scholarly journals Spindle microtubules generate tension-dependent changes in the distribution of inner kinetochore proteins

2011 ◽  
Vol 193 (1) ◽  
pp. 125-140 ◽  
Author(s):  
Aussie Suzuki ◽  
Tetsuya Hori ◽  
Tatsuya Nishino ◽  
Jiro Usukura ◽  
Atsushi Miyagi ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Light Microscopy ◽  
Immunoelectron Microscopy ◽  
Mitotic Spindle ◽  
Structural Changes ◽  
Live Cell ◽  
Dynamic Interface ◽  
Spindle Microtubules ◽  
Kinetochore Region

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle. Live-cell light microscopy–based observations on the dynamic structural changes within the kinetochore suggest that molecular rearrangements within the kinetochore occur upon microtubule interaction. However, the source of these rearrangements is still unclear. In this paper, we analyze vertebrate kinetochore ultrastructure by immunoelectron microscopy (EM) in the presence or absence of tension from spindle microtubules. We found that the inner kinetochore region defined by CENP-A, CENP-C, CENP-R, and the C-terminal domain of CENP-T is deformed in the presence of tension, whereas the outer kinetochore region defined by Ndc80, Mis12, and CENP-E is not stretched even under tension. Importantly, based on EM, fluorescence microscopy, and in vitro analyses, we demonstrated that the N and C termini of CENP-T undergo a tension-dependent separation, suggesting that CENP-T elongation is at least partly responsible for changes in the shape of the inner kinetochore.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

scholarly journals ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO

1971 ◽  
Vol 50 (2) ◽  
pp. 416-431 ◽  
Author(s):  
B. R. Brinkley ◽  
Joiner Cartwright
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Spindle Axis ◽  
Hamster Strain ◽  
Early Anaphase ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Elongation Process

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK1) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.

scholarly journals Implication of a novel multiprotein Dam1p complex in outer kinetochore function

2001 ◽  
Vol 155 (7) ◽  
pp. 1137-1146 ◽  
Author(s):  
Iain M. Cheeseman ◽  
Christine Brew ◽  
Michael Wolyniak ◽  
Arshad Desai ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Phosphorylation Event ◽  
Spindle Microtubules ◽  
Green Fluorescent ◽  
Kinetochore Function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

O-154 First mitotic spindle formation led by sperm centrosome-dependent microtubule organising centres may cause high incidence of zygotic division errors in humans

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Kai ◽  
H Kawano ◽  
N Yamashita
Keyword(s):  
Mitotic Spindle ◽  
High Frequency ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Immunofluorescence Assay ◽  
Live Cell ◽  
Microtubule Assembly ◽  
Cell Stage ◽  
Spindle Formation

