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.

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.

Oxidative Stress Induces Binding of FANCD2 to STAT5 and Facilitates STAT5-Dependent Survival Signals.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 33-33 ◽  
Author(s):  
Grover Bagby ◽  
Winifred Keeble ◽  
Tara Koretsky ◽  
Dylan Zodrow ◽  
Richard Jove ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ground State ◽  
Mutant Cell ◽  
Extracellular Proteins ◽  
Wild Type ◽  
Direct Role ◽  
Normal Cells ◽  
Jak2 Inhibitor ◽  
Stat Signaling ◽  
Mutant Cells

Abstract Fanconi anemia (FA) cells are hypersensitive to oxidative stress and exhibit aberrant STAT activation responses to defined extracellular proteins but whether these abnormalities are linked is unclear. Because oxidative stress is known to induce STAT activation, we hypothesized that proper STAT signaling responses in normal cells exposed to H2O2 require intact FA proteins. In fact, we found that FA-C, FA-G, and FA-D2 cells (fibroblasts) showed a significant increase in apoptosis after H2O2-exposure compared to retrovirally-complemented cells. H2O2 induced higher phospho-STAT5 (P-STAT5) expression in complemented cells than in mutant cells. Conversely, mutant cells expressed higher levels of P-STAT3 in both the ground state and after H2O2-induction than complemented cells. Aberrant STAT activation in FA mutant cells was shown to be both nucleus- and JAK2 kinase-dependent. Only low levels of STAT3 and STAT5 were induced in both mutant and complemented cytoplasts and AG490 (a Jak2 inhibitor) significantly suppressed H2O2-induced STAT5 responses. Seeking a direct role of FANCD2 in regulating proper STAT activation responses to H2O2, we carried out immunoprecipitation experiments (with an antibody to the N-terminal fragment of FANCD2) using PD20, a FA-D2 mutant cell line, and FANCD2 complemented PD20. In FANCD2-complemented and normal cells, anti-FANCD2 antibody immunoprecipitated STAT5. However, in mutant cells the same antibody immunoprecipitated STAT3, not STAT5. Thus, mutant (truncated) FANCD2 preferentially binds to and may activate STAT3 in the ground state. In fact, wild type FANCD2 also binds aberrantly to STAT3 in HSC536 (FA-C lymphoblasts) indicating that FANCC may influence the function of wild type FANCD2 and that binding of wild type FANCD2 to STAT3 does not require FANCD2 ubiquitinylation (FANCD2 is not ubiquitinylated in FA-C). Suspecting that in H2O2-exposed cells STAT5 signaling pathways lead to survival while STAT3 pathways lead to apoptosis, we transduced constitutively active mutants (*) of STATs 3 and 5 in mutant D2 and complemented cells. STAT3* increased apoptotic responses to H2O2 in complemented FA-D2 cells and STAT5* decreased apoptotic responses in H2O2-induced FA-D2 cells. In addition, the STAT5 inducible anti-apoptotic gene Bcl-XL was induced in H2O2-exposed complemented FA-D2 cells but not in FA-D2 cells. We conclude that FANCD2 functions to promote survival by ordering proper STAT signaling responses to oxidative stress and that this function of FANCD2 depends in part upon FA-C. We propose that FA cells are hypersensitive to oxidative stress in part because of imbalanced STAT signal transduction responses.

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.

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 Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

2014 ◽  
Vol 13 (5) ◽  
pp. 560-571 ◽  
Author(s):  
Pardeep Kumar ◽  
Christian Heiss ◽  
Felipe H. Santiago-Tirado ◽  
Ian Black ◽  
Parastoo Azadi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Morphology ◽  
Mutant Cell ◽  
Sequence Motifs ◽  
Cell Integrity ◽  
Metabolic Imbalance ◽  
Normal Mass ◽  
Capsule Assembly ◽  
Altered Cell ◽  
Mutant Cells

