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.

Structure-function analysis of anaphase spindle elongation using isolated diatom spindles

1991 ◽  
Vol 49 ◽  
pp. 206-207
Author(s):  
W. Z. Cande ◽  
C.J. Hogan ◽  
M. Lee
Keyword(s):  
Morphological Changes ◽  
Function Analysis ◽  
Light And Electron Microscopy ◽  
Near Neighbor ◽  
Model Systems ◽  
Central Spindle ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Anaphase B

Diatom spindles are important model systems for describing the morphological changes associated with anaphase chromosome movement because the fibrous systems responsible for anaphase A (chromosome-to-pole movement) and anaphase B (spindle elongation) are spatially separate and the central spindle is a paracrystalline array of microtubules. The diatom central spindle, which is responsible for anaphase B, is constructed of two sets of interdigiting microtubules that originate from plate-like spindle poles and display specific near-neighbor interactions in the zone of microtubule overlap. The microtubules of each half-spindle are of relatively unifrom length such that the plus ends are clustered together in narrow zones at each edge of the zone of microtubule overlap. This has allowed us to monitor changes in extent of microtubule overlap in the light microscope with polarization optics. We have isolated spindles from synchronized populations of several species of dividing diatom cells to study the mechanochemistry of anaphase spindle elongation in vitro and to analyze the rearrangement of spindle components by light and electron microscopy during reactivation.

scholarly journals Physiological and ultrastructural analysis of elongating mitotic spindles reactivated in vitro.

1986 ◽  
Vol 103 (2) ◽  
pp. 593-604 ◽  
Author(s):  
W Z Cande ◽  
K McDonald
Keyword(s):  
Cross Sections ◽  
Simple Procedure ◽  
Ultrastructural Level ◽  
Mitotic Spindles ◽  
Gap Formation ◽  
Microtubule Depolymerization ◽  
Central Spindle ◽  
Original Number ◽  
Spindle Elongation

We have developed a simple procedure for isolating mitotic spindles from the diatom Stephanopyxis turris and have shown that they undergo anaphase spindle elongation in vitro upon addition of ATP. The isolated central spindle is a barrel-shaped structure with a prominent zone of microtubule overlap. After ATP addition greater than 75% of the spindle population undergoes distinct structural rearrangements: the spindles on average are longer and the two half-spindles are separated by a distinct gap traversed by only a small number of microtubules, the phase-dense material in the overlap zone is gone, and the peripheral microtubule arrays have depolymerized. At the ultrastructural level, we examined serial cross-sections of spindles after 1-, 5-, and 10-min incubations in reactivation medium. Microtubule depolymerization distal to the poles is confirmed by the increased number of incomplete, i.e., c-microtubule profiles specifically located in the region of overlap. After 10 min we see areas of reduced microtubule number which correspond to the gaps seen in the light microscope and an overall reduction in the number of half-spindle microtubules to about one-third the original number. The changes in spindle structure are highly specific for ATP, are dose-dependent, and do not occur with nonhydrolyzable nucleotide analogues. Spindle elongation and gap formation are blocked by 10 microM vanadate, equimolar mixtures of ATP and AMPPNP, and by sulfhydryl reagents. This process is not affected by nocodazole, erythro-9-[3-(2-hydroxynonyl)]adenine, cytochalasin D, and phalloidin. In the presence of taxol, the extent of spindle elongation is increased; however, distinct gaps still form between the two half-spindles. These results show that the response of isolated spindles to ATP is a complex process consisting of several discrete steps including initiation events, spindle elongation mechanochemistry, controlled central spindle microtubule plus-end depolymerization, and loss of peripheral microtubules. They also show that the microtubule overlap zone is an important site of ATP action and suggest that spindle elongation in vitro is best explained by a mechanism of microtubule-microtubule sliding. Spindle elongation in vitro cannot be accounted for by cytoplasmic forces pulling on the poles or by microtubule polymerization.

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 Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2273
Author(s):  
Wan-Ying Huang ◽  
Norichika Hashimoto ◽  
Ryuhei Kitai ◽  
Shin-ichiro Suye ◽  
Satoshi Fujita
Keyword(s):  
Three Dimensional ◽  
Hollow Structure ◽  
Dimensional Structure ◽  
The Novel ◽  
Fibrous Scaffold ◽  
Hydrogel Beads ◽  
Inner Surface ◽  
Intracranial Epidermoid ◽  
Hollow Ball

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the “nanofiber-mâché ball.” This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

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 Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

scholarly journals Based on systems science methodology—— Research on the talent training system of hall three-dimensional structure

2020 ◽  
Vol 198 ◽  
pp. 01050
Author(s):  
Yong-Chen ◽  
Zhong Hua-Cheng ◽  
Qi-Xu
Keyword(s):  
Three Dimensional ◽  
Training System ◽  
Three Dimensions ◽  
Structure Theory ◽  
Dimensional Structure ◽  
Systems Science ◽  
Three Dimension ◽  
Time Dimension ◽  
Dimension Analysis ◽  
Science Methodology

Draw lessons from the basic idea of system science methodology, based on the hall three dimensions structure theory construction of research personnel training problems, from the time dimension, logic knowledge and three dimension analysis of all aspects of talent cultivation system, clear the talent training theory support, and solve the problem of a series of contradictions in the process of talent training, further enriched and developed the theoretical system of talents cultivation.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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