Abstract Study question Why do multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans? Summary answer Failure in microtubule assembly during the first mitotic spindle body formation by sperm centrosome-dependent microtubule organising centres (MTOCs) may lead to chromosomal instability. What is known already Unlike that in mice, multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans. However, the underlying mechanism remains elusive. In mice, multiple acentriolar MTOCs appear around the male and female pronuclei after pronuclear disappearance and contribute to dual-spindle formation, engulfing each parental chromosome. This spindle formation may ensure an error-free division, keeping the chromosomes stable during the first cleavage, as observed in mice, but it is unclear whether a similar mechanism exists in humans. Study design, size, duration To examine how sperm centrosomes contribute to MTOC formation in humans, two types of 3PN zygotes derived fromeither conventional in vitro fertilization (c-IVF, n = 30) or intracytoplasmic sperm injection (ICSI, n = 10) were used. The zygotes were collected from October 2018 to January 2020. MTOC and mitotic spindle formation at consecutive stages of development during the first cleavage were analysed under static and dynamic conditions using immunofluorescence assay and fluorescent live-cell imaging. Participants/materials, setting, methods Under ethics approval, 3PN zygotes were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. All participants provided informed consent. Immunofluorescence assay was performed using antibodies against α-tubulin, pericentrin, and H3K9me3 after fixation with MTSB-XF solution. Fluorescent live-cell imaging was performed using TagGFP2-H2B mRNA (chromosome marker) and FusionRed-MAP4 mRNA (microtubule marker). Main results and the role of chance Immunofluorescence revealed that while 3PN zygotes derived from c-IVF showed four pericentrin dots, those derived from ICSI exhibited two pericentrin dots. In pro-metaphase, an independent group of chromosomes derived from each pronucleus and MTOCs were formed by the sperm centrosome at the core. Microtubules from each MTOC extended toward the chromosomes in the early metaphase; a quadrupolar spindle was formed in the c-IVF-derived zygotes, and a bipolar spindle was formed in the ICSI-derived zygotes by the MTOCs at the zygote apex after chromosome alignment. In pro-metaphase, the microtubules extended from the MTOCs to the nearest chromosome. Since microtubule assembly was found on oocyte-derived chromosomes, we hypothesised that whether a chromosome is surrounded by microtubules depends on the location of the MTOCs, irrespective of its origin. Live-cell imaging of histone H2B and MAP4 revealed that four MTOCs appeared around the three pronuclei just before the disappearance of the pronuclear membrane; microtubules then extended from the MTOCs toward the chromosomes, beginning to form a mitotic spindle as the chromosomes moved to the centre of the oocyte. Interestingly, one of the three assembled chromosome groups showed no microtubule assembly in the pro-metaphase. Similar results were obtained in all six 3PN zygotes subjected. Limitations, reasons for caution We demonstrated the high risk of developing bare chromosomes not surrounded by microtubules during the formation of the first mitotic spindle, using human tripronuclear zygotes. However, owing to unavailability of normal fertilized oocytes for this study because of the clinical use, we were unable to confirm this in normal zygotes. Wider implications of the findings Although two sperm centrosome-dependent MTOCs are expected to be formed in normal fertilized oocytes, these MTOCs are not sufficient to completely enclose physically separated female and male chromosomes with the microtubules. This explains the high frequency of zygotic division errors that lead to unstable human chromosomes. Trial registration number not applicable

scholarly journals Mechanical impact stimulation platform tailored for high-resolution light microscopy

2019 ◽  
Vol 10 (1) ◽  
pp. 87-99 ◽  
Author(s):  
Heidi T. Halonen ◽  
Jari A.K. Hyttinen ◽  
Teemu O. Ihalainen
Keyword(s):  
High Resolution ◽  
Epithelial Cells ◽  
Light Microscopy ◽  
Mechanical Stimulation ◽  
Cellular Response ◽  
Mechanical Vibration ◽  
Stem Cell Differentiation ◽  
Live Cell ◽  
Mechanical Stimuli

Abstract High frequency (HF) mechanical vibration has been used in vitro to study the cellular response to mechanical stimulation and induce stem cell differentiation. However, detailed understanding of the effect of the mechanical cues on cellular physiology is lacking. To meet this limitation, we have designed a system, which enables monitoring of living cells by high-resolution light microscopy during mechanical stimulation by HF vibration or mechanical impacts. The system consists of a commercial speaker, and a 3D printed sample vehicle and frame. The speaker moves the sample in the horizontal plane, allowing simultaneous microscopy. The HF vibration (30–200 Hz) performances of two vehicles made of polymer and aluminum were characterized with accelerometer. The mechanical impacts were characterized by measuring the acceleration of the aluminum vehicle and by time lapse imaging. The lighter polymer vehicle produced higher HF vibration magnitudes at 30–50 Hz frequencies than the aluminum vehicle. However, the aluminum vehicle performed better at higher frequencies (60–70 Hz, 90–100 Hz, 150 Hz). Compatibility of the system in live cell experiments was investigated with epithelial cells (MDCKII, expressing Emerald-Occludin) and HF (0.56 Gpeak, 30 Hz and 60 Hz) vibration. Our findings indicated that our system is compatible with high-resolution live cell microscopy. Furthermore, the epithelial cells were remarkable stable under mechanical vibration stimulation. To conclude, we have designed an inexpensive tool for the studies of cellular biophysics, which combines versatile in vivo like mechanical stimuli with live cell imaging, showing a great potential for several cellular applications.

