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.

scholarly journals High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

1999 ◽  
Vol 10 (6) ◽  
pp. 2017-2031 ◽  
Author(s):  
Eileen T. O’Toole ◽  
Mark Winey ◽  
J. Richard McIntosh
Keyword(s):  
High Voltage ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Spindle Pole ◽  
Dimensional Structure ◽  
Microtubule Organizing Center ◽  
Median Length ◽  
Intact Cells ◽  
Voltage Electron ◽  
High Voltage Electron

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5–10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ∼150 nm. MTs growing from duplicated but not separated SPBs have a median length of ∼130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ∼300 nm and then decreases to ∼30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

scholarly journals Three-dimensional structure of the central mitotic spindle of Diatoma vulgare.

1979 ◽  
Vol 83 (2) ◽  
pp. 428-442 ◽  
Author(s):  
J R McIntosh ◽  
K L McDonald ◽  
M K Edwards ◽  
B M Ross
Keyword(s):  
Three Dimensional ◽  
Three Dimensions ◽  
Dimensional Structure ◽  
Mitotic Spindles ◽  
Stereo Images ◽  
Central Spindle ◽  
Spindle Poles ◽  
Length Changes ◽  
Spindle Elongation

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three-dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements.

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.

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 High-resolution three-dimensional probes of biomaterials and their interfaces

2012 ◽  
Vol 370 (1963) ◽  
pp. 1337-1351 ◽  
Author(s):  
Kathryn Grandfield ◽  
Anders Palmquist ◽  
Håkan Engqvist
Keyword(s):  
High Resolution ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Controlled Drug Release ◽  
Biological Tissues ◽  
Dimensional Structure ◽  
Bone Interface ◽  
Biomaterial Interfaces ◽  
Titania Coating

Interfacial relationships between biomaterials and tissues strongly influence the success of implant materials and their long-term functionality. Owing to the inhomogeneity of biological tissues at an interface, in particular bone tissue, two-dimensional images often lack detail on the interfacial morphological complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar length scale. Electron tomography (ET) can meet these challenges by enabling high-resolution three-dimensional imaging of biomaterial interfaces. In this article, we review the fundamentals of ET and highlight its recent applications in probing the three-dimensional structure of bioceramics and their interfaces, with particular focus on the hydroxyapatite–bone interface, titanium dioxide–bone interface and a mesoporous titania coating for controlled drug release.

scholarly journals Biophysical and biochemical investigations of RNA catalysis in the hammerhead ribozyme

1999 ◽  
Vol 32 (3) ◽  
pp. 241-284 ◽  
Author(s):  
William G. Scott
Keyword(s):  
Metal Ions ◽  
Conformational Change ◽  
Large Scale ◽  
Enzyme Inhibitor ◽  
Hammerhead Ribozyme ◽  
Three Dimensional ◽  
Chemical Mechanism ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Hammerhead Rna

1. How do ribozymes work? 2412. The hammerhead RNA as a prototype ribozyme 2422.1 RNA enzymes 2422.2 Satellite self-cleaving RNAs 2422.3 Hammerhead RNAs and hammerhead ribozymes 2443. The chemical mechanism of hammerhead RNA self-cleavage 2463.1 Phosphodiester isomerization via an SN2(P) reaction 2473.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 2513.3 The hammerhead ribozyme does not actually require metal ions for catalysis 2543.4 Hammerhead RNA enzyme kinetics 2574. Sequence requirements for hammerhead RNA self-cleavage 2604.1 The conserved core, mutagenesis and functional group modifications 2604.2 Ground-state vs. transition-state effects 2615. The three-dimensional structure of the hammerhead ribozyme 2625.1 Enzyme–inhibitor complexes 2625.2 Enzyme–substrate complex in the initial state 2645.3 Hammerhead ribozyme self-cleavage in the crystal 2645.4 The requirement for a conformational change 2655.5 Capture of conformational intermediates using crystallographic freeze-trapping 2665.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 2675.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 2685.8 Is the conformational change pH dependent? 2695.9 Isolating the structure of the cleavage product 2715.10 Evidence for and against additional large-scale conformation changes 2745.11 NMR spectroscopic studies of the hammerhead ribozyme 2786. Concluding remarks 2807. Acknowledgements 2818. References 2811. How do ribozymes work? 241The discovery that RNA can be an enzyme (Guerrier-Takada et al. 1983; Zaug & Cech, 1986) has created the fundamental question of how RNA enzymes work. Before this discovery, it was generally assumed that proteins were the only biopolymers that had sufficient complexity and chemical heterogeneity to catalyze biochemical reactions. Clearly, RNA can adopt sufficiently complex tertiary structures that make catalysis possible. How does the three- dimensional structure of an RNA endow it with catalytic activity? What structural and functional principles are unique to RNA enzymes (or ribozymes), and what principles are so fundamental that they are shared with protein enzymes?

scholarly journals Crystal structure of a tankyrase 1–telomere repeat factor 1 complex

2016 ◽  
Vol 72 (4) ◽  
pp. 320-327 ◽  
Author(s):  
Bo Li ◽  
Ruihong Qiao ◽  
Zhizhi Wang ◽  
Weihong Zhou ◽  
Xin Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Large Scale ◽  
Sparse Matrix ◽  
Critical Role ◽  
Three Dimensional ◽  
Mitotic Cell ◽  
Ankyrin Repeat ◽  
Dimensional Structure ◽  
Telomere Repeat ◽  
Tankyrase 1

Telomere repeat factor 1 (TRF1) is a subunit of shelterin (also known as the telosome) and plays a critical role in inhibiting telomere elongation by telomerase. Tankyrase 1 (TNKS1) is a poly(ADP-ribose) polymerase that regulates the activity of TRF1 through poly(ADP-ribosyl)ation (PARylation). PARylation of TRF1 by TNKS1 leads to the release of TRF1 from telomeres and allows telomerase to access telomeres. The interaction between TRF1 and TNKS1 is thus important for telomere stability and the mitotic cell cycle. Here, the crystal structure of a complex between the N-terminal acidic domain of TRF1 (residues 1–55) and a fragment of TNKS1 covering the second and third ankyrin-repeat clusters (ARC2-3) is presented at 2.2 Å resolution. The TNKS1–TRF1 complex crystals were optimized using an `oriented rescreening' strategy, in which the initial crystallization condition was used as a guide for a second round of large-scale sparse-matrix screening. This crystallographic and biochemical analysis provides a better understanding of the TRF1–TNKS1 interaction and the three-dimensional structure of the ankyrin-repeat domain of TNKS.

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 A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

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