scholarly journals The Internal Phosphodiesterase RegA Is Essential for the Suppression of Lateral Pseudopods duringDictyosteliumChemotaxis

2000 ◽  
Vol 11 (8) ◽  
pp. 2803-2820 ◽  
Author(s):  
Deborah J. Wessels ◽  
Hui Zhang ◽  
Joshua Reynolds ◽  
Karla Daniels ◽  
Paul Heid ◽  
...  
Keyword(s):  
Mutant Cell ◽  
Computer Assisted ◽  
Spatial Gradient ◽  
Wild Type ◽  
Gene Encoding ◽  
Camp Phosphodiesterase ◽  
Temporal Gradient ◽  
Spatial Gradients ◽  
Normal Manner ◽  
Analysis System

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system,regA−mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregatingregA−cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA−cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA−cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex ofregA−cells in an increasing temporal gradient of cAMP.

Phosphorylation of the myosin regulatory light chain plays a role in motility and polarity duringDictyosteliumchemotaxis

2002 ◽  
Vol 115 (8) ◽  
pp. 1733-1747 ◽  
Author(s):  
Hui Zhang ◽  
Deborah Wessels ◽  
Petra Fey ◽  
Karla Daniels ◽  
Rex L. Chisholm ◽  
...  
Keyword(s):  
Light Chain ◽  
Mutant Cell ◽  
Cell Aggregation ◽  
Regulatory Light Chain ◽  
Computer Assisted ◽  
Spatial Gradient ◽  
Myosin Regulatory Light Chain ◽  
Cellular Polarity ◽  
Wild Type ◽  
Mutant Cells

The myosin regulatory light chain (RLC) of Dictyostelium discoideum is phosphorylated at a single serine site in response to chemoattractant. To investigate the role of the phosphorylation of RLC in both motility and chemotaxis, mutants were generated in which the single phosphorylatable serine was replaced with a nonphosphorylatable alanine. Several independent clones expressing the mutant RLC in the RLC null mutant, mlcR-, were obtained. These S13A mutants were subjected to high resolution computer-assisted motion analysis to assess the basic motile behavior of cells in the absence of a chemotatic signal, and the chemotactic responsiveness of cells to the spatial, temporal and concentration components of natural cAMP waves. In the absence of a cAMP signal, mutant cells formed lateral pseudopods less frequently and crawled faster than wild-type cells. In a spatial gradient of cAMP, mutant cells chemotaxed more efficiently than wild-type cells. In the front of simulated temporal and natural waves of cAMP,mutant cells responded normally by suppressing lateral pseudopod formation. However, unlike wild-type cells, mutant cells did not lose cellular polarity at the peak and in the back of either wave. Since depolarization at the peak and in the descending phase of the natural wave is necessary for efficient chemotaxis, this deficiency resulted in a decrease in the capacity of S13A mutant cells to track natural cAMP waves relayed by wild-type cells, and in the fragmentation of streams late in mutant cell aggregation. These results reveal a regulatory pathway induced by the peak and back of the chemotactic wave that alters RLC phosphorylation and leads to cellular depolarization. We suggest that depolarization requires myosin II rearrangement in the cortex facilitated by RLC phosphorylation, which increases myosin motor function.

Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis

1995 ◽  
Vol 108 (3) ◽  
pp. 1105-1115 ◽  
Author(s):  
E. Shelden ◽  
D.A. Knecht
Keyword(s):  
Cell Morphology ◽  
Mutant Cell ◽  
Myosin Ii ◽  
Cell Phenotype ◽  
Computer Assisted ◽  
Wild Type ◽  
Isolated Cells ◽  
Multicellular Development ◽  
Periodic Movement ◽  
Mutant Cells

We have used fluorescent labeling, confocal microscopy and computer-assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dictyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5 +/- 4.9 microns/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0 +/- 4.8 microns/min, twice as fast as reported previously for isolated myosin II mutant cells, and fail to display regular periodic movement. When removed from aggregation streams myosin II mutant cells move at only 2.5 +/- 2.0 microns/min, while wild-type cells under these conditions move at 5.9 +/- 4.5 microns/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular development. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the cortex of such cells is too flacid to resist forces generated during movement. The increased rate of mutant cell movement and distortion of mutant cell morphology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a multicellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. In addition, the distortion of myosin II mutant cells within wild-type aggregation streams indicates that myosin is not required for the formation of cell-cell contacts. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium.

scholarly journals RasC Plays a Role in Transduction of Temporal Gradient Information in the Cyclic-AMP Wave of Dictyostelium discoideum

2004 ◽  
Vol 3 (3) ◽  
pp. 646-662 ◽  
Author(s):  
Deborah Wessels ◽  
Rebecca Brincks ◽  
Spencer Kuhl ◽  
Vesna Stepanovic ◽  
Karla J. Daniels ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Myosin Ii ◽  
Three Dimensional ◽  
Computer Assisted ◽  
Expression Array ◽  
Temporal Gradient ◽  
Spatial Gradients ◽  
Quantitative Studies ◽  
Myosin I ◽  
The Individual

ABSTRACT To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC −, in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC − cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC − cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC − cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC − cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA −/myoF − myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC − mutant are the immediate result of the absence of RasC function.

scholarly journals Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants.

