The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised

2002 ◽  
Vol 115 (14) ◽  
pp. 2847-2856 ◽  
Author(s):  
Jun-yong Huang ◽  
Jordan W. Raff
Keyword(s):  
Fusion Proteins ◽  
Cyclin B ◽  
Subcellular Localisation ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Mitotic Chromosomes ◽  
Nuclear Envelope Breakdown ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Drosophila Cells

In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularised embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos,only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction,but its subcellular localisation remains controversial. We constructed GFP fusions of two core APC/C subunits, Cdc16 and Cdc27. These fusion proteins were incorporated into the endogenous APC/C and were largely localised in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulated in the nucleus prior to nuclear envelope breakdown but only weakly associated with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly,we noticed that GFP-Cdc27 associated with mitotic chromosomes, whereas GFP-Cdc16 did not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90%in tissue culture cells led to a transient mitotic arrest that was both biochemically and morphologically distinct. Taken together, our results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localised and perform distinct functions.

Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells

1993 ◽  
Vol 51 ◽  
pp. 342-343
Author(s):  
Heide Schatten ◽  
Neidhard Paweletz ◽  
Ron Balczon
Keyword(s):  
Tissue Culture ◽  
Cell Cycle Progression ◽  
Group Formation ◽  
Sulfhydryl Group ◽  
Mammalian Tissue ◽  
Mitotic Chromosomes ◽  
Microtubule Network ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Transmission Electron

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.

Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster

1997 ◽  
Vol 110 (17) ◽  
pp. 1979-1988 ◽  
Author(s):  
H. Bousbaa ◽  
L. Correia ◽  
G.J. Gorbsky ◽  
C.E. Sunkel
Keyword(s):  
Drosophila Melanogaster ◽  
Metaphase Plate ◽  
Cell Types ◽  
Early Prophase ◽  
Mitotic Chromosomes ◽  
Microtubule Inhibitors ◽  
Phosphatase Inhibitor ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Late Telophase

The progression of cells from metaphase to anaphase is thought to be regulated by a checkpoint that delays entry into anaphase until all chromosomes reach a stable bi-polar attachment at the metaphase plate. Previous work has suggested that the 3F3/2 kinetochore phosphoepitopes are involved in this checkpoint system. We show that the 3F3/2 centromere phosphoepitopes are present in Kc cells, third instar larval neuroblasts and isolated chromosomes of Drosophila melanogaster. In tissue culture cells and neuroblasts isolated from third instar larvae, centromere labelling is detected from early prophase to the metaphase-anaphase transition but absent once cells center anaphase. During anaphase, the antibody stains the spindle mid zone and during telophase the midbody is labelled until cells separate. In both cell types, the 3F3/2 antibody stains the centrosome from prophase to late telophase. The 3F3/2 staining is retained in Kc cells and third instar larval neuroblasts arrested at the prometaphase state with microtubule inhibitors. Also, two mitotic mutants that show abnormal spindle morphology retain the centromere labelling in a metaphase-like configuration, suggesting that they activate the metaphase-anaphase checkpoint. Finally, mitotic chromosomes isolated in the presence of a phosphatase inhibitor show phosphoepitopes at the primary constriction on the surface of each chromatid, however, chromosomes isolated in the absence of a phosphatase inhibitor do not. Incubation of these chromosomes with ATP causes the rephosphorylation of the phosphoepitopes at the centromere.

scholarly journals Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line.

1982 ◽  
Vol 93 (3) ◽  
pp. 576-582 ◽  
Author(s):  
J V Kilmartin ◽  
B Wright ◽  
C Milstein
Keyword(s):  
Monoclonal Antibodies ◽  
Myeloma Cell ◽  
Yeast Cells ◽  
Immunofluorescent Staining ◽  
Mitotic Spindles ◽  
Spleen Cells ◽  
Microtubule Network ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Parental Lines

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.

scholarly journals The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

2008 ◽  
Vol 183 (2) ◽  
pp. 267-277 ◽  
Author(s):  
Evan C. Osmundson ◽  
Dipankar Ray ◽  
Finola E. Moore ◽  
Qingshen Gao ◽  
Gerald H. Thomsen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
E3 Ligase ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Carboxyl Terminus ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Anaphase Onset

