Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis

P.R. Andreassen; D.K. Palmer; M.H. Wener; R.L. Margolis

doi:10.1242/jcs.99.3.523

Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis

Journal of Cell Science ◽

10.1242/jcs.99.3.523 ◽

1991 ◽

Vol 99 (3) ◽

pp. 523-534 ◽

Cited By ~ 12

Author(s):

P.R. Andreassen ◽

D.K. Palmer ◽

M.H. Wener ◽

R.L. Margolis

Keyword(s):

Confocal Microscopy ◽

Mammalian Cells ◽

Three Dimensional ◽

Temperature Treatment ◽

Cold Temperature ◽

Cell Cortex ◽

Membrane Association ◽

Normal Cells ◽

Binucleate Cells ◽

Late Anaphase

We have discovered a novel mitosis-specific human autoantigen that arises at the centromeres of prophase chromosomes, but ultimately participates in formation of an organelle that bisects the cell at late anaphase and during telophase. The organelle, discernible as a three-dimensional disc by confocal microscopy, encompasses the entire midzone diameter, and its distribution survives disassembly of interpolar microtubules by cold temperature treatment and detergent lysis of cells. Cytokinetic furrow contraction proceeds normally in dihydrocytochalasin B (DCB)-treated cells, and antigen distribution in the furrow is unaltered. In DCB, the furrow retracts in early interphase, coincident with loss of normal membrane association with the disc, resulting in the formation of binucleate cells. The midzone disc in both drug-treated and normal cells is present at the correct time and position to play a central role in cytokinesis. By immunocytochemistry, the disc appears to contain myosin but not actin. The position of the disc and the possible presence of myosin suggest that cytokinesis may involve the interaction of the disc organelle with actin in the cell cortex to produce cleavage in mammalian cells.

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Role of the Midbody Matrix in Cytokinesis: RNAi and Genetic Rescue Analysis of the Mammalian Motor Protein CHO1

Molecular Biology of the Cell ◽

10.1091/mbc.e03-12-0888 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3083-3094 ◽

Cited By ~ 33

Author(s):

Jurgita Matuliene ◽

Ryoko Kuriyama

Keyword(s):

Late Stage ◽

Cell Separation ◽

Mammalian Cells ◽

Motor Protein ◽

Cell Cortex ◽

Intercellular Bridge ◽

Genetic Rescue ◽

Binucleate Cells ◽

Complete Cell

CHO1 is a kinesin-like motor protein essential for cytokinesis in mammalian cells. To analyze how CHO1 functions, we established RNAi and genetic rescue assays. CHO1-depleted cells reached a late stage of cytokinesis but fused back to form binucleate cells because of the absence of the midbody matrix in the middle of the intercellular bridge. Expression of exogenous CHO1 restored the formation of the midbody matrix and rescued cytokinesis in siRNA-treated cells. By analyzing phenotypes rescued with different constructs, it was shown that both motor and stalk domains function in midbody formation, whereas the tail is essential for completion of cytokinesis after the midbody matrix has formed. During the terminal stage of cytokinesis, different subregions of the tail play distinctive roles in stabilizing the midbody matrix and maintaining an association between the midbody and cell cortex. These results demonstrate that CHO1 consists of functionally differentiated subregions that act in concert to ensure complete cell separation.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue

Microscopy and Microanalysis ◽

10.1017/s1431927600008412 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 305-306

Author(s):

David W. Piston

Keyword(s):

Confocal Microscopy ◽

Collection Efficiency ◽

Three Dimensional ◽

Spatial Filter ◽

Input Power ◽

Photon Absorption ◽

Focal Volume ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. It provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. This results in inherent submicron optical sectioning by excitation alone. In practice, TPEM is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume.

