Localization of endogenous and recombinant Na+-driven anion exchanger protein NDAE1 fromDrosophila melanogaster

2001 ◽  
Vol 281 (2) ◽  
pp. C449-C463 ◽  
Author(s):  
Christopher M. Sciortino ◽  
Lamara D. Shrode ◽  
Bonnie R. Fletcher ◽  
Peter J. Harte ◽  
Michael F. Romero
Keyword(s):  
Xenopus Oocytes ◽  
Alimentary Tract ◽  
Regulatory Protein ◽  
Malpighian Tubule ◽  
Cell Types ◽  
Malpighian Tubules ◽  
Developmental Expression ◽  
Α Subunit ◽  
Vertebrate Cell ◽  
Exchange Activity

Na+-dependent Cl−/HCO[Formula: see text]exchange activity helps maintain intracellular pH (pHi) homeostasis in many invertebrate and vertebrate cell types. Our laboratory cloned and characterized a Na+-dependent Cl−/HCO[Formula: see text] exchanger (NDAE1) from Drosophila melanogaster (Romero MF, Henry D, Nelson S, Harte PJ, and Sciortino CM. J Biol Chem 275: 24552–24559, 2000). In the present study we used immunohistochemical and Western blot techniques to characterize the developmental expression, subcellular localization, and tissue distribution of NDAE1 protein in D. melanogaster. We have shown that a polyclonal antibody raised against the NH2terminus of NDAE1 (αCWR57) recognizes NDAE1 electrophysiologically characterized in Xenopus oocytes. Moreover, our results begin to delineate the NDAE1 topology, i.e., both the NH2and COOH termini are intracellular. NDAE1 is expressed throughout Drosophila development in the central and peripheral nervous systems, sensilla, and the alimentary tract (Malpighian tubules, gut, and salivary glands). Coimmunolabeling of larval tissues with NDAE1 antibody and a monoclonal antibody to the Na+-K+-ATPase α-subunit revealed that the majority of NDAE1 is located at the basolateral membranes of Malpighian tubule cells. These results suggest that NDAE1 may be a key pHi regulatory protein and may contribute to basolateral ion transport in epithelia and nervous system of Drosophila.

Fine Structure of the Malpighian Tubules of the Mosquito, Culex pipiens

1971 ◽  
Vol 29 ◽  
pp. 402-403
Author(s):  
Brendan Clifford
Keyword(s):  
Fine Structure ◽  
Culex Pipiens ◽  
Stellate Cell ◽  
Malpighian Tubule ◽  
Cell Types ◽  
Instar Larva ◽  
Malpighian Tubules ◽  
Calliphora Erythrocephala ◽  
Distinct Cell ◽  
Basal Plasma

An ultrastructural investigation of the Malpighian tubules of the fourth instar larva of Culex pipiens was undertaken as part of a continuing study of the fine structure of transport epithelia.Each of the five Malpighian tubules was found to be morphologically identical and regionally undifferentiated. Two distinct cell types, the primary and stellate, were found intermingled along the length of each tubule. The ultrastructure of the stellate cell was previously described in the Malpighian tubule of the blowfly, Calliphora erythrocephala by Berridge and Oschman.The basal plasma membrane of the primary cell is extremely irregular, giving rise to a complex interconnecting network of basal channels. The compartments of cytoplasm entrapped within this system of basal infoldings contain mitochondria, free ribosomes, and small amounts of rough endoplasmic reticulum. The mitochondria are distinctive in that the cristae run parallel to the long axis of the organelle.

A study of the Malpighian tubules of the plecopteran nymph Paragnetina media (Walker) (Plecoptera: Perlidae) by light, scanning electron, and transmission electron microscopy

1994 ◽  
Vol 72 (9) ◽  
pp. 1566-1575 ◽  
Author(s):  
N. N. Kapoor
Keyword(s):  
Malpighian Tubule ◽  
Stellate Cells ◽  
Basal Membrane ◽  
Cell Types ◽  
Distal Segment ◽  
Malpighian Tubules ◽  
Primary Cells ◽  
Transmission Electron ◽  
Total Cell Population ◽  
Paragnetina Media

