Wingless signaling generates pattern through two distinct mechanisms

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3727-3736 ◽  
Author(s):  
R. Hays ◽  
G.B. Gibori ◽  
A. Bejsovec
Keyword(s):  
Cell Fate ◽  
Cellular Response ◽  
Biological Activities ◽  
Essential Feature ◽  
Structural Features ◽  
Protein Distribution ◽  
Wild Type ◽  
Cell Fates ◽  
Amino Acid Region ◽  
Acid Region

wingless (wg) and its vertebrate homologues, the Wnt genes, play critical roles in the generation of embryonic pattern. In the developing Drosophila epidermis, wg is expressed in a single row of cells in each segment, but it influences cell identities in all rows of epidermal cells in the 10- to 12-cell-wide segment. Wg signaling promotes specification of two distinct aspects of the wild-type intrasegmental pattern: the diversity of denticle types present in the anterior denticle belt and the smooth or naked cuticle constituting the posterior surface of the segment. We have manipulated the expression of wild-type and mutant wg transgenes to explore the mechanism by which a single secreted signaling molecule can promote these distinctly different cell fates. We present evidence consistent with the idea that naked cuticle cell fate is specified by a cellular pathway distinct from the denticle diversity-generating pathway. Since these pathways are differentially activated by mutant Wg ligands, we propose that at least two discrete classes of receptor for Wg may exist, each transducing a different cellular response. We also find that broad Wg protein distribution across many cell diameters is required for the generation of denticle diversity, suggesting that intercellular transport of the Wg protein is an essential feature of pattern formation within the epidermal epithelium. Finally, we demonstrate that an 85 amino acid region not conserved in vertebrate Wnts is dispensable for Wg function and we discuss structural features of the Wingless protein required for its distinct biological activities.

Sectors expressing the homeobox gene liguleless3 implicate a time-dependent mechanism for cell fate acquisition along the proximal-distal axis of the maize leaf

Development ◽  
1997 ◽  
Vol 124 (24) ◽  
pp. 5097-5106 ◽  
Author(s):  
G.J. Muehlbauer ◽  
J.E. Fowler ◽  
M. Freeling
Keyword(s):  
Cell Fate ◽  
Homeobox Gene ◽  
Longitudinal Axis ◽  
Cell Types ◽  
Transverse Dimension ◽  
Leaf Sheath ◽  
Wild Type ◽  
Cell Fates ◽  
Maize Leaf ◽  
Gradual Process

The longitudinal axis of the maize leaf is composed of, in proximal to distal order, sheath, ligule, auricle and blade. The semidominant Liguleless3-O (Lg3-O) mutation disrupts leaf development at the ligular region of the leaf midrib by transforming blade to sheath. In a previous study, we showed that leaf sectors of Lg3 mutant activity are cell nonautonomous in the transverse dimension and can confer several alternative developmental fates (Fowler, Muehlbauer and Freeling (1996) Genetics 143, 489–503). In our present study we identify five Lg3 sector types in the leaf: sheath-like with displaced ligule (sheath-like), sheath-like with ectopic ligule (ectopic ligule), auricle-like, macro-hairless blade and wild-type blade. The acquisition of a specific sector fate depends on the timing of Lg3 expression. Early Lg3 expression results in adoption of the sheath-like phenotype at the ligule position (a proximal cell fate), whereas later Lg3 expression at the same position results in one of the more distal cell fates. Furthermore, sheath-like Lg3 sectors exhibit a graded continuum of phenotypes in the transformed blade region from the most proximal (sheath) to the most distal (wild-type blade), suggesting that cell fate acquisition is a gradual process. We propose a model for leaf cell fate acquisition based on a timing mechanism whereby cells of the leaf primordium progress through a maturation schedule of competency stages which eventually specify the cell types along the proximal to distal axis of the leaf. In addition, the lateral borders between Lg3 ‘on’ sectors and wild-type leaf sometimes provide evidence of no spreading of the transformed phenotype. In these cases, competency stages are inherited somatically.