ABSTRACTThe cryptococcal capsule is a critical virulence factor of an important pathogen, but little is known about how it is associated with the cell or released into the environment. Two mutants lackingPBX1andPBX2were found to shed reduced amounts of the capsule polysaccharide glucuronoxylomannan (GXM). Nuclear magnetic resonance, composition, and physical analyses showed that the shed material was of normal mass but was slightly enriched in xylose. In contrast to previous reports, this material contained no glucose. Notably, the capsule fibers ofpbxΔ mutant cells grown under capsule-inducing conditions were present at a lower than usual density and were loosely attached to the cell wall. Mutant cell walls were also defective, as indicated by phenotypes including abnormal cell morphology, reduced mating filamentation, and altered cell integrity. All observed phenotypes were shared between the two mutants and exacerbated in a double mutant. Consistent with a role in surface glycan synthesis, the Pbx proteins localized to detergent-resistant membrane domains. These results, together with the sequence motifs in the Pbx proteins, suggest that Pbx1 and Pbx2 are redundant proteins that act in remodeling the cell wall to maintain normal cell morphology and precursor availability for other glycan synthetic processes. Their absence results in aberrant cell wall growth and metabolic imbalance, which together impact cell wall and capsule synthesis, cell morphology, and capsule association. The surface changes also lead to increased engulfment by host phagocytes, consistent with the lack of virulence ofpbxmutants in animal models.

scholarly journals Genetic and Molecular Analysis of cglB, a Gene Essential for Single-Cell Gliding in Myxococcus xanthus

1999 ◽  
Vol 181 (14) ◽  
pp. 4381-4390 ◽  
Author(s):  
Ana M. Rodriguez ◽  
Alfred M. Spormann
Keyword(s):  
Single Cell ◽  
Myxococcus Xanthus ◽  
Gliding Motility ◽  
Open Reading Frames ◽  
Wild Type Allele ◽  
Wild Type ◽  
Isolated Cells ◽  
Type Allele ◽  
C Terminus ◽  
Mutant Cells

ABSTRACT Gliding movements of individual isolated Myxococcus xanthus cells depend on the genes of the A-motility system (agl and cgl genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of cgl mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the cglB wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed ΔcglBmutant were fully restored upon complementation with thecglB wild-type allele. Experiments with a cglBallele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate ΔcglB cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.

The effect of TAE226 on the non-small cell lung cancer including Japanese origin lung cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22103-e22103
Author(s):  
H. Otani ◽  
M. Jida ◽  
M. Takaoka ◽  
T. Kubo ◽  
T. Hayashi ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Lines ◽  
Egfr Mutation ◽  
Mutant Cell ◽  
Small Cell ◽  
Nsclc Cell ◽  
Wild Type ◽  
Small Cell Lung ◽  
Egfr Mutant ◽  
Mutant Cells

e22103 Background: Mutations in the epidermal growth factor receptor (EGFR) gene is the predictive factor for sensitivity of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer. Focal adhesion kinase (FAK) that is the downstream molecule of EGFR has been reported to be highly expressed in NSCLC suggesting novel therapeutic target of NSCLC. TAE226, dual inhibitor for FAK and insulin like growth factor-I receptor (IGF-IR), have been developed as anticancer reagent. In this study, we examined the effect of TAE226 on NSCLC from the view point of EGFR mutation status. Methods: We used NSCLC cell lines consisting of 4 EGFR mutant cell lines (PC9, H3255, HCC827, H1975) and 3 EGFR wild type cell lines (H1819, H1299, A549). We also used PC9 derived resistant cell line (RPC9). Antiproliferative effect of TAE226 on NSCLC cell lines was examined with MTS assay. The status of EGFR related molecules including its downstream signal pathway was investigated by western blotting analysis. The effect of TAE226 on xenograft mouse models was also examined. Results: TAE226 was effective on NSCLC cell lines with EGFR mutation including T790M mutation, compared to those with EGFR wild type. The value of IC50 (μmol/L) for PC-9, H3255, HCC827, H1975, RPC-9 and H1819, H1299, A549 was 0.16, 0.12, 0.086, 0.17, 0.31 and 4.7, 2.8, 1.4, respectively. Western blotting assay showed that TAE226 preferentially inhibited phosphor-EGFR and its downstream signaling mediators. We could confirm the anticancer effect of TAE226 on EGFR mutant cells was confirmed in xenograft mouse models. Conclusions: We indicated that TAE226 showed antitumor effect on EGFR mutant cell lines even T790M mutant cells. Further study is necessary to understand the mechanism of TAE226 effect on EGFR mutant cell lines. Our results suggest that TAE226 will be expected as the novel strategy for NSCLC. No significant financial relationships to disclose.

