The progeny of wingless-expressing cells deliver the signal at a distance in Drosophila embryos

Pattern formation in developing animals requires that cells exchange signals mediated by secreted proteins. How these signals spread is still unclear. It is generally assumed that they reach their target site either by diffusion or active transport (reviewed in [1] [2]). Here, we report an alternative mode of transport for Wingless (Wg), a member of the Wnt family of signaling molecules. In embryos of the fruit fly Drosophila, the wingless (wg) gene is transcribed in narrow stripes of cells abutting the source of Hedgehog protein. We found that these cells or their progeny are free to roam towards the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wg. The cells leaving the expression domain retain inherited Wg protein in secretory vesicles, however, and carry it forwards over a distance of up to four cell diameters. Experiments using a membrane-tethered form of Wg showed that this mechanism is sufficient to account for the normal range of Wg. Nevertheless, evidence exists that Wg can also reach distant target cells independently of protein inheritance, possibly by restricted diffusion. We suggest that both transport mechanisms operate in wild-type embryos.     

Producing cells retain and recycle Wingless in Drosophila embryos

There is considerable interest in the mechanisms that drive and control the spread of morphogens in developing animals. Although much attention is given to events occurring after release from expressing cells, release itself could be an important modulator of range. Indeed, a dedicated protein, Dispatched, is needed to release Hedgehog from the surface of expressing cells. We find that, in Drosophila embryos, much Wingless (as well as a GFP-Wingless fusion protein) remains tightly associated with secreting cells. Retention occurs both within the secretory pathway and at the cell surface and requires functional heparan sulfate proteoglycans. As a further means of retention, secreting cells readily endocytose Wingless protein that does reach the cell surface. Such endocytosed Wingless can in turn be sent back to the cell surface (the first direct observation of ligand recycling in live embryos). Recycling may serve to sustain high-level signaling in this region of the epidermis.     

Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% +/- 12.5% of the total virus yield compared with 0.23% +/- 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms.     

Rotavirus is released from the apical surface of cultured human intestinal cells through nonconventional vesicular transport that bypasses the Golgi apparatus.

Rotaviruses are nonenveloped viruses that infect enterocytes of the small intestine and cause severe infantile gastroenteritis. It was previously thought that rotavirus exits cells by lysis, but this behavior does not match the local pathogenesis of the virus. In this study, we have investigated the release of the simian rotavirus strain (RRV) from the polarized intestinal Caco-2 cells. We found that RRV is released almost exclusively from the apical pole of Caco-2 cells before any cells lyse. Using confocal laser scanning microscopy and drugs that inhibit vesicular transport, we studied the RRV transport route from the endoplasmic reticulum (ER) to the apical side of intestinal cells. We demonstrated that RRV exits from the ER through a carbonyl cyanide m-chlorophenylhydrazone-sensitive vesicular transport. RRV staining was never found within the Golgi apparatus or lysosomes, suggesting that the RRV intracellular pathway does not involve these organelles. This finding was confirmed by treatment with monensin or NH4Cl, which do not affect release of RRV. Electron microscopic analysis revealed RRV containing small smooth vesicles in the apical area and free virions outside the cell in the brush border, consistent with a vesicular vectorial transport of virus. These results may provide, for the first time, a cellular explanation of the pathogenesis of rotavirus.     

The endocytic pathway and formation of the Wingless morphogen gradient

Controlling the spread of morphogens is crucial for pattern formation during development. In the Drosophila wing disc, Wingless secreted at the dorsal-ventral compartment boundary forms a concentration gradient in receiving tissue, where it activates short- and long-range target genes. The glypican Dally-like promotes Wingless spreading by unknown mechanisms, while Dynamin-dependent endocytosis is thought to restrict Wingless spread. We have utilized short-term expression of dominant negative Rab proteins to examine the polarity of endocytic trafficking of Wingless and its receptors and to determine the relative contributions of endocytosis, degradation and recycling to the establishment of the Wingless gradient. Our results show that Wingless is internalized via two spatially distinct routes: one on the apical, and one on the basal, side of the disc. Both restrict the spread of Wingless, with little contribution from subsequent degradation or recycling. As previously shown for Frizzled receptors, depleting Arrow does not prevent Wingless from entering endosomes. We find that both Frizzled and Arrow are internalized mainly from the apical membrane. Thus, the basal Wingless internalization route must be independent of these proteins. We find that Dally-like is not required for Wingless spread when endocytosis is blocked, and propose that Dally-like promotes the spread of Wingless by directing it to lateral membranes, where its endocytosis is less efficient. Thus, subcellular localization of Wingless along the apical-basal axis of receiving cells may be instrumental in shaping the Wingless gradient.     