The Regulatory Function Of Amino Acid Region 473-487 Of Prothrombin During Coagulation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3574-3574
Author(s):  
Joesph R Wiencek ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Fold Increase ◽  
The United States ◽  
Regulatory Function ◽  
Wild Type ◽  
Amino Acid Region ◽  
Initial Cleavage ◽  
Acid Region ◽  
Thrombin Formation

Abstract Background In the United States, every thirty nine seconds an individual dies from complications from Cardiovascular Diseases. Persistent thrombus formation at the genesis of these diseases, such as stroke and other coagulation disorders, has no full model to date. Intrinsically blood clots are produced due to excessive/unnecessary thrombin formation, which leads to the conversion of fibrinogen to fibrin. As a result the regulation of thrombin formation is critical in controlling clot generation. Upon vasculature damage, the proteolytic conversion of prothrombin (Pro) to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of calcium ions. Although fXa is capable of activating Pro through the initial cleavage at Arg271 followed by the cleavage at Arg320 (pre2 pathway), it would take approximately six months to form a clot. However, the incorporation of fVa into prothrombinase results in a five-fold increase in the catalytic efficiency of fXa for thrombin generation and the order of cleavages reversed (meizo pathway). Thus the timely arrest of unwarranted bleeding is due to the assembly of prothrombinase at the site of injury. Inevitably the presence and absence of fVa dictates the pathway of Pro activation and previous studies have suggested that fXa interacts with Pro within amino acid region 473-487 in a fVa-dependent manner. Aim To evaluate the role amino acid region 473-487 of Pro has in coagulation. Methods A recombinant Pro molecule with the region 473-487 was deleted (rProΔ473-487) using site-directed mutagenesis. Methotrexate was used for selection to stably transfect BHK-21 cells with rProΔ473-487 and wild-type Pro (rProWT). The two recombinant molecules were purified according to a well-established protocol and, at the last step, Fast Performance Liquid Chromatography was used equipped with a strong anionic Mono-Q 5/50 column. Properly carboxylated rProΔ473-487 and rProWT was isolated and removed from the column by utilizing a calcium gradient. Subsequently Pro deficient plasma was used to assess the molecules clotting activities on a Diagnostica Stago STart® 4 Hemostasis Analyzer. Gel electrophoresis was used to evaluate both recombinant molecules and their ability to generate active thrombin by either the multifaceted prothrombinase or fXa alone. Further studies were then performed using generated recombinant thrombin from the recombinant Pro molecules to investigate in their ability to activate procofactors V (fV) & VIII (fVIII). Results The investigation into the Activated Partial Thromboplastin Time [APTT] revealed clotting activity for human Pro and rProWT to be comparable, whereas rProΔ473-487 was substantially limited in the process of forming a fibrin clot. Next gel electrophoresis and scanning densitometry indicated the consumption of rProΔ473-487 by prothrombinase and subsequent thrombin formation was decreased 24-fold when compared to rProWT. In contrast membrane-bound fXa alone, in the absence of fVa, exhibited a 6-fold increase in the rate of initial cleavage Arg271 and subsequent activation of rProΔ473-487. Both recombinant Pro molecules demonstrated a similar cleavage pattern of activation equivalent with human Pro suggesting no structural alterations took place in rProΔ473-487following the mutation. Furthermore, generated human thrombin and recombinant wild-type thrombin were found to activate fV and fVIII within five minutes while the recombinant mutant thrombin was impeded in the activation process out to three hours. Conclusion Overall the data demonstrate that amino acid sequence 473-487 of Pro plays a preeminent role in 1) timely activation of Pro at initial cleavage Arg320 by prothrombinase, and 2) suitable macromolecular procofactor activation. Thus there is incisive rationale why no major mutations have been identified in this dynamic region which would be problematic for inherent physiological hemostasis. Disclosures: No relevant conflicts of interest to declare.