The present study concerns the structural details of the Malpighian tubules in the nymph of the stonefly Paragnetina media. There is no external segmentation except for a distal short hyaline segment. The tubules are composed of two cell types: primary and stellate. Primary cells in the proximal and middle portions of the tubule have short infoldings of the basal membrane and the cytosol is packed with laminate spheres. Cells of the distal segment possess long and tightly packed membrane folds but are devoid of laminate spheres. The stellate cells are sparsely distributed in the middle region and make up 12% of the total cell population in the Malpighian tubule; they lack laminate spheres. Long processes of the stellate cells extend between adjacent primary cells to the luminal and outer surfaces of the tubule.

scholarly journals A cell atlas of the fly kidney

2021 ◽  
Author(s):  
Jun Xu ◽  
Yifang Liu ◽  
Hongjie Li ◽  
Alexander J. Tarashansky ◽  
Colin H. Kalicki ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Kidney Cell ◽  
Single Cells ◽  
Malpighian Tubule ◽  
Cell Types ◽  
Malpighian Tubules ◽  
Disease Models ◽  
Marker Genes ◽  
Waste Products ◽  
Renal Stem Cells

Like humans, insects rely on precise regulation of their internal environments to survive. The insect renal system consists of Malpighian tubules and nephrocytes that share similarities to the mammalian kidney. Studies of the Drosophila Malpighian tubules and nephrocytes have provided many insights into our understanding of the excretion of waste products, stem cell regeneration, protein reabsorption, and as human kidney disease models. Here, we analyzed single-nucleus RNA sequencing (snRNA-seq) data sets to characterize the cell types of the adult fly kidney. We identified 11 distinct clusters representing renal stem cells (RSCs), stellate cells (SCs), regionally specific principal cells (PCs), garland nephrocyte cells (GCs) and pericardial nephrocytes (PNs). Analyses of these clusters revealed many new interesting features. For example, we found a new, previously unrecognized cell cluster: lower segment PCs that express Esyt2. In addition, we find that the SC marker genes RhoGEF64c, Frq2, Prip and CG10939 regulate their unusual cell shape. Further, we identified transcription factors specific to each cluster and built a network of signaling pathways that are potentially involved in mediating cell-cell communication between Malpighian tubule cell types. Finally, cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia - knowledge that will help the generation of kidney disease models. To visualize this dataset, we provide a web-based resource for gene expression in single cells (https://www.flyrnai.org/scRNA/kidney/). Altogether, our study provides a comprehensive resource for addressing gene function in the fly kidney and future disease studies.

A Malpighian Tubule Lime Gland in an Insect Inhabiting Alkaline Salt Lakes

1989 ◽  
Vol 145 (1) ◽  
pp. 63-78 ◽  
Author(s):  
DAVID B. HERBST ◽  
TIMOTHY J. BRADLEY
Keyword(s):  
Malpighian Tubule ◽  
Cell Types ◽  
Chemical Precipitation ◽  
Malpighian Tubules ◽  
Salt Lakes ◽  
Alkaline Salt ◽  
X Ray ◽  
Posterior Pair ◽  
High Concentrations ◽  
Scanning Electron

The alkali fly, Ephydra hians Say, inhabits alkaline salt lakes which can contain concentrations of dissolved carbonate and bicarbonate as high as 500 mmol l−1. Larvae of the alkali fly possess two pairs of Malpighian tubules. The posterior pair has a morphology similar to that of the tubules of most other insects, but the anterior pair is modified into an enlarged gland containing white microsphere concretions. We describe the ultrastructure of all cell types in both pairs of tubules. Using scanning electron microscope (SEM) X-ray microanalysis and chemical CO2 quantification, we demonstrate that the concretions in the lime glands are composed of nearly pure calcium carbonate. Isolated preparations of lime gland tubules accumulate 45Ca significantly more rapidly than do normal tubules. Although similar to the rime concretions found in the Malpighian tubules of other Diptera, the lime glands of this insect may function to regulate the high concentrations of carbonate and bicarbonate encountered in their aquatic environment. It is proposed that the mechanism of this regulation may be chemical precipitation of carbonate/bicarbonate with calcium in the lumen of these specialized lime gland tubules.

scholarly journals A SLC4-like anion exchanger from renal tubules of the mosquito (Aedes aegypti): evidence for a novel role of stellate cells in diuretic fluid secretion

2010 ◽  
Vol 298 (3) ◽  
pp. R642-R660 ◽  
Author(s):  
Peter M. Piermarini ◽  
Laura F. Grogan ◽  
Kenneth Lau ◽  
Li Wang ◽  
Klaus W. Beyenbach
Keyword(s):  
Aedes Aegypti ◽  
Anion Exchanger ◽  
Xenopus Oocytes ◽  
Malpighian Tubule ◽  
Stellate Cells ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Renal Tubules ◽  
Basal Region ◽  
Principal Cells

