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.

Action of a slowly hydrolysable cyclic AMP analogue on developing cells of Dictyostelium discoideum

1979 ◽  
Vol 35 (1) ◽  
pp. 321-338
Author(s):  
C. Rossier ◽  
G. Gerisch ◽  
D. Malchow
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Agar Plate ◽  
Cell Aggregation ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Camp Analogue ◽  
Order Of Magnitude

Adenosine 3′,5′-cyclic phosphorothioate (cAMP-S) is a cyclic AMP (cAMP) analogue which is only slowly hydrolysed by phosphodiesterases of Dictyostelium discoideum. The affinity of cAMP-S to cAMP receptors at the cell surface is only one order of magnitude lower than that of cAMP. cAMP-S can replace cAMP as a stimulant with respect to all receptor-mediated responses tested, including chemotaxis and the induction of cAMP pulses. cAMP-S does not affect growth of D. discoideum but it blocks cell aggregation at a uniform concentration of 5 × 10(−7) M in agar plate cultures of strain NC-4 as well as its axenically growing derivative, Ax-2. Another wild-type strain of D. discoideum, v-12, is able to aggregate on agar plates supplemented with 1 mM cAMP-S. The development of Polysphondylium pallidum and P. violaceum is also highly cAMP-S resistant. In Ax-2 both differentiation from the growth phase to the aggregation-competent stage and chemotaxis are cAMP-S sensitive, whereas in v-12 only chemotaxis is inhibited. v-12 can still form streams of cohering cells and fruiting bodies when chemotaxis is inhibited by cAMP-S. Whereas cAMP induces differentiation into stalk cells at concentrations of 10(−3) or 10(−4) M, cAMP-S has the same effect in strain v-12 at the much lower concentration of 10(−6) M.

scholarly journals Isoprenylcysteine Carboxy Methylation Is Essential for Development in Dictyostelium discoideum

2007 ◽  
Vol 18 (10) ◽  
pp. 4106-4118 ◽  
Author(s):  
Ying Chen ◽  
Kyle J. McQuade ◽  
Xiao-Juan Guan ◽  
Peter A. Thomason ◽  
Michael S. Wert ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Intercellular Signaling ◽  
Wild Type ◽  
Protein Subunit ◽  
Encoding Gene ◽  
Carboxy Terminal ◽  
Mutant Cells ◽  
Do So

Members of the Ras superfamily of small GTPases and the heterotrimeric G protein γ subunit are methylated on their carboxy-terminal cysteine residues by isoprenylcysteine methyltransferase. In Dictyostelium discoideum, small GTPase methylation occurs seconds after stimulation of starving cells by cAMP and returns quickly to basal levels, suggesting an important role in cAMP-dependent signaling. Deleting the isoprenylcysteine methyltransferase-encoding gene causes dramatic defects. Starving mutant cells do not propagate cAMP waves in a sustained manner, and they do not aggregate. Motility is rescued when cells are pulsed with exogenous cAMP, or coplated with wild-type cells, but the rescued cells exhibit altered polarity. cAMP-pulsed methyltransferase-deficient cells that have aggregated fail to differentiate, but mutant cells plated in a wild-type background are able to do so. Localization of and signaling by RasG is altered in the mutant. Localization of the heterotrimeric Gγ protein subunit was normal, but signaling was altered in mutant cells. These data indicate that isoprenylcysteine methylation is required for intercellular signaling and development in Dictyostelium.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals An Adenylyl Cyclase, CyaB, Acts as an Osmosensor in Myxococcus xanthus

2005 ◽  
Vol 187 (10) ◽  
pp. 3593-3598 ◽  
Author(s):  
Yoshio Kimura ◽  
Mika Ohtani ◽  
Kaoru Takegawa
Keyword(s):  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Growth Retardation ◽  
Spore Germination ◽  
Germination Rate ◽  
Myxococcus Xanthus ◽  
Receptor Type ◽  
Wild Type ◽  
Vegetative Cells ◽  
Mutant Cells

