez ◽  
R.D. Saunders ◽  
J. Casal ◽  
I. Molina ◽  
M. Carmena ◽  
...  
Keyword(s):  
Mitotic Index ◽  
Centrosome Duplication ◽  
Mitotic Spindles ◽  
Female Meiosis ◽  
Metaphase Chromosomes ◽  
Meiotic Spindles ◽  
Spindle Microtubules ◽  
As If

Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilised eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilised homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularisation, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure.

scholarly journals Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

2019 ◽  
Author(s):  
K. Tanuj Sapra ◽  
Zhao Qin ◽  
Anna Dubrovsky-Gaupp ◽  
Ueli Aebi ◽  
Daniel J. Müller ◽  
...  
Keyword(s):  
Mechanical Response ◽  
Mechanical Stability ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Small World ◽  
Nuclear Lamina ◽  
Integrative Approach ◽  
Graph Theory Analysis ◽  
Dynamics Simulations

AbstractThe nuclear lamina – a meshwork of intermediate filaments termed lamins – functions as a mechanotransduction interface between the extracellular matrix and the nucleus via the cytoskeleton. Although lamins are primarily responsible for the mechanical stability of the nucleus in multicellular organisms, in situ characterization of lamin filaments under tension has remained elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in its three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior – they deform reversibly under a force of a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough, similar to natural silk and superior to the synthetic polymer Kevlar®. Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but the meshwork topology follows ‘small world’ properties. Our results suggest that the lamin filaments arrange to form a robust, emergent meshwork that dictates the mechanical properties of individual lamin filaments. The combined approach provides quantitative insights into the structure-function organization of lamins in situ, and implies a role of meshwork topology in laminopathies.

Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis

1992 ◽  
Vol 102 (3) ◽  
pp. 401-416 ◽  
Author(s):  
M.A. Jordan ◽  
D. Thrower ◽  
L. Wilson
Keyword(s):  
Hela Cells ◽  
Mitotic Arrest ◽  
Mitotic Spindles ◽  
Microtubule Depolymerization ◽  
Metaphase Chromosomes ◽  
Low Concentrations ◽  
Subtle Effect ◽  
Full Complement ◽  
Spindle Microtubules

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.

Mitotic spindles of Drosophila melanogaster: a phase-contrast and scanning electron-microscope study

1977 ◽  
Vol 23 (1) ◽  
pp. 43-55
Author(s):  
A. Milsted ◽  
W.D. Cohen ◽  
N. Lampen
Keyword(s):  
Drosophila Melanogaster ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Phase Contrast ◽  
Mitotic Spindles ◽  
Scanning Electron Microscope Study ◽  
Cytoplasmic Material ◽  
Spindle Structure ◽  
Scanning Electron

Mitotic spindles have been isolated from the blastema stage of Drosophila melanogaster embryos using modified tubulin-polymerizing medium. ‘Clean’ spindles, relatively free of contaminating cytoplasmic material, are obtained. Under phase contrast, mitotic stages appear remarkably similar to those seen in situ, as reported in early literature. This preservation of morphological integrity, coupled with relative structural simplicity due to low chromosome number (2n = 8), makes these spindles ideal subjects for study. Use of the scanning electron microscope provides excellent visulization of their general structural organization, changes in whole spindle structure during the course of mitosis, and higher resolution viewing of surface detail than is permitted with light microscopy.

scholarly journals C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

2016 ◽  
Author(s):  
Stefanie Redemann ◽  
Johannes Baumgart ◽  
Norbert Lindow ◽  
Sebastian Fürthauer ◽  
Ehssan Nazockdast ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Large Scale ◽  
Cell Imaging ◽  
Electron Tomography ◽  
Mitotic Spindles ◽  
C Elegans ◽  
Microtubule Growth ◽  
Segregation Of Chromosomes

AbstractThe mitotic spindle ensures the faithful segregation of chromosomes. To discover the nature of the crucial centrosome-to-chromosome connection during mitosis, we combined the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging. Using tomography, we reconstructed the positions of all microtubules in 3D, and identified their plus- and minus-ends. We classified them as kinetochore (KMTs), spindle (SMTs), or astral microtubules (AMTs) according to their positions, and quantified distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs are directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

scholarly journals Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
K. Tanuj Sapra ◽  
Zhao Qin ◽  
Anna Dubrovsky-Gaupp ◽  
Ueli Aebi ◽  
Daniel J. Müller ◽  
...  
Keyword(s):  
Mechanical Response ◽  
Mechanical Stability ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Small World ◽  
Nuclear Lamina ◽  
Integrative Approach ◽  
Graph Theory Analysis ◽  
Dynamics Simulations ◽  
Multicellular Organisms

AbstractThe nuclear lamina—a meshwork of intermediate filaments termed lamins—is primarily responsible for the mechanical stability of the nucleus in multicellular organisms. However, structural-mechanical characterization of lamin filaments assembled in situ remains elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior – they deform reversibly at a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough similar to natural silk and superior to the synthetic polymer Kevlar®. Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but exhibits small-world properties. Our results suggest that lamin filaments arrange to form an emergent meshwork whose topology dictates the mechanical properties of individual filaments. The quantitative insights imply a role of meshwork topology in laminopathies.

scholarly journals Self-organization of kinetochore-fibers in human mitotic spindles

2021 ◽  
Author(s):  
William Conway ◽  
Robert Kiewisz ◽  
Gunar Fabig ◽  
Colm P Kelleher ◽  
Hai-Yin Wu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
De Novo ◽  
Electron Tomography ◽  
Polarized Light ◽  
Eukaryotic Cell ◽  
The Self ◽  
Self Organization ◽  
Macromolecular Complex ◽  
Biophysical Modeling ◽  
Mitotic Spindles

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

scholarly journals Three-dimensional structure of the kinetochore-fibers in human mitotic spindles

2021 ◽  
Author(s):  
Robert Kiewisz ◽  
Gunar Fabig ◽  
William Conway ◽  
Daniel Needleman ◽  
Thomas Muller-Reichert
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Spindle Pole ◽  
Dimensional Structure ◽  
Biophysical Modeling ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Physical Linkage

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the spindle poles and the chromosomes. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers in detail, and we find that they exhibit remarkable variation. K-fibers display differences in circumference and KMT density along their length, with the pole-facing side showing a splayed-out appearance. We further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for models of KMT behavior and k-fiber self-organization as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2021). The presented data will also serve as a resource for further studies on mitosis in human cells.

Sign in / Sign up

Export Citation Format

Share Document