scholarly journals Localization of T25 glycoprotein in wild-type and Thy 1- mutant cells by immunofluorescence and immunoelectron microscopy.

1978 ◽  
Vol 147 (5) ◽  
pp. 1348-1354 ◽  
Author(s):  
L Y Bourguignon ◽  
R Hyman ◽  
I Trowbridge ◽  
S J Singer
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Immunoelectron Microscopy ◽  
Mutant Cell ◽  
Wild Type Cell ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Mutant Derivative ◽  
Mutant Cells

The wild-type BW5147 (Thy 1+) cell line and its Thy 1- mutant derivative BW5147 (Thy 1-a) were examined by immunofluorescence and immunoelectron microscopy for the presence of T25, the glycoprotein which bears the Thy 1 alloantigen. The wild-type cell had T25 predominantly localized on the cell surface. In the mutant cell line, T25 accumulated intracellularly and was present in a clustered distribution throughout the cytoplasm. T25 was not present on the surface of the mutant cell line in significant amount.

scholarly journals Extracellular Reduction of Hexavalent Chromium by Cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

2011 ◽  
Vol 77 (12) ◽  
pp. 4035-4041 ◽  
Author(s):  
Sara M. Belchik ◽  
David W. Kennedy ◽  
Alice C. Dohnalkova ◽  
Yuanmin Wang ◽  
Papatya C. Sevinc ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Outer Membrane ◽  
Initial Rate ◽  
Mutant Cell ◽  
Shewanella Oneidensis ◽  
Wild Type ◽  
Content Type ◽  
Transmission Electron ◽  
Mutant Cells

ABSTRACTTo characterize the roles of cytochromes MtrC and OmcA ofShewanella oneidensisMR-1 in Cr(VI) reduction, the effects of deleting themtrCand/oromcAgene on Cr(VI) reduction and the cellular locations of reduced Cr(III) precipitates were investigated. Compared to the rate of reduction of Cr(VI) by the wild type (wt), the deletion ofmtrCdecreased the initial rate of Cr(VI) reduction by 43.5%, while the deletion ofomcAor bothmtrCandomcAlowered the rate by 53.4% and 68.9%, respectively. In wt cells, Cr(III) precipitates were detected by transmission electron microscopy in the extracellular matrix between the cells, in association with the outer membrane, and inside the cytoplasm. No extracellular matrix-associated Cr(III) precipitates, however, were found in the cytochrome mutant cell suspension. In mutant cells without either MtrC or OmcA, most Cr(III) precipitates were found in association with the outer membrane, while in mutant cells lacking both MtrC and OmcA, most Cr(III) precipitates were found inside the cytoplasm. Cr(III) precipitates were also detected by scanning election microscopy on the surfaces of the wt and mutants without MtrC or OmcA but not on the mutant cells lacking both MtrC and OmcA, demonstrating that the deletion ofmtrCandomcAdiminishes the extracellular formation of Cr(III) precipitates. Furthermore, purified MtrC and OmcA reduced Cr(VI) with apparentkcatvalues of 1.2 ± 0.2 (mean ± standard deviation) and 10.2 ± 1 s−1andKmvalues of 34.1 ± 4.5 and 41.3 ± 7.9 μM, respectively. Together, these results consistently demonstrate that MtrC and OmcA are the terminal reductases used byS. oneidensisMR-1 for extracellular Cr(VI) reduction where OmcA is a predominant Cr(VI) reductase.

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