ABSTRACT We have previously reported that a receptor-type adenylyl cyclase (CyaA) of Myxococcus xanthus undergoes an osmosensor mainly during spore germination (Y. Kimura et al., J. Bacteriol. 184:3578-3585, 2002). In the present study, we cloned another receptor-type adenylyl cyclase gene (cyaB) and characterized the function of the cyaB-encoded protein. Disruption of cyaB generates a mutant that showed growth retardation at high ionic (NaCl) or high nonionic (sucrose) osmolarity. When vegetative cells were stimulated with 0.15 M NaCl, the increases in intracellular cyclic AMP levels of cyaB mutant cells were lower than those of wild-type cells. Under nonionic osmostress, the cyaB mutant exhibited reduced spore germination; however, the germination rate of the cyaB mutant was significantly higher than that of the cyaA mutant.

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.

Effects of calcium antagonists on cyclic AMP phosphodiesterase induction in Dictyostelium discoideum

1987 ◽  
Vol 88 (3) ◽  
pp. 379-388
Author(s):  
M.B. Coukell ◽  
A.M. Cameron
Keyword(s):  
Cyclic Amp ◽  
Cell Viability ◽  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Cell Shape ◽  
Second Messengers ◽  
Channel Blockers ◽  
Cyclic Amp Phosphodiesterase ◽  
Dose Dependent Fashion ◽  
Altered Cell

Previous studies have suggested that cyclic GMP and/or Ca2+ might function as second messengers in the induction by exogenous cyclic AMP of the cyclic AMP phosphodiesterase (PD) in Dictyostelium discoideum. To assess further the role of Ca2+ in PD induction we examined the effect on this process of a number of putative Ca2+-channel blockers. At relatively low micromolar concentrations, TMB-8, nicardipine, nifedipine, diltiazem and verapamil all altered cell shape and inhibited PD induction in a similar dose-dependent fashion. Concentrations of these drugs that abolished PD induction had no effect on cell viability; however, higher concentrations reduced viability and caused cell lysis. All effects of these compounds on the cells were antagonized at least partially by 5–10 mM-Ca2+. Other cations tested were considerably less effective. Like the organic inhibitors, La3+ also altered cell shape, inhibited PD induction and reduced cell viability at elevated concentrations, but its effect on the cells appeared to be more complex. Inhibition of PD induction by the organic antagonists could not be attributed solely to an impaired uptake of extracellular Ca2+, a reduction of ATP pools in the cells or a direct effect on calmodulin. Concentrations of TMB-8 that inhibited PD induction had little effect on the cyclic GMP response. Therefore, this compound did not inhibit PD induction indirectly by blocking cyclic GMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

A mechanical function of myosin II in cell motility

1995 ◽  
Vol 108 (1) ◽  
pp. 387-393 ◽  
Author(s):  
P.Y. Jay ◽  
P.A. Pham ◽  
S.A. Wong ◽  
E.L. Elson
Keyword(s):  
Cell Shape ◽  
Myosin Ii ◽  
Mechanical Function ◽  
Null Mutant ◽  
Wild Type ◽  
Cytoskeletal Network ◽  
Cytoskeletal Tension ◽  
Tractional Forces ◽  
Contractile Forces ◽  
Mutant Cells

Myosin II mutant Dictyostelium amoebae crawl more slowly than wild-type cells. Thus, myosin II must contribute to amoeboid locomotion. We propose that contractile forces generated by myosin II help the cell's rear edge to detach from the substratum and retract, allowing the cell to continue forward. To test this hypothesis, we measured the speed of wild-type and myosin II null mutant Dictyostelium cells on surfaces of varying adhesivity. As substratum adhesivity increased, the speed of myosin II null mutant cells decreased substantially compared to wild-type cells, suggesting that the mutant is less able to retract from sticky surfaces. Furthermore, interference reflection microscopy revealed a myosin-II-dependent contraction in wild-type but not null mutant cells that is consistent with a balance of adhesive and contractile forces in retraction. Although myosin II null mutant cells have a defect in retraction, pseudopod extension does not cause the cells to become elongated on sticky surfaces. This suggests a mechanism, based possibly on cytoskeletal tension, for regulating cell shape in locomotion. The tension would result from the transmission of tractional forces through the cytoskeletal network, providing the myosin II null mutant with a limited means of retraction and cell division on a surface.

scholarly journals Translational control of discoidin lectin expression in drsA suppressor mutants of Dictyostelium discoideum.