New Derivatives of Ascomycin with Modifications in the Amino Acid Region - Synthesis and Biological Activities, and X-Ray Crystal Structure of 5,6-Dehydroascomycin

2009 ◽  
Vol 92 (5) ◽  
pp. 839-889 ◽  
Author(s):  
Murty A. R. C. Bulusu ◽  
Peter Waldstätten ◽  
Thomas Tricotet ◽  
Christophe Rochais ◽  
Andrea Steck ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Biological Activities ◽  
X Ray ◽  
Amino Acid Region ◽  
Derivatives Of ◽  
Acid Region

scholarly journals Polycomb and Notch signaling regulate cell proliferation potential duringCaenorhabditis eleganslife cycle

2018 ◽  
Vol 2 (1) ◽  
pp. e201800170 ◽  
Author(s):  
Francesca Coraggio ◽  
Ringo Püschel ◽  
Alisha Marti ◽  
Peter Meister
Keyword(s):  
Cell Proliferation ◽  
Notch Signaling ◽  
Cell Fate ◽  
Essential Feature ◽  
Wild Type ◽  
H3k27 Methylation ◽  
Histone 3 ◽  
Proliferation And Differentiation ◽  
Multicellular Organisms ◽  
Proliferation Potential

Stable cell fate is an essential feature for multicellular organisms in which individual cells achieve specialized functions.Caenorhabditis elegansis a great model to analyze the determinants of cell fate stability because of its invariant lineage. We present a tractable cell fate challenge system that uses the induction of fate-specifying transcription factors. We show that wild-type differentiated animals are highly resistant to fate challenge. Removal of heterochromatin marks showed marked differences: the absence of histone 3 lysine 9 methylation (H3K9) has no effect on fate stability, whereas Polycomb homologmes-2mutants lacking H3K27 methylation terminally arrest larval development upon fate challenge. Unexpectedly, the arrest correlated with widespread cell proliferation rather than transdifferentiation. Using a candidate RNAi larval arrest-rescue screen, we show that the LIN-12Notchpathway is essential for hyperplasia induction. Moreover, Notch signaling appears downstream of food-sensing pathways, as dauers and first larval stage diapause animals are resistant to fate challenge. Our results demonstrate an equilibrium between proliferation and differentiation regulated by Polycomb and Notch signaling in the soma during the nematode life cycle.

Identification of Sulfo-Tyrosines of Factor V.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1731-1731
Author(s):  
Evrim Erdogan ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Factor Xa ◽  
Factor V ◽  
Wild Type ◽  
Single Chain ◽  
Amino Acid Region ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Cofactor Activity ◽  
Acid Region

Abstract The factor Va molecule is the essential cofactor of the prothrombinase complex. This complex composed of factor Xa and factor Va assembled on a platelet membrane-surface in the presence of Ca2+ ions converts membrane-bound prothrombin to thrombin. Single chain factor V does not bind factor Xa. Single-chain factor V is cleaved by thrombin first at Arg709 followed by cleavages at Arg1018 and Arg1545 to produce the heavy and light chains of the active cofactor (factor Va) and two activation peptides. Efficient thrombin cleavage and activation of factor V is essential for cofactor function and requires tyrosine sulfation. Tyrosine sulfation of factor V also appears to regulate its activity. Seven tyrosine residues in factor V, Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, and Tyr1565 have been identified as potential sites of sulfation. However, which residues are sulfated and their contribution to procofactor activation and cofactor function still remain to be elucidated. Two of the sulfation sites Tyr696 and Tyr698 are located in the acidic amino acid region near to the first required thrombin cleavage site at Arg709. Recent data demonstrated that these residues are essential for factor V activation and cofactor activity. Another acidic amino acid region, 1490–1520 is adjacent to the thrombin cleavage site at Arg1545 required for light chain formation. This region also contains three potential sulfation sites at residues 1494, 1510, and 1515 and was shown to be required for optimum procofactor activation. To ascertain which of these three residues is important for procofactor activation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations 1493DY1494→AF, 1508DDY1510→AAF and 1514DY1515→AF. The clotting and cofactor activity of the 1493DY1494→AF and 1514DY1515→AF mutants was similar to the clotting activity observed with the wild type recombinant factor Va molecule following activation by thrombin or RVV-V activator. In contrast, under similar experimental conditions recombinant factor V with the substitution 1508DDY1510→AAF was deficient in its clotting activity and had impaired cofactor activity. Moreover, following prolonged incubation with thrombin, no light chain formation was observed in the factor V molecule bearing the 1508DDY1510→AAF mutation. Thus, amino acid residues 1508–1510 of factor V are required for thrombin interaction with the procofactor which in turn appears necessary for cleavage at Arg1545. Studies of sulfated proteins have shown that the effect of sulfo-tyrosines on protein structure/function can be preserved by replacing them with glutamic acid. To explicitly identify the sulfated tyrosines on the factor V molecule, we mutated Tyr696, Tyr698 and Tyr1510 to glutamic acid and transfected them into COS-7L cells. Expression was performed in the presence of media containing or devoid of sulfate. In the presence of sulfate, the cofactor and clotting activities of the DY696DY698→DEDE and DDY1510→DDE mutants, separately were similar to the wild type recombinant factor Va molecule. However, in the absence of sulfate, the wild type and the mutant recombinant factor V molecules had both impaired cofactor activity and clotting activity following their activation with thrombin. However, their respective activity was higher than the activity of the factor V molecule bearing the 1508DDY1510→AAF mutation. Our data suggest that residues 696, 698, and 1510 of factor V appear to be sulfated and might be important for procofactor activation and cofactor function.

