Differentiation of Dictyostelium discoideum mutant cells in a shaken suspension culture and the effect of cyclic AMP

1981 ◽  
Vol 51 (1) ◽  
pp. 131-142
Author(s):  
K. Abe ◽  
Y. Saga ◽  
H. Okada ◽  
K. Yanagisawa
Keyword(s):  
Cyclic Amp ◽  
Suspension Culture ◽  
Dictyostelium Discoideum ◽  
Solid Surface ◽  
Enzyme Activities ◽  
Parental Strain ◽  
Mutant Cell ◽  
Specific Activities ◽  
Mutant Cells ◽  
Kinetics Of

In Dictyostelium discoideum, 16 mutants in which cells differentiate into spores and stalk cells without normal morphogenesis were isolated. All these mutants are rapidly developing and capable of differentiating in a shaken suspension of phosphate buffer.The developmental kinetics of specific activities of enzymes in one of the mutants, HTY 1851, cultured in the suspension was compared with that in the parental strain, X2, developed on a solid surface. Most of the enzyme activities appeared much earlier and the peaks of the activities were lower in HTY1851 than X2, but the order or appearance of the activities was the same in both the strains cultured under the conditions described above. These results suggest that the biochemical steps in the development of the mutant in a shaken suspension are essentially the same as those of the parental strain X2 on a solid surface. It was also found that addition of cyclic AMP (2.5 X 10-5 M to 1 X 10-4 M) to the mutant cell suspension 6–8 h after the initiation of development induced an increase in the number of spores and the specific activities of some enzymes to values twice as high as those of an untreated control.

The role of cyclic GMP in regulating myosin during chemotaxis of Dictyostelium: evidence from a mutant lacking the normal cyclic GMP response to cyclic AMP

1993 ◽  
Vol 106 (2) ◽  
pp. 591-595 ◽  
Author(s):  
G. Liu ◽  
H. Kuwayama ◽  
S. Ishida ◽  
P.C. Newell
Keyword(s):  
Cyclic Amp ◽  
Heavy Chain ◽  
Cyclic Gmp ◽  
Parental Strain ◽  
Response Curve ◽  
Myosin Ii ◽  
Cellular Slime ◽  
Mutant Cells ◽  
Slight Displacement

Evidence has previously been reported that, during chemotaxis of the cellular slime mould Dictyostelium discoideum, cyclic GMP regulates the association of myosin II with the cytoskeleton and that this regulation is effected by inhibiting myosin II heavy chain phosphorylation (Liu and Newell, J. Cell Sci., 90, 123–129, 1988; 98, 483–490, 1991). Here we provide further evidence in support of this hypothesis using a mutant (KI-10) that is defective in chemotaxis and lacks the normal cyclic AMP-induced cyclic GMP response. We found that the cyclic AMP-induced cytoskeletal actin response was similar to that of the parental strain in this mutant (although showing a slight displacement in the dose-response curve) but the cytoskeletal myosin II heavy chain response was abolished. Moreover, the mutant showed no phosphorylation of myosin II heavy chain in response to cyclic AMP. Compared to the parental strain XP55, the mutant cells contained approximately 40% more protein and their doubling time was 30% longer. These differences could be due to differences in the efficiency of cell division, a process in which the proper regulation of myosin function is essential and in which cyclic GMP may therefore play a role.

Influence of cyclic AMP and hydrolysis products on cell type regulation in Dictyostelium discoideum

Development ◽  
1985 ◽  
Vol 86 (1) ◽  
pp. 19-37
Author(s):  
Cornelis J. Weijer ◽  
Antony J. Durston
Keyword(s):  
Cyclic Amp ◽  
Suspension Culture ◽  
Dictyostelium Discoideum ◽  
Double Negative ◽  
Cell Type ◽  
Camp Phosphodiesterase ◽  
Feedback Model ◽  
Hydrolysis Products ◽  
Low Concentrations ◽  
Extracellular Camp

We describe the effect of cyclic AMP on regulation of the proportion of prespore and prestalk cells in Dictyostelium discoideum. Prespore and prestalk cells from slugs were enriched on Percoll density gradients and allowed to regulate in suspension culture under 100% oxygen. The transition of prespore to prestalk cells is blocked by cAMP, while cAMP phosphodiesterase and caffeine cause a decrease in the number of prespore cells. This suggests that extracellular cAMP plays a role in cell type proportioning by inhibiting the conversion of prespore to prestalk cells. Low concentrations of cAMP prevent the conversion of prestalk to prespore cells; the same effect is seen with hydrolysis products of cAMP, 5 AMP, adenosine and also adenine. We suggest that, when low concentrations of cAMP are added to regulating cells, the cAMP itself is quickly broken down and the breakdown products thereafter inhibit the prestalk-to-prespore conversion. The relevance of these findings is discussed in the context of an non-positional double-negative feedback model for cell type homeostasis.

scholarly journals Macrocyst development in Dictyostelium discoideum. I. Induction of synchronous development by giant cells and biochemical analysis

1982 ◽  
Vol 55 (1) ◽  
pp. 341-352
Author(s):  
Y. Saga ◽  
K. Yanagisawa
Keyword(s):  
Dictyostelium Discoideum ◽  
Enzyme Activities ◽  
Biochemical Analysis ◽  
Giant Cells ◽  
Mating Types ◽  
Fruiting Body Formation ◽  
Single Strain ◽  
Synchronous Development ◽  
A Cell ◽  
Kinetics Of