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

scholarly journals Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

1995 ◽  
Vol 128 (5) ◽  
pp. 849-862 ◽  
Author(s):  
K Ookata ◽  
S Hisanaga ◽  
J C Bulinski ◽  
H Murofushi ◽  
H Aizawa ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
Cyclin B ◽  
Microtubule Associated Proteins ◽  
Mitotic Kinase ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Detergent Extraction ◽  
M Phase ◽  
Associated Proteins ◽  
Free Microtubules

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

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.

scholarly journals Integrating Light and TEM Information with F-TEM images

2005 ◽  
Vol 13 (5) ◽  
pp. 16-19
Author(s):  
P.A. Sims ◽  
C.A. Lockwood ◽  
J.D. Hardin
Keyword(s):  
Tissue Culture ◽  
High Pressure ◽  
Light Microscopy ◽  
Fusion Proteins ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Thin Sections ◽  
Embedding Method ◽  
Culture Cells ◽  
Tissue Culture Cells

Fluorescent fusion proteins are widely used to visualize the localization of proteins in worms, fish, flies and tissue culture cells. We have used two different methods that use high pressure freezing (HPF) combined with correlative light microscopy (LM) and TEM. The first method uses fluorescence from live organisms immobilized in agarose followed by HPF and standard freeze substitution in dry solvent with osmium. This pre-embedding method optimizes ultrastructural preservation. A second, post-embedding method preserves fluorescence and immunoreactivity from embedded and polymerized thin sections. Here we describe post-embedding fluorescent integrated TEM images (F-TEM).

scholarly journals REPAIR OF UV-INDUCED PYRIMIDINE DIMERS IN DROSOPHILA MELANOGASTER CELLS IN VITRO

Genetics ◽  
1973 ◽  
Vol 73 (2) ◽  
pp. 297-302
Author(s):  
James E Trosko ◽  
Kathy Wilder
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Ultraviolet Light ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Pyrimidine Dimers ◽  
Drosophila Cells

ABSTRACT Tissue culture cells of Drosophila melanogaster were given various doses of ultraviolet light. The results indicate that Drosophila cells do have a darkrepair excision mechanism which is not sensitive to caffeine. Pyrimidine dimers were destroyed by photoreactivating illumination in these cells and this destruction probably represents monomerization of the pyrimidine dimers.

Sharpening the anaphase switch

2015 ◽  
Vol 43 (1) ◽  
pp. 19-22 ◽  
Author(s):  
John C. Meadows ◽  
Jonathan B.A. Millar
Keyword(s):  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Mitotic Chromosomes ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Substantial Progress ◽  
A Cell ◽  
Spindle Elongation ◽  
Anaphase B

The segregation of sister chromatids during mitosis is one of the most easily visualized, yet most remarkable, events during the life cycle of a cell. The accuracy of this process is essential to maintain ploidy during cell duplication. Over the past 20 years, substantial progress has been made in identifying components of both the kinetochore and the mitotic spindle that generate the force to move mitotic chromosomes. Additionally, we now have a reasonable, albeit incomplete, understanding of the molecular and biochemical events that are involved in establishing and dissolving sister-chromatid cohesion. However, it is less well-understood how this dissolution of cohesion occurs synchronously on all chromosomes at the onset of anaphase. At the centre of the action is the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that, in association with its activator cell-division cycle protein 20 homologue (Cdc20), is responsible for the destruction of securin. This leads to the activation of separase, a specialized protease that cleaves the kleisin-subunit of the cohesin complex, to relieve cohesion between sister chromatids. APC/C–Cdc20 is also responsible for the destruction of cyclin B and therefore inactivation of the cyclin B–cyclin-dependent kinase 1 (Cdk1). This latter event induces a change in the microtubule dynamics that results in the movement of sister chromatids to spindle poles (anaphase A), spindle elongation (anaphase B) and the onset of cytokinesis. In the present paper, we review the emerging evidence that multiple, spatially and temporally regulated feedback loops ensure anaphase onset is rapid, co-ordinated and irreversible.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

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