scholarly journals Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

2013 ◽  
Vol 24 (20) ◽  
pp. 3238-3250 ◽  
Author(s):  
Elena Nazarova ◽  
Eileen O'Toole ◽  
Susi Kaitna ◽  
Paul Francois ◽  
Mark Winey ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Dynamical Behavior ◽  
Three Dimensional ◽  
Spindle Assembly ◽  
Intermediate Step ◽  
High Angle ◽  
Normal Number ◽  
Spindle Poles ◽  
Evolutionarily Conserved ◽  
Microtubule Formation

During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles.

scholarly journals Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein, Eg5, and Microtubule Flux

2010 ◽  
Vol 21 (6) ◽  
pp. 979-988 ◽  
Author(s):  
Nan Ma ◽  
U. S. Tulu ◽  
Nick P. Ferenz ◽  
Carey Fagerstrom ◽  
Andrew Wilde ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Fiber Formation ◽  
Aurora A ◽  
Mitotic Progression ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
C Terminus ◽  
Assembly Factor ◽  
Microtubule Formation

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

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.

Practical electron tomography

1995 ◽  
Vol 53 ◽  
pp. 742-743
Author(s):  
C.L. Woodcock
Keyword(s):  
Electron Tomography ◽  
Tomographic Reconstruction ◽  
Three Dimensional ◽  
3D Shape ◽  
Computational Resources ◽  
Shape Of Particles

Despite the potential of the technique, electron tomography has yet to be widely used by biologists. This is in part related to the rather daunting list of equipment and expertise that are required. Thanks to continuing advances in theory and instrumentation, tomography is now more feasible for the non-specialist. One barrier that has essentially disappeared is the expense of computational resources. In view of this progress, it is time to give more attention to practical issues that need to be considered when embarking on a tomographic project. The following recommendations and comments are derived from experience gained during two long-term collaborative projects.Tomographic reconstruction results in a three dimensional description of an individual EM specimen, most commonly a section, and is therefore applicable to problems in which ultrastructural details within the thickness of the specimen are obscured in single micrographs. Information that can be recovered using tomography includes the 3D shape of particles, and the arrangement and dispostion of overlapping fibrous and membranous structures.

Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

1994 ◽  
Vol 52 ◽  
pp. 310-311
Author(s):  
M.B. Braunfeld ◽  
M. Moritz ◽  
B.M. Alberts ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Vital Role ◽  
Complex Nature ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Nucleation Sites ◽  
Dimensional Reconstruction ◽  
Organizing Center ◽  
Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

Three-Dimensional Nanoparticle Distribution and Local Curvature of Heterogeneous Catalysts Revealed by Electron Tomography

2007 ◽  
Vol 111 (31) ◽  
pp. 11501-11505 ◽  
Author(s):  
Edmund P. W. Ward ◽  
Timothy J. V. Yates ◽  
José-Jesús Fernández ◽  
David E. W. Vaughan ◽  
Paul A. Midgley
Keyword(s):  
Heterogeneous Catalysts ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Local Curvature ◽  
Nanoparticle Distribution

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

The Prospect of Three-Dimensional Induction Mapping Inside Magnetic Nanostructures by Combining Electron Holography with Electron Tomography

2004 ◽  
Vol 10 (S02) ◽  
pp. 1010-1011 ◽  
Author(s):  
Rafal E Dunin-Borkowski ◽  
Takeshi Kasama
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Magnetic Nanostructures ◽  
Electron Holography

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Nonlinear Dynamics and Application of the Four Dimensional Autonomous Hyper-Chaotic System

2014 ◽  
Author(s):  
Ge Kai ◽  
Wei Zhang
Keyword(s):  
Nonlinear Dynamics ◽  
Numerical Simulation ◽  
Differential Equations ◽  
Chaotic System ◽  
Dynamical Behavior ◽  
Three Dimensional ◽  
Phase Portraits ◽  
State Variables ◽  
Chaotic Motions ◽  
Finance System

In this paper, we establish a dynamic model of the hyper-chaotic finance system which is composed of four sub-blocks: production, money, stock and labor force. We use four first-order differential equations to describe the time variations of four state variables which are the interest rate, the investment demand, the price exponent and the average profit margin. The hyper-chaotic finance system has simplified the system of four dimensional autonomous differential equations. According to four dimensional differential equations, numerical simulations are carried out to find the nonlinear dynamics characteristic of the system. From numerical simulation, we obtain the three dimensional phase portraits that show the nonlinear response of the hyper-chaotic finance system. From the results of numerical simulation, it is found that there exist periodic motions and chaotic motions under specific conditions. In addition, it is observed that the parameter of the saving has significant influence on the nonlinear dynamical behavior of the four dimensional autonomous hyper-chaotic system.

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