Mammalian Wnt3a is Released on Lipoprotein Particles

Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid-linked morphogens Wingless and Hedgehog in Drosophila . To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high-density lipoproteins (HDL) and low-density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein-independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.     

Glypicans shunt the Wingless signal between local signalling and further transport

The two glypicans Dally and Dally-like have been implicated in modulating the activity of Wingless, a member of the Wnt family of secreted glycoprotein. So far, the lack of null mutants has prevented a rigorous assessment of their roles. We have created a small deletion in the two loci. Our analysis of single and double mutant embryos suggests that both glypicans participate in normal Wingless function, although embryos lacking maternal and zygotic activity of both genes are still capable of transducing the signal from overexpressed Wingless. Genetic analysis of dally-like in wing imaginal discs leads us to a model whereby, at the surface of any given cell of the epithelium, Dally-like captures Wingless but instead of presenting it to signalling receptors expressed in this cell, it passes it on to neighbouring cells, either for paracrine signalling or for further transport. In the absence of dally-like, short-range signalling is increased at the expense of long-range signalling (reported by the expression of the target gene distalless) while the reverse is caused by Dally-like overexpression. Thus, Dally-like act as a gatekeeper, ensuring the sharing of Wingless among cells along the dorsoventral axis. Our analysis suggests that the other glypican, Dally, could act as a classical co-receptor.     

Some considerations on a carrier mechanism as a model for ion accumulation

A mechanism is described which accounts for the active transport of Na⁺ ions through a membrane. It is assumed that at one side of the membrane the ion combines with a carrier ion, the resulting carrier compound then diffuses through the membrane and decomposes at the other side of the membrane. The free diffusion of the ions is also taken into account. The time rate of accumulation of the ion in question at the latter side of the membrane is calculated in terms of the concentrations of the ion at both sides of the membrane.     

The Electrical Characteristics of Active Sodium Transport in the Toad Bladder

The mechanism responsible for active sodium transport in the urinary bladder of the toad appears to be located at the serosal boundary of the epithelial cell layer of the bladder. Studies of the potential step observed at the serosal boundary in the open-circuited state were undertaken in an attempt to define the factors responsible for its production. Glass micropipettes were used to measure the serosal potential step in bladders exposed on the serosal side to solutions of high potassium or of high potassium and low chloride concentration. Observed potentials exceed the maximum values which would have been expected if the serosal potential step were a potassium or chloride diffusion potential. Measurements of net cation flux exclude the possibility of a diffusion potential at this border due to the passive movement of any anionic species. The observed independence of transbladder potential and short-circuit current from the pH of the serosal medium over a wide range of pH makes it unlikely that the observed serosal potential step is a hydrogen ion diffusion potential. We conclude that the active sodium transport mechanism in toad bladder is "electrogenic."     

Drosophila S2 Cells Secrete Wingless on Exosome‐Like Vesicles but the Wingless Gradient Forms Independently of Exosomes

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless‐expressing Drosophila S2 cells, Wingless is present on exosome‐like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless‐containing exosome‐like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome‐like vesicles. Using these exosome markers and a cell‐based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless‐expressing cells.     

LDL transcytosis by protein membrane diffusion

Endothelial cell (EC) cultures of different, selected vascular beds and/or organs were screened for receptor-mediated transport of proteins with a semipermeable filter assay. In SVEC4-10 cells, a mouse lymphoid endothelial cell line, orosomucoid, albumin, insulin and LDL were transcytosed from the apical (luminal) to basal (abluminal) side by a receptor-mediated pathway. Specific LDL transcytosis involved transport of intact LDL. A pathway of degradation of LDL and basal release involved vesicles in transport to lysosomes and amino acid merocrine secretion. This newly described transcellular passage of LDL via lysosomes, as well as the standard pathway, were reduced to 70% by PEG(50)-cholesterol (PEG-Chol). Combined results of temperature-dependence analysis and PEG(50)-cholesterol sensitivity show that two pathways contribute to general LDL transcellular passage. We suggest a mechanism of domain hopping by protein membrane diffusion of receptors as the pathway for intact LDL delivery. Based on theoretical considerations we propose that active transport by protein membrane diffusion can be facilitated by an organizational structure of lipid microdomains and polar cellular organization.     