scholarly journals A tetratricopeptide repeat mutation in yeast transcription factor IIIC131 (TFIIIC131) facilitates recruitment of TFIIB-related factor TFIIIB70.

1997 ◽  
Vol 17 (12) ◽  
pp. 7119-7125 ◽  
Author(s):  
R D Moir ◽  
I Sethy-Coraci ◽  
K Puglia ◽  
M D Librizzi ◽  
I M Willis
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Tetratricopeptide Repeat ◽  
Related Factor ◽  
Wild Type ◽  
Dna Complexes ◽  
Amino Acid Region ◽  
Pol Iii ◽  
Acid Region

Transcription factor IIIC (TFIIIC) plays an important role in assembling the initiation factor TFIIIB on genes transcribed by RNA polymerase III (Pol III). In Saccharomyces cerevisiae, assembly of the TFIIIB complex by promoter-bound TFIIIC is thought to be initiated by its tetratricopeptide repeat (TPR)-containing subunit, TFIIIC131, which interacts directly with the TFIIB-related factor, TFIIIB70/Brf1. In this work, we have identified 10 dominant mutations in TFIIIC131 that increase Pol III gene transcription. All of these mutations are found within a discrete 53-amino-acid region of the protein encompassing TPR2. Biochemical studies of one of the mutations (PCF1-2) show that the increase in transcription is due to an increase in the recruitment of TFIIIB70 to TFIIC-DNA. The PCF1-2 mutation does not affect the affinity of TFIIIC for DNA, and the differential in both transcription and TFIIIB complex assembly is observed at saturating levels of TFIIIB70. This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB70 and suggests that the increased recruitment of this factor is achieved by a nonequilibrium binding mechanism. A novel mechanism of activation in which the TPR mutations facilitate a conformational change in TFIIIC that is required for TFIIIB70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.

scholarly journals Stability and dynamics interrelations in a Lipase: Mutational and MD simulations based investigations

2019 ◽  
Author(s):  
Tushar Ranjan Moharana ◽  
Virendra Kumar ◽  
N. Madhusudhana Rao
Keyword(s):  
Amino Acid ◽  
Outer Region ◽  
Md Simulations ◽  
Parameter Analysis ◽  
Amide Bond ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Amino Acid Region ◽  
Non Local ◽  
Acid Region