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 Organization of Microtubules and Endoplasmic Reticulum During Mitosis and Cytokinesis in Wheat Meristems

1966 ◽  
Vol 1 (1) ◽  
pp. 109-120
Author(s):  
J. D. PICKETT-HEAPS ◽  
D. H. NORTHCOTE
Keyword(s):  
Endoplasmic Reticulum ◽  
Mitotic Spindle ◽  
Structural Changes ◽  
Preprophase Band ◽  
Cell Plate ◽  
Late Prophase ◽  
Daughter Cells ◽  
Initial Stage ◽  
Spindle Microtubules ◽  
Mother Cell Wall

The fine-structural changes accompanying mitosis in meristematic cells of the roots and coleoptile tissue of wheat have been studied. A band of microtubules encircling the nucleus appeared in the cytoplasm before the cells entered prophase. These microtubules were oriented at right angles to the direction of the mitotic spindle and were located at the position on the mother cell wall where the future cell plate dividing the daughter cells would have joined it. During prophase the number of microtubules in this preprophase band decreased and eventually disappeared, while microtubules were found to be aligned along the spindle axis. These spindle microtubules appeared as a cone-shaped array of units radiating from the polar zones of the spindle and passing very close tangentially to the nucleus. At late prophase they penetrated the disintegrating nuclear envelope and were seen between the chromosomes. During metaphase and anaphase many microtubules were present running throughout the length of the spindle, and others were found to be attached to chromosomes. Paired sister chromosomes were found joined to microtubules from opposite poles of the spindle. The position and orientation of the lamellae of the endoplasmic reticulum which invaded the spindle from the two poles was closely related to the position and alignment of the microtubules. During the formation of the cell plate vesicles were seen to be collected between the microtubules. As the vesicles fused to form the plate the microtubules were found only at its growing edge, where the vesicles were still being aligned. At the initial stage of its formation the microtubules passed right through the plate, but as it extended they appeared to end at the plate region. The results of the investigation are discussed in relation to the descriptions of mitosis and cytokinesis based on optical microscopy of living cells.

Microtubule motors associated with kinetochores in mitotic cells

1993 ◽  
Vol 51 ◽  
pp. 76-77
Author(s):  
Linda Wordeman
Keyword(s):  
Mitotic Spindle ◽  
Centromere Region ◽  
Mitotic Chromosomes ◽  
Directed Movement ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Microtubule Motors ◽  
Double Label ◽  
Spindle Microtubules ◽  
Kinetochore Region

Chromosomes in dividing tissue culture cells exhibit three types of movement along mitotic spindle microtubules: l)Fast minus-end directed movement (prometaphase), 2)Plus-end directed movement, and 3) Slow minus-end directed movement (anaphase) . In all cases these movements are mediated by the kinetochore region of the centromere of mitotic chromosomes. This region consists of three domains based on both immunocytochemistry and electron microscopy. The outermost or kinetochore domain is composed of the distal fibrous corona and trilamminar plate. The central and pairing domains are located in the chromatin beneath the kinetochore. Both plus- and minus-end directed microtubule motors have been localized to the kinetochore region of mitotic CHO chromosomes. I have used double-label immunocytochemistry to map the location of these motors within the centromere region at the level of the light microscope. Furthermore, I have cloned and expressed a number of novel kinesin-related motors, two of which (Clone 26 and Clone 14) are localized to kinetochores and kinetochore microtubules, respectively.

Differential Lability of Spindle Microtubules in Crane-Fly Spermatocytes

1977 ◽  
Vol 35 ◽  
pp. 458-459
Author(s):  
Marie A. Janicke ◽  
James R. LaFountain
Keyword(s):  
Epoxy Resin ◽  
Mitotic Spindle ◽  
Osmium Tetroxide ◽  
Cold Shock ◽  
Ringer’S Solution ◽  
Crane Flies ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules

A well-established property of cytoplasmic microtubules is that they rapidly depolymerize in vivo and in vitro when the temperature of the medium is dropped to 0-4°C. In the case of the mitotic spindle, which contains hundreds of microtubules, it has been shown that microtubules attached to the kinetochores of chromosomes appear to be more stable to cold shock than are the more numerous non-kinetochore microtubules, which are not attached to chromosomes (1). These results led us to inquire into the possible universality of differential lability of spindle microtubules as well as to speculate on possible factors that could give rise to this phenomenon.Our approach was to incubate spermatocytes of crane flies (Nephrotoma suturalis) in cold Ringer's solution, fix them in cold, buffered glutaralde- hyde at intervals of 7, 15, 30 and 60 minutes after the start of cold shock, post-fix in osmium tetroxide and then process them for embedding in epoxy resin.

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