scholarly journals Dictyostelium RasG Is Required for Normal Motility and Cytokinesis, But Not Growth

1997 ◽  
Vol 138 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Richard I. Tuxworth ◽  
Janet L. Cheetham ◽  
Laura M. Machesky ◽  
George B. Spiegelmann ◽  
Gerald Weeks ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Cell Movement ◽  
Ras Proteins ◽  
Wild Type ◽  
Principal Role ◽  
Ras Protein ◽  
Wide Range ◽  
And Control ◽  
Mutant Cells ◽  
Lamellipodia Formation

RasG is the most abundant Ras protein in growing Dictyostelium cells and the closest relative of mammalian Ras proteins. We have generated null mutants in which expression of RasG is completely abolished. Unexpectedly, RasG− cells are able to grow at nearly wild-type rates. However, they exhibit defective cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, absence of normal lamellipodia, formation of unusual small, punctate polymerized actin structures, and a large number of abnormally long filopodia. Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG− cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture. Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton.

Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5071-5076
Author(s):  
C A Hrycyna ◽  
S Clarke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Direct Result ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Ras Protein ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Mutant Cells

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

scholarly journals A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

1989 ◽  
Vol 108 (3) ◽  
pp. 985-995 ◽  
Author(s):  
E André ◽  
M Brink ◽  
G Gerisch ◽  
G Isenberg ◽  
A Noegel ◽  
...  
Keyword(s):  
Cell Function ◽  
Polyclonal Antibodies ◽  
Deae Cellulose ◽  
Cell Movement ◽  
Processing System ◽  
Chemical Mutagenesis ◽  
Deficient Mutant ◽  
Wild Type ◽  
Coding Region ◽  
Mutant Cells

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

scholarly journals The number of cytokinesis nodes in mitotic fission yeast scales with cell volume

2021 ◽  
Author(s):  
Wasim A Sayyad ◽  
Thomas D Pollard
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Large Population ◽  
Yeast Cells ◽  
Wild Type ◽  
Contractile Ring ◽  
Node Number ◽  
Nmt1 Promoter ◽  
Wide Range ◽  
Mutant Cells

Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here we used the ~140 nm resolution of Airyscan confocal microscopy to resolve a large population of dim, unitary cytokinesis nodes in 3D reconstructions of whole fission yeast cells. Wild-type fission yeast cells make about 200 unitary cytokinesis nodes. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although wide rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The surface density of Pom1 kinase on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, varying protein concentrations with the nmt1 promoter showed that the numbers of nodes increase above a baseline of about 200 with the total cellular concentration of either Pom1 or the kinase Cdr2.

scholarly journals Gliding Mutants of Myxococcus xanthuswith High Reversal Frequencies and Small Displacements

1999 ◽  
Vol 181 (8) ◽  
pp. 2593-2601 ◽  
Author(s):  
Alfred M. Spormann ◽  
Dale Kaiser
Keyword(s):  
Single Cell ◽  
Cell Movement ◽  
Gliding Motility ◽  
Wild Type ◽  
Epistasis Analysis ◽  
Type Iv ◽  
Reversal Frequency ◽  
Order Of Magnitude ◽  
Dependent Cell ◽  
Mutant Cells

ABSTRACT Myxococcus xanthus cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the mgl and frz genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective inmglAB or in cglB, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min−1for ΔmglAB mutants and 2.7 min−1 forcglB mutants, compared to 0.17 min−1 for wild-type cells). The average gliding speed of ΔmglABmutant cells was 40% of that of wild-type cells (on average 1.9 μm/min for ΔmglAB mutants, compared to 4.4 μm/min for wild-type cells). The mglA-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein: mglB mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min−1 and an average speed of 2.6 μm/min. These values range between those exhibited by wild-type cells and by ΔmglAB mutant cells. Epistasis analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation, but not a frzE mutation, partially suppressed themglA phenotype. In contrast to mgl mutants,cglB mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern ofmglAB cells was only partially reduced by apilR mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S motility.

The RHO GTPASE CDC42 Regulator CDC42GAP Plays a Critical Role in Neutrophil Migration Via Podosome-Like Formation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 651-651 ◽  
Author(s):  
Marie-Dominique Filippi ◽  
Haiming Xu ◽  
Jason Towe ◽  
Chad E. Harris ◽  
Kathleen Szczur ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Rho Gtpase ◽  
Critical Role ◽  
Neutrophil Migration ◽  
Cell Movement ◽  
Directed Migration ◽  
Mutant Cells

