Kinetic analysis of a signal-transduction pathway by time-resolved somatic complementation of mutants.

1998 ◽  
Vol 201 (13) ◽  
pp. 1991-1999 ◽  
Author(s):  
C Starostzik ◽  
W Marwan
Keyword(s):  
Signal Transduction ◽  
Blue Light ◽  
Red Light ◽  
Signal Transduction Pathway ◽  
Intermediate Phenotype ◽  
Regulatory Function ◽  
Transduction Pathway ◽  
Wild Type ◽  
Time Resolved ◽  
Small Change

Sensory control of sporulation in Physarum polycephalum plasmodia is mediated by a branched signal-transduction pathway that integrates blue light, far-red light, heat shock and the starvation state. Mutants defective in the pathway were isolated and three phenotypes obtained: blue-blind, general-blind and light-independent sporulating. When plasmodia of the blue-blind mutant Blu1 were exposed to a pulse of blue light and subsequently fused to non-induced wild-type plasmodia, the resulting heterokaryons sporulated, indicating a functional blue- light photoreceptor in the mutant. When the general-blind mutant Nos1 was fused to a wild-type plasmodium which had been induced by light, sporulation of the heterokaryon was blocked. However, the dominant inhibition of sporulation by Nos1 was gradually lost with increasing time between induction by light and time of fusion, suggesting that Nos1 can be bypassed by the time-dependent formation of a downstream signal-transduction intermediate. Phenotype expression in constitutively sporulating (Cos) mutants depended on starvation. The Cos2 product was titrated by fusing mutant plasmodia of different sizes to wild-type plasmodia of constant size and analysing the sporulation probability of the resulting heterokaryon. The titration curve indicates that a small change in the amount of Cos2 product can cause sporulation. We conclude that somatic complementation analysis allows the time-resolved evaluation of the regulatory function of mutations in a signal-transduction pathway without prior cloning of the gene. This shortcut allows us to characterize many mutants quickly and to select those for molecular analysis that display a well-defined regulatory function.

scholarly journals Mating in Saccharomyces cerevisiae: The Role of the Pheromone Signal Transduction Pathway in the Chemotropic Response to Pheromone

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 19-32 ◽  
Author(s):  
Kathrin Schrick ◽  
Barbara Garvik ◽  
Leland H Hartwell
Keyword(s):  
Signal Transduction ◽  
Map Kinase ◽  
Transcriptional Activation ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Wild Type ◽  
Mating Partner ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Wild Type Control

Abstract The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the β or γ subunit (ste4 and stel8) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

scholarly journals Function of a Chemotaxis-Like Signal Transduction Pathway in Modulating Motility, Cell Clumping, and Cell Length in the Alphaproteobacterium Azospirillum brasilense

2008 ◽  
Vol 190 (19) ◽  
pp. 6365-6375 ◽  
Author(s):  
Amber N. Bible ◽  
Bonnie B. Stephens ◽  
Davi R. Ortega ◽  
Zhihong Xie ◽  
Gladys Alexandre
Keyword(s):  
Signal Transduction ◽  
Signal Transduction Pathway ◽  
Cell Aggregation ◽  
Cell Length ◽  
Transduction Pathway ◽  
Wild Type ◽  
Cellular Functions ◽  
Swimming Pattern ◽  
A Minor ◽  
Nutritional Imbalance

ABSTRACT A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium Azospirillum brasilense. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the cheA1 (strain AB101) and the cheY1 (strain AB102) genes and a newly constructed mutant lacking the entire operon [Δ(cheA1-cheR1)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional cheB1 and cheR1 genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the cheA1 or cheY1 genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional cheA1 or cheY1 genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking cheA1 or cheY1 genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

2007 ◽  
Vol 292 (1) ◽  
pp. C423-C431 ◽  
Author(s):  
Li Liu ◽  
Yukisato Ishida ◽  
Gbolahan Okunade ◽  
Gail J. Pyne-Geithman ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Contractile Response ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Wild Type ◽  
Bladder Smooth Muscle

We previously showed that plasma membrane Ca2+-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle ( 8 ). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca2+ ([Ca2+]i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1+/−, Pmca4+/−, Pmca4−/−, and Pmca1+/− Pmca4−/− mice. There were no differences in basal [Ca2+]i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca2+]i (130–180%) in smooth muscle from Pmca1+/− and Pmca1+/− Pmca4−/− bladders than those in WT or Pmca4−/−. The responses to carbachol (CCh: 10 μM) were also greater in Pmca1+/− (120–150%) than in WT bladders. In contrast, the responses in Pmca4−/− and Pmca1+/− Pmca4−/− bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca2+]i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4−/− (130–190%) and Pmca1+/− Pmca4−/− (120–250%) bladders, but not in Pmca1+/− bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca2+ clearance, while PMCA4 is essential for the [Ca2+]i increase and contractile response to the CCh receptor-mediated signal transduction pathway.

