The
Drosophila genes
fat (
ft) and
dachsous (
ds) encode large atypical cadherins that collaborate to coordinately polarize cells in the plane of the epithelium (planar cell polarity) and to affect ...growth via the Warts/Hippo pathway
1–3. Ft and Ds form heterodimeric bridges that convey polarity information from cell to cell
4–7.
four-jointed (
fj) is a modulator of Ft/Ds activity that acts in a graded fashion in the abdomen, eye, and wing
8–11. Genetic evidence indicates that Fj acts via Ds and/or Ft
4, 6–9, 12, and here we demonstrate that Fj can act independently on Ds and on Ft. It has been reported that Fj has kinase activity and can phosphorylate a subset of cadherin domains of both Ft and Ds in vitro
13. We have used both cell and in vitro assays to measure binding between Ft and Ds. We find that phosphorylation of Ds reduces its affinity for Ft in both of these assays. By expressing forms of Ds that lack the defined phosphorylation sites or have phosphomimetic amino acids at these positions, we demonstrate that effects of Fj on wing size and planar polarity can be explained by Fj phosphorylating these sites.
► Four-jointed can modify the activity of both Dachsous and Fat in vivo ► Mutation of conserved residues in the Four-jointed kinase domain blocks its activity ► Four-jointed phosphorylates Dachsous at 3 specific sites to reduce its binding to Ft ► Four-jointed affects growth and planar polarity via phosphorylation at these 3 sites
The larval ventral belts of Drosophila consist of six to seven rows of denticles that are oriented, some pointing forwards, some backwards. We present evidence that denticle orientation is determined ...almost entirely by Dachsous and Fat, one of two planar cell polarity systems. If we change the distribution of Dachsous we can alter the polarity of denticles. We suggest that the orientation of the individual denticle rows, in both the anterior compartment (which mostly point backwards) and the posterior compartment (which point forwards), is determined by the opposing slopes of a Dachsous/Fat gradient. We show, by altering the concentration gradients of Dachsous during development, that we can change the polarity of the denticles made by larval cells as they progress between the first and third larval instars without mitosis.
To understand the cellular parameters that govern Drosophila wing disc regeneration, we genetically eliminated specific stripes of the wing disc along the proximodistal axis and used vein and ...intervein markers to trace tissue regeneration. We found that veins could regenerate interveins and vice versa, indicating respecification of cell fates. Moreover, respecification occurred in cells close to the wound. The newly generated domains were intercalated to fill in the missing parts. This intercalation was driven by increased proliferation, accompanied by changes in the orientation of the cell divisions. This reorientation depended on Fat (Ft) and Crumbs (Crb), which acted, at least partly, to control the activity of the effector of the Hippo pathway, Yorkie (Yki). Increased Yki, which promotes proliferation, affected the final shape and size. Heterozygous ft or crb, which normally elicit size and shape defects in regenerated wings, could be rescued by yki heterozygosity. Thus, Ft and Crb act as sensors to drive cell orientation during intercalary regeneration and control Yki levels to ensure a proper balance between proliferation and cell reorientation. We propose a model based on intercalation of missing cell identities, in which a coordinated balance between orientation and proliferation is required for normal organ shape and size.
In a patient with clinical diagnosis of Hereditary Spherocytosis and partial deficiency (50%) of red blood cell phosphoglycerate mutase (PGAM) activity, we have recently reported A. Repiso, P. Pérez ...de la Ossa, X. Avilés, B. Oliva, J. Juncá, R. Oliva, E. Garcia, J.L.L. Vives-Corrons, J. Carreras, F. Climent, Red blood cell phosphoglycerate mutase. Description of the first human BB isoenzyme mutation, Haematologica 88 (2003) (03) ECR07 the first described mutation of type B PGAM subunit that as a dimer constitutes the PGAM (EC 5.4.2.1) isoenzyme present in red blood cells. The mutation is the substitution c.690G>A (p.Met230Ile). In this report, we show that the mutated PGAM possesses an abnormal behaviour on ion-exchange chromatography and is more thermo-labile that the native enzyme. We also confirm that, similar to the PGAM isoenzymes from other sources, the BB-PGAM from human erythrocytes has a ping pong or phosphoenzyme mechanism, and that the mutation does not significantly change the
K
m and
K
i values, and the optimum pH of the enzyme. The increased instability of the mutated enzyme can account for the decreased PGAM activity in patient's red blood cells. However, the implication of a change of the
k
cat produced by the mutation cannot be discarded, since we could not determine the
k
cat value of the mutated PGAM.
Recently, we reported a new mutation of phosphoglycerate kinase (PGK), called PGK-Barcelona, which causes chronic hemolytic anemia associated with progressive neurological impairment. We found a ...140T→A substitution that produces an Ile46Asn change located at the N-domain of the enzyme and we suggested that the decrease of the PGK activity is probably related to a loss of enzyme stability. In this paper, by analyzing whole hemolysates and cloned enzymes, we show that both enzymes possess similar kinetic properties (although some differences are observed in the Km values) and the same electrophoretic mobility. However, PGK-Barcelona has higher thermal instability. Therefore, we confirm that the decrease of the red blood cell (RBC) PGK activity caused by the PGK-Barcelona mutation is more closely related to a loss of enzyme stability than to a decrease of enzyme catalytic function. Furthermore, we have measured the levels of glycolytic metabolites and adenine nucleotides in the RBC from controls and from the patient. The increase of 2,3-bisphosphoglycerate and the decrease of ATP RBC levels are the only detected metabolic changes that could cause hemolytic anemia.
Homozygous glucose phosphate isomerase (GPI) deficiency is one of the most important genetic disorders responsible for chronic non-spherocytic hemolytic anemia (CNSHA), a red blood cell autosomal ...recessive genetic disorder which causes severe metabolic alterations. In this work, we studied a patient with CNSHA due to an 82% loss of GPI activity resulting from the homozygous missense replacement in cDNA position 1040G>A, which leads to substitution of the protein residue A346H mutation. The enzyme is present in a dimeric form necessary for normal activity; the A346H mutation causes a loss of GPI capability to dimerize, which renders the enzyme more susceptible to thermolability and produces significant changes in erythrocyte metabolism.
Glycolysis is one of the principle pathways of ATP generation in cells and is present in all cell tissues; in erythrocytes, glycolysis is the only pathway for ATP synthesis since mature red cells ...lack the internal structures necessary to produce the energy vital for life. Red cell deficiencies have been detected in all erythrocyte glycolytic pathways, although their frequencies differ owing to diverse causes, such as the affected enzyme and severity of clinical manifestations. The number of enzyme deficiencies known is endless. The most frequent glycolysis abnormality is pyruvate kinase deficiency, since around 500 cases are known, the first of which was reported in 1961. However, only approximately 200 cases were due to mutations. In contrast, only one case of phosphoglycerate mutase BB type mutation, described in 2003, has been detected. Most mutations are located in the coding sequences of genes, while others, missense, deletions, insertions, splice defects, premature stop codons and promoter mutations, are also frequent. Understanding of the crystal structure of enzymes permits molecular modelling studies which, in turn, reveal how mutations can affect enzyme structure and function.
We previously reported the first case of red blood cell phosphoglycerate mutase (PGAM) isozyme BB deficiency due to the homozygous point mutation cDNA 690G->A, which causes a substitution of ...methionine 230 by isoleucine. In the present work we analyzed the changes in glycolytic intermediates caused by this mutation. With the exception of hexose phosphates, all other intermediates were decreased. In contrast, lactate levels were increased. The methionine 230 isoleucine change did not alter the mutated PGAM levels.
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