AbstractDynamics plays crucial role in the function and stability of proteins. Earlier studies have provided ambivalent nature of these interrelations. Epistatic effects of amino acid substitutions on dynamics are an interesting strategy to investigate such relations. In this study we investigated the interrelation between dynamics with that of stability and activity ofBacillus subtilislipase (BSL) using experimental and molecular dynamics simulation (MDS) approaches. Earlier we have identified many stabilising mutations in BSL using directed evolution. In this study these stabilizing mutations were clustered based on their proximity in the sequence into four groups (CM1 to 4). Activity, thermal stability, protease stability and aggregations studies were performed on these four mutants, along with the wild type BSL, to conclude that the mutations in each region contributed additively to the overall stability of the enzyme without suppressing the activity. Root mean square fluctuation and amide bond squared order parameter analysis from MDS revealed that dynamics has increased for CM1, CM2 and CM3 compared to the wild type in the amino acid region 105 to 112 and for CM4 in the amino acid region 22 to 30. In all the mutants core regions dynamics remained unaltered, while the dynamics in the rigid outer region (RMSF <0.05 nm) has increased. Alteration in dynamics, took place both in the vicinity (CM2, 0.41 nm) as well as far away from the mutations (CM1, 2.6 nm; CM3 1.5 nm; CM4 1.7 nm). Our data suggests that enhanced dynamics in certain regions in a protein may actually improve stability.Statement of SignificanceHow does a protein readjust its dynamics upon incorporation of an amino acid that improved its stability? Are the stabilizing effects of a substitution being local or non-local in nature? While there is an excellent documentation (from x-ray studies) of both local and non-local adjustments in interactions upon incorporation of a stabilizing mutations, the effect of these on the protein dynamics is less investigated. The stability and MD data presented here on four mutants, stabilized around four loop regions of a lipase, suggests that stabilizing effects of these mutations influence two specific regions leaving rest of the protein unperturbed. In addition, our data supports, observations by others, wherein enhancement in stability in a protein need not result in dampening of dynamics of a protein.

scholarly journals A 70 Amino Acid Region within the Semaphorin Domain Activates Specific Cellular Response of Semaphorin Family Members

Neuron ◽  
1997 ◽  
Vol 19 (3) ◽  
pp. 531-537 ◽  
Author(s):  
Adam M Koppel ◽  
Leonard Feiner ◽  
Hiroaki Kobayashi ◽  
Jonathan A Raper
Keyword(s):  
Amino Acid ◽  
Cellular Response ◽  
Family Members ◽  
Amino Acid Region ◽  
Acid Region

scholarly journals A gene expression atlas of abicoid-depleted Drosophila embryo reveals early canalization of cell fate

2014 ◽  
Author(s):  
Max V Staller ◽  
Charless C Fowlkes ◽  
Meghan D.J. Bragdon ◽  
Zeba B. Wunderlich ◽  
Angela DePace
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Early Stage ◽  
Expression Patterns ◽  
Drosophila Embryo ◽  
Wild Type ◽  
Cell Fates ◽  
Gene Expression Atlas ◽  
Expression Atlas ◽  
Gene Regulatory

In developing embryos, gene regulatory networks canalize cells towards discrete terminal fates. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos depleted of a key maternal input, bicoid (bcd), by building a cellular- resolution gene expression atlas containing measurements of 12 core patterning genes over 6 time points in early development. With this atlas, we determine the precise perturbation each cell experiences, relative to wild type, and observe how these cells assume cell fates in the perturbed embryo. The first zygotic layer of the network, consisting of the gap and terminal genes, is highly robust to perturbation: all combinations of transcription factor expression found in bcd depleted embryos were also found in wild type embryos, suggesting that no new cell fates were created even at this very early stage. All of the gap gene expression patterns in the trunk expand by different amounts, a feature that we were unable to explain using two simple models of the effect of bcd depletion. In the second layer of the network, depletion of bcd led to an excess of cells expressing both even skipped and fushi tarazu early in the blastoderm stage, but by gastrulation this overlap resolved into mutually exclusive stripes. Thus, following depletion of bcd, individual cells rapidly canalize towards normal cell fates in both layers of this gene regulatory network. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further modeling of canalization in this transcriptional network.

scholarly journals The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.

1993 ◽  
Vol 4 (9) ◽  
pp. 871-884 ◽  
Author(s):  
L Spinardi ◽  
Y L Ren ◽  
R Sanders ◽  
F G Giancotti
Keyword(s):  
Cell Surface ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Type Iii ◽  
Amino Acid Region ◽  
C Terminus ◽  
Mutant Forms ◽  
Terminal Pair ◽  
Acid Region

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.

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