Regulation of Microtubule Assembly-Disassembly in Mitotic Mammalian Cells: Role of Calcium, Calmodulin and MTOCs

1981 ◽  
Vol 39 ◽  
pp. 572-575
Author(s):  
B. R. Brinkley ◽  
S. L. Brenner ◽  
D. A. Pepper ◽  
R. L. Pardue
Keyword(s):  
Cell Cycle ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Microtubule Assembly ◽  
Free Calcium ◽  
Mitotic Apparatus ◽  
Cytoplasmic Microtubule ◽  
Dividing Cells ◽  
Late Telophase

Two microtubule arrays exist in cultured mammalian cells during their progression through the cell cycle; the cytoplasmic microtubule complexes (CMTC) of interphase cells (Figure 1) and the mitotic apparatus (MA) of dividing cells (Figure 2). As chromosomes are segregated to opposite poles of the spindle during telophase, the microtubules of the MA are disassembled. During late telophase -G1 phase the tubulin subunits from the spindle are recycled into the microtubules of the CMTC which forms an elaborate network throught the cytoplasm. When cells progress into late G2 -Prophase, the CMTC is disassembled and the tubulin is converted into microtubules of the MA. our research has been aimed at defining the mechanism whereby cells regulate the alternating patterns of microtubule assembly-disassembly during the cell cycle.In one series of experiments, we have investigated the role of calcium in microtubule assembly. Several laboratories have shown that cytoplasmic and spindle microtubules are unstable in the presence of elevated free calcium levels. Using monospecific antibodies and indirect immunofluorescence, we have demonstrated the presence of the ubiquitous calcium-binding protein calmodulin in the mitotic spindle of mammalian cells in vitro (Figure 3).

The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans

1997 ◽  
Vol 110 (5) ◽  
pp. 623-633 ◽  
Author(s):  
M.A. Martin ◽  
S.A. Osmani ◽  
B.R. Oakley
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Cycle Progression ◽  
Spindle Microtubules ◽  
Gamma Tubulin

gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of gamma-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a gamma-tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that gamma-tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that gamma-tubulin is involved in spindle microtubule nucleation. In the absence of functional gamma-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that gamma-tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

Calcium and cell cycle control

Development ◽  
1990 ◽  
Vol 108 (4) ◽  
pp. 525-542 ◽  
Author(s):  
M. Whitaker ◽  
R. Patel
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Cell Cycle Regulation ◽  
Sea Urchins ◽  
Cell Cycle Control ◽  
Control Point ◽  
Control Cell ◽  
Free Calcium ◽  
Cycle Regulation

The cell division cycle of the early sea urchin embryo is basic. Nonetheless, it has control points in common with the yeast and mammalian cell cycles, at START, mitosis ENTRY and mitosis EXIT. Progression through each control point in sea urchins is triggered by transient increases in intracellular free calcium. The Cai transients control cell cycle progression by translational and post-translational regulation of the cell cycle control proteins pp34 and cyclin. The START Cai transient leads to phosphorylation of pp34 and cyclin synthesis. The mitosis ENTRY Cai transient triggers cyclin phosphorylation. The motosis EXIT transient causes destruction of phosphorylated cyclin. We compare cell cycle regulation by calcium in sea urchin embryos to cell cycle regulation in other eggs and oocytes and in mammalian cells.

scholarly journals Role of spindle microtubules in the control of cell cycle timing.

1979 ◽  
Vol 80 (3) ◽  
pp. 674-691 ◽  
Author(s):  
G Sluder
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Control Cell ◽  
Microtubule Assembly ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Cells ◽  
Spindle Microtubules

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

scholarly journals Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

2014 ◽  
Vol 25 (5) ◽  
pp. 606-619 ◽  
Author(s):  
Zhen Zheng ◽  
Qingwen Wan ◽  
Gerry Meixiong ◽  
Quansheng Du
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Membrane Binding ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Α Subunit ◽  
Definitive Evidence ◽  
C Terminus ◽  
Chromosome Separation ◽  
Spindle Elongation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein–generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated—it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)–mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle–regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.

scholarly journals RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

2016 ◽  
Vol 27 (8) ◽  
pp. 1346-1357 ◽  
Author(s):  
Pavol Cekan ◽  
Keisuke Hasegawa ◽  
Yu Pan ◽  
Emily Tubman ◽  
David Odde ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Progression ◽  
Nucleotide Exchange ◽  
Normal Cells ◽  
Cytoplasmic Transport ◽  
Dividing Cells ◽  
Ran Gtp

The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage.