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Toxicity of Combinations of Kinase Pathway Inhibitors to Normal Human Cells in a Three-Dimensional Culture

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/24726303211008858 ◽

2021 ◽

pp. 247263032110088

Author(s):

Pouria Rafsanjani Nejad ◽

Pradip Shahi Thakuri ◽

Sunil Singh ◽

Astha Lamichhane ◽

Jacob Heiss ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Trials ◽

Protein Kinase ◽

Single Agent ◽

Three Dimensional ◽

Drug Combinations ◽

Toxic Effects ◽

Combination Drug ◽

Normal Cells ◽

Colon Cells

Resistance to single-agent chemotherapy and molecularly targeted drugs prevents sustained efficacy of treatments. To address this challenge, combination drug treatments have been used to improve outcomes for patients. Potential toxicity of combination treatments is a major concern, however, and has led to the failure of several clinical trials in different cancers. The use of cell-based models of normal tissues in preclinical studies enables testing and identifying toxic effects of drug combinations and facilitates an informed decision-making process for advancing the treatments to animal models and clinical trials. Recently, we established that combinations of molecular inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase–protein kinase B (PI3K/Akt) pathways effectively and synergistically inhibit growth of BRAFmut and KRASmut colorectal tumor spheroids by blocking feedback signaling of downstream kinase pathways. These pathways are important for cell proliferation, however, and their simultaneous inhibition may cause toxicity to normal cells. We used a cellular spheroid model to study toxicities of drug combinations to human bone marrow and colon. Our results indicated that MAPK and PI3K/Akt inhibitors used simultaneously were only moderately toxic to bone marrow cells but significantly more toxic to colon cells. Our molecular analysis of proliferative cell activities and housekeeping proteins further corroborated these results. Overall, our approach to identify toxic effects of combinations of cancer drugs to normal cells in three-dimensional cultures will facilitate more informed treatment selections for subsequent animal studies.

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Preparation of Porous Organically-Modified Silicate Hybrids for Cell Culture Matrix

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.330-332.1177 ◽

2007 ◽

Vol 330-332 ◽

pp. 1177-1180 ◽

Cited By ~ 3

Author(s):

Kanji Tsuru ◽

Satoshi Hayakawa ◽

Yuki Shirosaki ◽

T. Okayama ◽

K. Kataoka ◽

...

Keyword(s):

Cell Culture ◽

Mammalian Cells ◽

Pore Diameter ◽

Sol Gel ◽

Three Dimensional ◽

Culture Methods ◽

Organically Modified ◽

Dimensional Cell

Porous & rubbery organic-inorganic hybrids were synthesized from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) through a sol-gel route using sieved sucrose granules as a porogen. The porous hybrids with a high content of PDMS behaved like polymer sponge. The porosity was over 90% irrespective of the hybrid composition and the pore diameter ranged from 100 to 500 μm. In the three-dimensional cell culture, mammalian cells were well cultured in the porous hybrids. The present results indicate that the hybrids may be a promising scaffold for developing such functional culture methods.

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Three-dimensional ultrastructural imaging reveals the nanoscale architecture of mammalian cells

Microscopy and Microanalysis ◽

10.1017/s1431927621005778 ◽

2021 ◽

Vol 27 (S1) ◽

pp. 1566-1569

Author(s):

Shengkun Yao ◽

Jiadong Fan ◽

Zhiyun Chen ◽

Yunbing Zong ◽

Jianhua Zhang ◽

...

Keyword(s):

Mammalian Cells ◽

Three Dimensional

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When rDNA transcription is arrested during mitosis, UBF is still associated with non-condensed rDNA

Journal of Cell Science ◽

10.1242/jcs.110.19.2429 ◽

1997 ◽

Vol 110 (19) ◽

pp. 2429-2440 ◽

Cited By ~ 4

Author(s):

J. Gebrane-Younes ◽

N. Fomproix ◽

D. Hernandez-Verdun

Keyword(s):

Secondary Constriction ◽

Three Dimensional ◽

Ribosomal Gene ◽

Rdna Transcription ◽

Transcription Machinery ◽

Late Anaphase ◽

Early Anaphase ◽

Dimensional Distribution ◽

Open Question ◽

Kinetics Of

The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.

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Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.4128-4134.1991 ◽

1991 ◽

Vol 11 (8) ◽

pp. 4128-4134

Author(s):

J Venema ◽

A van Hoffen ◽

V Karcagi ◽

A T Natarajan ◽

A A van Zeeland ◽

...

Keyword(s):

Xeroderma Pigmentosum ◽

Mammalian Cells ◽

Excision Repair ◽

Complementation Group ◽

C Cells ◽

Dhfr Gene ◽

Normal Cells ◽

Pyrimidine Dimers ◽

Normal Human ◽

Active Genes

We have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

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