Transepithelial fluid secretion across the renal (Malpighian) tubule epithelium of the mosquito ( Aedes aegypti ) is energized by the vacuolar-type (V-type) H+-ATPase and not the Na+-K+-ATPase. Located at the apical membrane of principal cells, the V-type H+-ATPase translocates protons from the cytoplasm to the tubule lumen. Secreted protons are likely to derive from metabolic H2CO3, which raises questions about the handling of HCO3−by principal cells. Accordingly, we tested the hypothesis that a Cl/HCO3anion exchanger (AE) related to the solute-linked carrier 4 (SLC4) superfamily mediates the extrusion of HCO3−across the basal membrane of principal cells. We began by cloning from Aedes Malpighian tubules a full-length cDNA encoding an SLC4-like AE, termed AeAE. When expressed heterologously in Xenopus oocytes, AeAE is both N- and O-glycosylated and mediates Na+-independent intracellular pH changes that are sensitive to extracellular Cl−concentration and to DIDS. In Aedes Malpighian tubules, AeAE is expressed as two distinct forms: one is O-glycosylated, and the other is N-glycosylated. Significantly, AeAE immunoreactivity localizes to the basal regions of stellate cells but not principal cells. Concentrations of DIDS that inhibit AeAE activity in Xenopus oocytes have no effects on the unstimulated rates of fluid secretion mediated by Malpighian tubules as measured by the Ramsay assay. However, in Malpighian tubules stimulated with kinin or calcitonin-like diuretic peptides, DIDS reduces the diuretic rates of fluid secretion to basal levels. In conclusion, Aedes Malpighian tubules express AeAE in the basal region of stellate cells, where this transporter may participate in producing diuretic rates of transepithelial fluid secretion.

scholarly journals CELLULAR SPECIALIZATION IN THE EXCRETORY EPITHELIA OF AN INSECT, Macrosteles fascifrons STÅL (HOMOPTERA)

1960 ◽  
Vol 8 (1) ◽  
pp. 103-133 ◽  
Author(s):  
David S. Smith ◽  
Virginia C. Littau
Keyword(s):  
Malpighian Tubule ◽  
Cell Types ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Malpighian Tubules ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Distinct Cell ◽  
Protein Components ◽  
Cellular Specialization

An electron microscopic investigation of the Malpighian tubules of a leaf hopper, Macrosteles fascifrons, shows that these organs comprise three quite distinct cell types, and the structure of these and of the mid- and hindgut epithelial cells is described. In particular, a comparison is made between the organization of the basal and apical surfaces of cells in the Malpighian tubule and in the vertebrate kidney, and it is suggested that similarities between these excretory epithelia reflect functional parallels between them. While the midgut and one region of the Malpighian tubule bear a typical microvillar brush border, elsewhere in the tubule and in the hindgut the apical surface bears cytoplasmic leaflets or lamellae. The sole solid excretory material of these insects consists of the brochosomes, secreted by cells of one region of the Malpighian tubule. The structure, geometry, and development of these unusual bodies, apparently formed within specialized Golgi regions, has been investigated, and histochemical tests indicate that they contain lipid and protein components.

Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization

1995 ◽  
Vol 268 (1) ◽  
pp. C201-C209 ◽  
Author(s):  
B. D. Humphreys ◽  
L. Jiang ◽  
M. N. Chernova ◽  
S. L. Alper
Keyword(s):  
Anion Exchanger ◽  
Cellular Response ◽  
Xenopus Oocytes ◽  
Chloride Transport ◽  
Cell Types ◽  
Dependent Manner ◽  
Hypertonic Stress ◽  
Anion Transporter ◽  
Hypertonic Stimulation ◽  
Exchange Activity