1996 ◽  
Vol 16 (4) ◽  
pp. 1519-1526 ◽  
Author(s):  
A Errami ◽  
V Smider ◽  
W K Rathmell ◽  
D M He ◽  
E A Hendrickson ◽  
...  
Keyword(s):  
Mutant Cell ◽  
Genetic Defect ◽  
Strand Break ◽  
Complementation Group ◽  
X Rays ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Gene Encoding ◽  
X Ray ◽  
Group 5

X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.

scholarly journals Role of CheB and CheR in the Complex Chemotactic and Aerotactic Pathway of Azospirillum brasilense

2006 ◽  
Vol 188 (13) ◽  
pp. 4759-4768 ◽  
Author(s):  
Bonnie B. Stephens ◽  
Star N. Loar ◽  
Gladys Alexandre
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Azospirillum Brasilense ◽  
Model Organism ◽  
Spatial Gradient ◽  
Wild Type ◽  
Temporal Gradient ◽  
E Coli ◽  
Significant Difference

ABSTRACT It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.

scholarly journals Ponticulin plays a role in the positional stabilization of pseudopods.

1995 ◽  
Vol 131 (6) ◽  
pp. 1495-1506 ◽  
Author(s):  
D C Shutt ◽  
D Wessels ◽  
K Wagenknecht ◽  
A Chandrasekhar ◽  
A L Hitt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Three Dimensional ◽  
Computer Assisted ◽  
Spatial Gradient ◽  
Actin Network ◽  
Wild Type ◽  
Cortical Actin ◽  
High Affinity

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer-assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.

scholarly journals Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 517-526
Author(s):  
R Gudenus ◽  
S Mariotte ◽  
A Moenne ◽  
A Ruet ◽  
S Memet ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Essential Gene ◽  
Mutant Cell ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Gene Encoding

Abstract A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO3 and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pcil-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

The regulation of chemotaxis and chemokinesis in Dictyostelium amoebae by temporal signals and spatial gradients of cyclic AMP

1994 ◽  
Vol 107 (2) ◽  
pp. 659-667 ◽  
Author(s):  
M.G. Vicker
Keyword(s):  
Cyclic Amp ◽  
Molecular Diffusion ◽  
Gradient Field ◽  
Computer Assisted ◽  
Spatial Gradient ◽  
Spatial Gradients ◽  
Cell Accumulation ◽  
Initial Effect ◽  
Gradient Orientation ◽  
Infusion System

The tactic and kinetic locomotion of Dictyostelium discoideum amoebae were examined in cyclic AMP (cAMP) spatial gradient and temporal signal fields. The distributions of migrating cells were examined within 150 microns-thick micropore filters after incubation with different cAMP concentrations, [cAMP], applied in three ways across the fields: as positively or negatively developing gradients, generated either by increasing or decreasing the [cAMP] on one side of the filter, respectively, or as static, linear gradients after negative development. Chemotaxis was only induced by oriented, temporally increasing [cAMP]. Pulses propagated by molecular diffusion or mechanical flow were equally effective. Negatively developing cAMP gradients had no initial effect on cell accumulation. However, if the subsequent static spatial gradient was maintained by an infusion system, some gradients also induced cell accumulation, whose degree and direction depended on the gradient [cAMP]. The basis of this new effect was examined by tracking individual cells by computer-assisted videomicroscopy during locomotion in different [cAMP]. Cells produced a triphasic [cAMP]-dependent response, with optimal cell motility induced by 10–30 nM. The results demonstrate that cell accumulation either up-field or down-field in spatial gradients is governed by the field locations of the attractant concentrations that induce the relative locomotory maxima and minima in the gradient field. Cells perceive the ambient [cAMP], but cannot read the spatial gradient orientation in static or yet steeper regions of developing gradients. Accumulation in static spatial gradients is a function of klino- and orthokinesis, but chemotaxis requires an oriented cAMP pulse or impulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 519d-519 ◽  
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Height ◽  
Dry Weight ◽  
Nicotiana Alata ◽  
Wild Type ◽  
Gene Encoding ◽  
Delayed Senescence ◽  
Gene Construct ◽  
Shoot Dry Weight ◽  
Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

scholarly journals Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1885
Author(s):  
José Néstor Caamaño ◽  
Carolina Tamargo ◽  
Inmaculada Parrilla ◽  
Felipe Martínez-Pastor ◽  
Lorena Padilla ◽  
...  
Keyword(s):  
Genetic Resource ◽  
Sperm Quality ◽  
Genetic Material ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Kinematic Parameters ◽  
Linear Mixed Effects ◽  
Sperm Analysis ◽  
Computer Assisted Sperm Analysis ◽  
Analysis System

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8–71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

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