1991 ◽  
Vol 11 (6) ◽  
pp. 3171-3179 ◽  
Author(s):  
S Alexander ◽  
S Leone ◽  
E Ostermeyer
Keyword(s):  
Dictyostelium Discoideum ◽  
Translational Control ◽  
Suppressive Effect ◽  
Developmental Expression ◽  
Wild Type ◽  
Rna Analysis ◽  
Type Pattern ◽  
Bona Fide ◽  
Lectin Protein ◽  
Mutant Cells

Genetic analysis in Dictyostelium discoideum has identified regulatory genes which control the developmental expression of the discoidin lectin multigene family. Among these, the drsA mutation is a dominant second-site suppressor of another mutation, disB, which has the discoidinless phenotype. We now demonstrate a novel mechanism by which the drsA allele exerts its suppressive effect on the disB mutation. Interestingly, drsA does not merely bypass the disB mutation and restore the wild-type pattern of lectin expression. Rather, drsA mutant cells have high levels of discoidin lectin synthesis during growth but do not express lectins during aggregation. In contrast, wild-type cells only express lectin protein during the aggregation period of development. Phenocopies of the drsA mutation show a pattern of discoidin expression similar to that seen in the bona fide mutant. These data suggest that there may be a mechanism of negative feedback, resulting from the high levels of discoidin lectin made during growth, which inhibits further discoidin lectin expression during development. Northern (RNA) analysis of developing drsA mutant cells shows that these cells contain high levels of discoidin mRNA, although no discoidin lectin protein is being translated from these messages. Therefore, expression of the discoidin gene family can be controlled at the level of translation as well as transcription.

scholarly journals A Novel Membrane Protein Influencing Cell Shape and Multicellular Swarming of Proteus mirabilis

1999 ◽  
Vol 181 (7) ◽  
pp. 2008-2016 ◽  
Author(s):  
Nicole A. Hay ◽  
Donald J. Tipper ◽  
Daniel Gygi ◽  
Colin Hughes
Keyword(s):  
Electron Microscopy ◽  
Proteus Mirabilis ◽  
Cell Shape ◽  
Null Mutant ◽  
Wild Type ◽  
Surface Migration ◽  
Multiple Copies ◽  
Type Gene ◽  
Mutant Cells ◽  
Major Domain

ABSTRACT Swarming in Proteus mirabilis is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called ccmA, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type P. mirabilis; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of accmA null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the ccmA gene caused cells of both Escherichia coli and P. mirabilis to become enlarged and ellipsoidal.

scholarly journals Comparative study of contact repulsion in control and mutant macrophages using a novel interaction detection

2020 ◽  
Author(s):  
JA Solís-Lemus ◽  
BJ Sánchez-Sánchez ◽  
S Marcotti ◽  
M Burki ◽  
B Stramer ◽  
...  
Keyword(s):  
Cell Shape ◽  
Cell Tracking ◽  
Statistical Significance ◽  
Specific Protein ◽  
Cell Segmentation ◽  
Wild Type ◽  
Detection Mechanism ◽  
Interaction Detection ◽  
Motion Characteristics ◽  
Mutant Cells

AbstractThis paper compares the contact-repulsion movement of mutant and wild-type macrophages using a novel interaction detection mechanism. The migrating macrophages are observed in Drosophila embryos. The study is carried out by a framework called macrosight, which analyses the movement and interaction of migrating macrophages. The framework incorporates a segmentation and tracking algorithm into analysing motion characteristics of cells after contact. In this particular study, the interactions between cells is characterised in the case of control embryos and Shot3 mutants, where the cells have been altered to suppress a specific protein, looking to understand what drives the movement. Statistical significance between control and mutant cells was found when comparing the direction of motion after contact in specific conditions. Such discoveries provide insights for future developments in combining biological experiments to computational analysis. Cell Segmentation, Cell Tracking, Macrophages, Cell Shape, Contact Analysis

Sign in / Sign up

Export Citation Format

Share Document