In Dictyostelium discoideum, cytological and physiological studies on macrocyst formation revealed that this process consists of at least two steps: the production of giant cells, which are believed to be formed from the fusion of cells of two opposite mating types, and the subsequent induction of macrocyst development by the giant cells. The conditions that had been considered formerly to be required for macrocyst formation, such as darkness at the presence of two cells of complementary mating types in heterothallic strains, were actually required only for the production of the giant cells. Once giant cells are produced, the surrounding cells can aggregate and form macrocysts even in the light. Furthermore, it was demonstrated that giant cells can switch the developmental mode of the surrounding cells to macrocyst formation. That is, if a critical number of the isolated giant cells are introduced into a cell population of a single strain of NC4, which normally would produce only fruiting-bodies, macrocysts are formed instead. When in the presence of giant cells, the development of macrocysts may be initiated by starvation. Therefore, if all cells are made to starve simultaneously development begins and proceeds synchronously. Using this technique of synchronous development, the developmental kinetics of enzyme activities were assayed during macrocyst and fruiting-body formation. Considerable differences in the patterns of those enzyme activities were demonstrated between the two developmental modes of D. discoideum.

Cytoskeletons from a mutant of Dictyostelium discoideum with flattened cells

1986 ◽  
Vol 86 (1) ◽  
pp. 69-82
Author(s):  
M. Claviez ◽  
M. Brink ◽  
G. Gerisch
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cell Shape ◽  
Wild Type ◽  
Aggregation Pattern ◽  
Mutant Cells ◽  
Moving Front ◽  
Microtubular System

Development of a mutant of Dictyostelium discoideum, HG403, is described whose cells spread strongly on a substratum. Although the mutant cells were less clearly polarized into the front and rear ends, and usually less extensively elongated than wild-type cells, their aggregation pattern was only slightly less regular. Cells of the mutant responded well to cyclic AMP by chemotaxis, although their capability of stabilizing cell shape and maintaining dominance of a single moving front appeared to be reduced. Mutant HG403 proved to be ideal for the preparation of cytoskeletons in which the organization of the microtubular system, the network of filaments between them, the dense texture of the microfilament network at the periphery of the cells, as well as the bundling of microfilaments in spike-like extensions, could be observed.

Role of cyclic AMP and ammonia in induction and maintenance of post-aggregative differentiation in a suspension culture of Dictyostelium discoideum

1988 ◽  
Vol 38 (1) ◽  
pp. 11-16 ◽  
Author(s):  
Masakazu Oyama ◽  
Yuzuru Kubohara ◽  
Akiko A. Oohata ◽  
Koji Okamoto
Keyword(s):  
Cyclic Amp ◽  
Suspension Culture ◽  
Dictyostelium Discoideum

Myosin II-independent processes in mitotic cells of Dictyostelium discoideum: redistribution of the nuclei, re-arrangement of the actin system and formation of the cleavage furrow

1997 ◽  
Vol 110 (2) ◽  
pp. 123-137 ◽  
Author(s):  
R. Neujahr ◽  
C. Heizer ◽  
G. Gerisch
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Solid Surface ◽  
Myosin Ii ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Multinucleated Cells ◽  
Dynamic Cell ◽  
Separated Sets ◽  
Mutant Cells

Mitosis was studied in multinucleated and mononucleated mutant cells of Dictyostelium discoideum that lack myosin II (Manstein et al. (1989) EMBO J. 8, 923–932). Multinucleated cells were produced by culture in suspension, mononucleated cells were enriched by growth on a solid surface (DeLozanne and Spudich (1987) Science 236, 1086–1091). The multinucleated cells disclosed interactions of mitotic complexes with the cell cortex that were not apparent in normal, mononucleated cells. During the anaphase stage, entire mitotic complexes consisting of spindle, microtubule asters, and separated sets of chromosomes were translocated to the periphery of the cells. These complexes were appended at a distance of about 3 microns from the cell surface, in a way that the spindle became orientated in parallel to the surface. Subsequently, lobes of the cell surface were formed around the asters of microtubules. These lobes were covered with tapered protrusions rich in coronin, an actin associated protein that typically accumulates in dynamic cell-surface projections (DeHostos et al. (1991) EMBO J. 10, 4097–4104). During their growth on a solid surface, mononucleated myosin II-null cells passed through the mitotic cleavage stages with a speed comparable to wild-type cells. Cytokinesis as linked to mitosis is distinguishable by several parameters from traction mediated cytofission, which results in the pinching off of pieces of a multinucleated cell in the interphase. The possibility is discussed that cells can cleave during mitosis without forming a contractile ring at the site of the cleavage furrow.

scholarly journals A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. I. Properties.

1984 ◽  
Vol 259 (1) ◽  
pp. 654-661 ◽  
Author(s):  
I H Majerfeld ◽  
B H Leichtling ◽  
J A Meligeni ◽  
E Spitz ◽  
H V Rickenberg
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i6-i7
Author(s):  
Alişan Kayabölen ◽  
Gizem Nur Sahin ◽  
Fidan Seker ◽  
Ahmet Cingöz ◽  
Bekir Isik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mutant Cell ◽  
Glioma Cells ◽  
Epigenetic Therapy ◽  
Low Grade ◽  
Orthotopic Model ◽  
Cycle Arrest ◽  
Mutant Cells

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

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