The blue light signal transduction pathway is involved in anthocyanin accumulation in ‘Red Zaosu’ pear

Planta ◽  
2018 ◽  
Vol 248 (1) ◽  
pp. 37-48 ◽  
Author(s):  
Ruiyan Tao ◽  
Songling Bai ◽  
Junbei Ni ◽  
Qinsong Yang ◽  
Yuan Zhao ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Blue Light ◽  
Signal Transduction Pathway ◽  
Light Signal ◽  
Transduction Pathway ◽  
Anthocyanin Accumulation ◽  
Light Signal Transduction

Dissecting blue light signal transduction pathway in leaf epidermis using a pharmacological approach

Planta ◽  
2015 ◽  
Vol 242 (4) ◽  
pp. 813-827 ◽  
Author(s):  
Branka D. Živanović ◽  
Lana I. Shabala ◽  
Theo J. M. Elzenga ◽  
Sergey N. Shabala
Keyword(s):  
Signal Transduction ◽  
Blue Light ◽  
Signal Transduction Pathway ◽  
Light Signal ◽  
Transduction Pathway ◽  
Leaf Epidermis ◽  
Light Signal Transduction ◽  
Pharmacological Approach

A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis

Development ◽  
1998 ◽  
Vol 125 (10) ◽  
pp. 1909-1920 ◽  
Author(s):  
P. Qiu ◽  
P.C. Pan ◽  
S. Govind
Keyword(s):  
Signal Transduction ◽  
Constitutive Expression ◽  
Signal Transduction Pathway ◽  
Lymph Gland ◽  
Transduction Pathway ◽  
Wild Type ◽  
In The Wild ◽  
Drosophila Larva ◽  
Common Signal ◽  
Almost All

In the Drosophila larva, blood cells or hemocytes are formed in the lymph gland. The major blood cell type, called plasmatocyte, is small, non-adhesive and phagocytic. Plasmatocytes differentiate into adhesive lamellocytes to form multilayered capsules around foreign substances or, in mutant melanotic tumor strains, around self tissue. Mutations in cactus or Toll, or constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic capsules. As maternally encoded proteins, Toll, Cactus and Dorsal, along with Tube and Pelle, participate in a common signal transduction pathway to specify the embryonic dorsal-ventral axis. Using the maternal pathway as a paradigm, we investigated if these proteins have additional roles in larval hemocyte formation and differentiation. Analysis of cactus mutants that lack Cactus protein revealed that almost all of these animals have an overabundance of hemocytes, carry melanotic capsules and die before reaching pupal stages. In addition, the lymph glands of cactus larvae are considerably enlarged. The number of mitotic cells in the cactus and TollD hemolymph is higher than that in the wild-type hemolymph. The hemocyte density of mutant Toll, tube or pelle hemolymph is significantly lower than that of the wild type. Lethality of mutant cactus animals could be rescued either by the selective expression of wild-type Cactus protein in the larval lymph gland or by the introduction of mutations in Toll, tube or pelle. Cactus, Toll, Tube and Pelle proteins are expressed in the nascent hemocytes of the larval lymph gland. Our results suggest that the Toll/Cactus signal transduction pathway plays a significant role in regulating hemocyte proliferation and hemocyte density in the Drosophila larva. These findings are discussed in light of similar hematopoietic functions of Rel/I(kappa)B-family proteins in mice.

scholarly journals Different Evolutionary Constraints on Chemotaxis Proteins CheW and CheY Revealed by Heterologous Expression Studies and Protein Sequence Analysis

2003 ◽  
Vol 185 (2) ◽  
pp. 544-552 ◽  
Author(s):  
Gladys Alexandre ◽  
Igor B. Zhulin
Keyword(s):  
Signal Transduction ◽  
Sequence Analysis ◽  
Heterologous Expression ◽  
Protein Sequence ◽  
Signal Transduction Pathway ◽  
Evolutionary Constraints ◽  
Transduction Pathway ◽  
Protein Sequence Analysis ◽  
Wild Type ◽  
E Coli

ABSTRACT CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis. In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons. We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense. We analyzed the functions of divergent CheW and CheY proteins from A. brasilense by heterologous expression in E. coli wild-type and mutant strains. Both proteins were able to specifically inhibit chemotaxis of a wild-type E. coli strain; however, only CheW from A. brasilense was able to restore signal transduction in a corresponding mutant of E. coli. Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins. Multiple, but conservative, substitutions were found in CheW homologs. No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important. Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway. Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions.

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