Abstract Neutrophils (PMN) are a critical cell in inflammatory processes. In response to environmental stimuli, they activate various signal transduction pathways allowing them to move rapidly to a site of microbial invasion and to perform phagocytosis, cytokine and oxygen substrate release. Rho GTPase proteins, Rac1, Rac2, CDC42 and Rho, are central regulators of cell movement via actin rearrangement. We have demonstrated the specific role of Rac1 and Rac2 in PMN functions (Gu and Filippi et al, Science 2003; Filippi et al. Nat Immuol., 2004) which raises the question of the specificity of the other Rho GTPases. CDC42 primarily regulates the formation of filopodia. CDC42 controls cell polarity and migration in hematopoietic cell lines. Most of previous studies have utilized dominant active or negative mutants which lack specificity and cannot be easily used to define in vivo cell biology. Here, we used mice genetically deficient in the CDC42 negative regulator CDC42 GTPase Activating Protein (GAP) to study the role of CDC42 in neutrophil functions in vitro and in vivo. Heterozygote (CDC42GAP+/−) or homozygote (CDC42GAP−/−) mutant mice displayed normal neutrophil differentiation in vitro and in vivo. PMN deficient in CDC42GAP displayed 2-fold increased in CDC42 activity. In vivo recruitment of PMN in peritoneal cavities after thioglycollate exposure was significantly impaired in CDC42GAP+/− mice compared with wild type (WT) mice (25.5±0.76 x 105 vs 35.7±0.38 x 105, p<0.05). Both CDC42GAP+/− and CDC42GAP−/− PMN demonstrated defective directed migration in vitro in response to fMLP in a Boyden chamber assay compared with WT (248±31 and 199±20 versus 314±29 migrated cells, p<0.05), suggesting that CDC42 plays a critical role in neutrophil migration in vitro and in vivo. To further understand the role of CDC42GAP in neutrophil migration, single-cell analysis by time-lapse videomicroscopy was performed. Surprisingly, CDC42GAP+/− PMN demonstrated higher migration velocity compared with WT cells in response to fMLP, but this increased speed was associated with an abnormal shape. Upon beta-2 integrin ligation, CDC42GAP+/− PMN demonstrated abnormal elongated trailing tail associated with increased tail filopodia. Importantly, the podosome-like structures identified by a vinculin ring surrounding F-actin at the ventral plasma membrane that are present in the leading edge of WT PMN was absent in the mutant cells. CDC42GAP−/− PMN demonstrated more dramatic F-actin impairment upon integrin ligation compared with CDC42GAP+/− and WT cells and remarkably showed complete loss of cell polarity, consistent with the known role of CDC42 in cell polarity. We hypothesize that the lack of podosome formation in mutant cells could account for the increased speed observed in CDC42GAP+/− cells and therefore result in ineffective directed migration in vivo. Altogether, this suggests that regulation of CDC42 activity plays a pivotal role in neutrophil migration likely via integrin-dependent podosome-like formation. This reinforces the importance of turnover of attachment structures during cell movement and suggests a new role for CDC42 in actin-based attachment structure in neutrophils.

scholarly journals Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (10) ◽  
pp. 5071-5076 ◽  
Author(s):  
C A Hrycyna ◽  
S Clarke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Direct Result ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Ras Protein ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Mutant Cells

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

scholarly journals Mutations in the SHR5 gene of Saccharomyces cerevisiae suppress Ras function and block membrane attachment and palmitoylation of Ras proteins.

1995 ◽  
Vol 15 (3) ◽  
pp. 1333-1342 ◽  
Author(s):  
V Jung ◽  
L Chen ◽  
S L Hofmann ◽  
M Wigler ◽  
S Powers
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Current Data ◽  
Ras Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Protein ◽  
Membrane Attachment ◽  
Extragenic Suppressors

We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins.

scholarly journals Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (8) ◽  
pp. 3051-3057 ◽  
Author(s):  
K Mbonyi ◽  
M Beullens ◽  
K Detremerie ◽  
L Geerts ◽  
J M Thevelein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transmission ◽  
Adenyl Cyclase ◽  
Gtpase Activity ◽  
Ras Proteins ◽  
Wild Type ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Camp Synthesis

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

Expression of a mutated ras gene in Dictyostelium discoideum alters the binding of cyclic AMP to its chemotactic receptor

1988 ◽  
Vol 90 (4) ◽  
pp. 701-706
Author(s):  
M.E. Luderus ◽  
C.D. Reymond ◽  
P.J. Van Haastert ◽  
R. Van Driel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cell Types ◽  
Ras Proteins ◽  
Wild Type ◽  
Ras Gene ◽  
Physiological Level ◽  
Ras Protein ◽  
Type Gene

Dictyostelium discoideum cells contain a ras gene that codes for a polypeptide that is highly homologous to the human ras proteins. Extra copies of the wild-type gene or a gene carrying a missense mutation in codon 12 (ras-Gly12 and ras-Thr12, respectively) have been introduced into Dictyostelium cells by transformation. We have investigated the properties of the chemotactic cell surface cyclic AMP receptor in crude membrane preparations of wild-type Dictyostelium cells and ras-Gly12 and ras-Thr12 transformants. In vitro, an ATP- and Ca2+-dependent reduction of the number of cyclic AMP receptors was observed in membranes from all three cell types. The number of available receptors was decreased maximally by about 50%. In the presence of ATP the half-maximal Ca2+ concentration required for this process was about 10(−5) M in wild-type and ras-Gly12 membranes, and less than 10(−7) M in ras-Thr12 membranes. Addition of GTP (but not GDP) or the phorbol ester PMA (phorbol-12-myristate-13-acetate) reduced the Ca2+ requirement of the process in wild-type and ras-Gly12 membranes to the physiological level of less than 10(−7) M. In membranes derived from ras-Thr12 cells addition of GTP or PMA had no effect. The results indicate that D. discoideum cells contain a cyclic AMP receptor-controlling pathway that can be activated in vitro and involves a GTP-binding protein and a Ca2+ plus ATP-dependent activity, possibly protein kinase C. It is concluded that the ras protein specifically interacts with this pathway; the pathway appears to be constitutively activated by the mutated ras gene product.

A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent

2021 ◽  
Author(s):  
Sara A. Kirolos ◽  
Richard H. Gomer
Keyword(s):  
G Protein ◽  
Cell Movement ◽  
Cortical Activation ◽  
Cell Cortex ◽  
Ras Proteins ◽  
Ras Protein ◽  
Chemical Gradients ◽  
Ras Activation ◽  
Extracellular Signal ◽  
A Cell

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein-coupled receptor GrlH, the G protein subunits Gβ and Gα8, Ras protein RasG, protein kinase B, the p-21 activated kinase PakD, and the extracellular signal-regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allow cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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