Xenopus oocytes express endogenous Na+/H+ exchange activity but lack significant endogenous Cl-/HCO3- exchange activity. Coupled operation of Na+/H+ exchange and Cl-/HCO3- exchange contributes in many cell types to the cellular response to hypertonic stress. We therefore examined in Xenopus oocytes the osmotic regulation of chloride transport mediated by recombinant anion exchanger proteins AE2 and AE1. Hypotonicity was without effect on either anion transporter. Hypertonicity activated AE2-associated 36Cl- influx and efflux in a time- and osmolarity-dependent manner, whether incremental osmoles were charged or uncharged, but had no measurable effect on AE1 function. Hypertonic stimulation of AE2 was completely inhibited by Na+ removal or by addition of amiloride. In contrast, neither maneuver altered isotonic activity of AE2. Hypertonicity also induced amiloride-sensitive elevation of oocyte intracellular pH (pHi), and shifted the sigmoidal relationship of extracellular pH vs. AE2 activity > or = 0.5 units to the acid. Injection of pH 7.4 buffer into oocytes attenuated both hypertonic alkalinization and activation of AE2-associated 36Cl- influx, without inhibition of isotonic AE2 function. These data demonstrate that recombinant AE2 expressed in Xenopus oocytes is activated by increased pHi and that hypertonic activation of AE2 is secondary to hypertonic activation of Na+/H+ exchange.

scholarly journals Infection, dissemination, and transmission efficiencies of Zika virus in Aedes aegypti after serial passage in mosquito or mammalian cell lines or alternating passage in both cell types

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lourdes G. Talavera-Aguilar ◽  
Reyes A. Murrieta ◽  
Sungmin Kiem ◽  
Rosa C. Cetina-Trejo ◽  
Carlos M. Baak-Baak ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Cell Lines ◽  
Zika Virus ◽  
Cell Types ◽  
Mosquito Cell ◽  
Cell Passage ◽  
Genetic Changes ◽  
Synonymous Substitutions ◽  
Vertebrate Cell

Abstract Background Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti. Methods An isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection. Results Genome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage. Conclusions Mosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies. Graphic abstract "Image missing"

Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-subunit of RNA polymerase

2021 ◽  
Author(s):  
Helen Camakaris ◽  
Ji Yang ◽  
Tadashi Fujii ◽  
James Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Protein ◽  
Aromatic Amino Acids ◽  
Regulation Of Transcription ◽  
Plant Interactions ◽  
Α Subunit ◽  
Partial Suppression ◽  
K 12

A novel selection was developed for RpoA α-CTD mutants altered in activation by the TyrR regulatory protein of E. coli K-12. This allowed the identification of an aspartate to asparagine substitution in residue 250 (DN250) as an Act - mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and substitutions DR250, RE310 and RD310 were all shown to be defective in activation. None of these mutations caused defects in UP regulation. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. TyrR Mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77 and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act - tyrR mutants were isolated in an rpoA + strain, adding positions R77, D97, K101, D118, R119, R121 and E141 to known residues, S95 and D103, and defining the ‘activation patch’ on the NTD of TyrR. These results support a model for activation of TyrR-regulated genes where the ‘activation patch’ on the TyrR NTD interacts with the ‘TyrR-specific patch’ on the αCTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed, and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants, as Activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene TyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence, and important plant interactions.

Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: the peptide CAP2b and cyclic GMP inhibit Malpighian tubule fluid secretion.

1997 ◽  
Vol 200 (17) ◽  
pp. 2363-2367 ◽  
Author(s):  
M C Quinlan ◽  
N J Tublitz ◽  
M J O'Donnell
Keyword(s):  
Cyclic Gmp ◽  
Physiological Role ◽  
Malpighian Tubule ◽  
Fluid Secretion ◽  
Blood Feeding ◽  
Rhodnius Prolixus ◽  
Malpighian Tubules ◽  
Tubule Fluid ◽  
Secretion Rates

Rhodnius prolixus eliminates NaCl-rich urine at high rates following its infrequent but massive blood meals. This diuresis involves stimulation of Malpighian tubule fluid secretion by diuretic hormones released in response to distention of the abdomen during feeding. The precipitous decline in urine flow that occurs several hours after feeding has been thought until now to result from a decline in diuretic hormone release. We suggest here that insect cardioacceleratory peptide 2b (CAP2b) and cyclic GMP are part of a novel mechanism of anti-diuresis. Secretion rates of 5-hydroxytryptamine-stimulated Malpighian tubules are reduced by low doses of CAP2b or cyclic GMP. Maximal secretion rates are restored by exposing tubules to 1 mmol l-1 cyclic AMP. Levels of cyclic GMP in isolated tubules increase in response to CAP2b, consistent with a role for cyclic GMP as an intracellular second messenger. Levels of cyclic GMP in tubules also increase as urine output rates decline in vivo, suggesting a physiological role for this nucleotide in the termination of diuresis.

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