Towards understanding the control of the division cycle in animal cells

1992 ◽  
Vol 70 (10-11) ◽  
pp. 920-945 ◽  
Author(s):  
Yoshio Masui
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Mammalian Cells ◽  
Microtubule Assembly ◽  
Nuclear Transplantation ◽  
Animal Cells ◽  
Cytoplasmic Factor ◽  
Molecular Control ◽  
Cytostatic Factor ◽  
Maturation Promoting Factor

The author reviewed the historical process by which classical knowledge of cell division accumulated, to give rise to the molecular biology of the cell cycle, and discussed the perspective of this field of research. The study of the control of cell division began at the turn of the century. It was hypothesized that cell division was a physiological regulation necessary for growing cells to maintain a proper nucleocytoplasmic ratio to survive, which was later substantiated by the finding that amoeba cells could be prevented from dividing by repeated excision of the cytoplasm. However, the observation in Tetrahymena that heat-shocked cells grow exceedingly, but fail to divide, suggested that the cell required the accumulation of a labile "division protein" to initiate division. Mechanisms that control the cell cycle were studied in oocytes by nuclear transplantation and cytoplasmic transfer, and in cultured mammalian cells, protozoa, and Physarum Plasmodia by cell fusion. These experiments demonstrated the existence of cytoplasmic factors that control the cell cycle. Maturation promoting factor (MPF) thus discovered in frog oocytes became known to be an ubiquitous cytoplasmic factor that causes the transition from interphase to metaphase in all organisms. The insight into the molecular control of cell growth and division was gained from yeast cell genetics. For biochemical analysis of the cell cycle control, the method to observe the cell cycle in vitro was developed using frog egg extracts. Thus, MPF was identified as a cdc2 – cyclin protein complex. Its activity was found to depend on synthesis and phosphorylation of these proteins. However, recently it was found that there were cell cycle phenomena that were difficult to explain in these terms. Various other cellular factors, including nucleocytoplasmic ratio and microtubule assembly, were also found to control MPF, as well as the cell cycle. It remained open to future investigation how these factors control MPF to alter the pattern of the cell cycle.Key words: cell cycle, cytostatic factor, maturation promoting factor, nucleocytoplasmic relation.

scholarly journals Intracellular Dynamics of the Ubiquitin-Proteasome-System

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 367 ◽  
Author(s):  
Maisha Chowdhury ◽  
Cordula Enenkel
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Current Knowledge ◽  
Ubiquitin Proteasome System ◽  
Yeast Cells ◽  
Ubiquitin Chain ◽  
Dividing Cells ◽  
Ubiquitin Proteasome

The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum ( ER) membranes. In prolonged quiescence, proteasome granules drop off the nuclear envelopeNE / ER membranes and migrate as droplet-like entitiesstable organelles  throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells, which comprise the majority of our body’s cells.

320. THE ROLE OF FIZZY RELATED 1 IN MALE MEIOSIS

2010 ◽  
Vol 22 (9) ◽  
pp. 120
Author(s):  
L. Hopkins ◽  
V. Pye ◽  
B. Fraser ◽  
J. Holt ◽  
K. Jones ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Binding Capacity ◽  
Cyclin B1 ◽  
Male Meiosis ◽  
Conditional Knockout ◽  
Wild Type ◽  
Cell Cycle Protein ◽  
Sperm Analysis

Accurate chromosome segregation during mitosis and meiosis is facilitated by a regulatory complex known as the Anaphase Promoting Cyclosome (APC), an ubiquitin ligase complex that tags proteins with ubiquitin. Subsequently targeted proteins are recognised by the 26S proteosome and degraded. In mammalian cells, two temporally regulated co-activators are required for the APC to function; fizzy and fzr1. In studies of female oocyte development fzr1 has been demonstrated to play an important role in maintaining G2 arrest during meiosis by controlling spatial levels of the cell cycle protein Cyclin B1 but the role of Fzr1 in spermatogenesis remains unknown. Germ cell specific conditional knockout fzr1mice were generated using the DDX4-Cre and flox/flox fzr1 mouse lines and initial gross morphological analysis indicated that at 7 weeks of age null mice possessed significantly smaller testes (21.81mg ± 0.23mg) when compared to heterozygote (99.86mg ± 1.58mg) and wildtype littermates (93.06mg ± 1.16mg) n = 3 P < 0.0001. Quantitative gene expression analysis confirmed almost complete absence of fzr1 transcript in testes (20-fold decrease) in comparison to wild-type. Immunoblotting and immunohistochemistry revealed no expression of Fzr1 protein in meiotic and post meiotic germ cells when compared to heterozygote and wild type littermates. Histomorphological analysis of testes tissue sections revealed Fzr1 null males exhibited spermatogenic arrest and a complete absence of round spermatids with concomitant apoptosis in the residual spermatocytes. Epididymal examination confirmed a complete lack of mature spermatozoa in the cauda epididymis of null males. In contrast, both wild type and heterozygote mice displayed normal spermatogenesis and epididymal sperm analysis indicated no distinguishable differences in seminal characteristics with normal motility, morphology and sperm-zona binding capacity. Based on these observations we hypothesise that Fzr1 plays a significant role in the establishment and maintenance of meiosis possibly through regulation of key cell cycle proteins.

Sign in / Sign up

Export Citation Format

Share Document