Steep differences in wingless signaling trigger Myc-independent competitive cell interactions

Wnt signaling is a key regulator of development that?is often associated with cancer. Wingless,?a Drosophila Wnt homolog, has been reported to be a survival factor in wing imaginal discs. However, we found that prospective wing cells survive in the absence of Wingless as long as they are not surrounded by Wingless-responding cells. Moreover, local autonomous overactivation of Wg signaling (as a result of a mutation in APC or axin) leads to the elimination of surrounding normal cells. Therefore, relative differences in Wingless signaling lead to competitive cell interactions. This process does not involve Myc, a well-established cell competition factor. It does, however, require Notum, a conserved secreted feedback inhibitor of Wnt signaling. We suggest that Notum could amplify local differences in Wingless signaling, thus serving as an early trigger of Wg signaling-dependent competition.     

Exchange of cystine and glutamate across plasma membrane of human fibroblasts

It is found that both the inward and outward transport of cystine and glutamate through the plasma membrane of cultured human fibroblasts is mediated mostly by a single transport system. Cystine and glutamate at one side of the membrane stimulate the passage of these amino acids present at the other side of the membrane. When the concentration of intracellular glutamate is reduced to near zero, cystine hardly enters the cell, and likewise the release of glutamate from the cell ceases when cystine is absent in the medium. Homocysteate and alpha-aminoadipate share this transport system and, when added, similarly participate in the transport process. Since the intracellular pool of cystine is negligibly small whereas that of glutamate is very large, the physiologic flows via this system are the entry of cystine and the exodus of glutamate coupled together. Measurements of the rate of uptake of cystine into the cells and the rate of release of glutamate from the cells indicate that the entry of cystine and the exodus of glutamate occur at a ratio close to 1:1. Since cystine is known to behave as an anionic form in this transport, it is concluded that the transport system for cystine and glutamate in plasma membrane of human fibroblasts is a kind of an anion-exchanging agency.     

Chloroquine-sensitive transplasmalemma electron transport in Tetrahymena pyriformis: a hypothesis for control of parasite protozoa through transmembrane redox

Plasma membrane electron transport was studied in a protozoan cell, Tetrahymena pyriformis, by assaying transmembrane ferricyanide reduction and the reduction of iron compounds. The rates of ferricyanide reduction varied between 0.5 and 2.5 mumol/g dry wt. per min, with a pH optimum at 7.0-7.5. Other active non-permeable electron acceptors, with redox potentials from +360 to -125 mV, were cytochrome c, hexaammine ruthenium chloride, ferric-EDTA, ammonium ferric citrate, and indigo di-, tri- and tetrasulfonates. It was found that Tetrahymena cells can reduce external electron acceptors with redox potentials at pH 7.0 down to -125 mV. Ferricyanide stimulates ciliary action. Transmembrane ferricyanide reduction by Tetrahymena was not inhibited by such mitochondrial inhibitors as antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide, or potassium cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Tetrahymena appears to involve a plasma membrane electron transport chain similar to those of other animal cells. As in other cells, the transmembrane electron transport is associated with proton release which may be involved in internal pH control. The transmembrane redox system differs from that of mammalian cells in a 20-fold greater sensitivity to chloroquine and quinacrine. The Tetrahymena ferricyanide reduction is also inhibited by chlorpromazine and suramin. Sensitivity to these drugs indicates that the transplasma membrane electron transport and associated proton pumping may be a target for drugs used against malaria, Trypanosomes and other protozoa.     

Wingless signaling and the control of cell shape in Drosophila wing imaginal discs

The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer β-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression.     

Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.     

The application of rate dialysis to the determination of free steroids in plasma

Rate dialysis is used to obtain the free steroid fraction in undiluted plasma at 37°C. The free steroid fraction is determined from the rate at which a small amount of tritiated steroid diffuses from plasma on one side of a semipermeable membrane into an identical plasma sample on the other side which lacks radioactive steroid. The method may be generally applicable to steroids since the cell permeability constant, which is a function of the volume of the dialysis cell and the area and diffusion properties of the membrane, was similar for seven steroids tested. The method requires only 0.3 ml of plasma, is simple and economical to perform, and enables up to 120 determinations to be made in one day. The free fractions of cortisol, progesterone, and estradiol-17β were measured in plasma pooled from pregnant and non-pregnant women and pregnant and lactating sows. The results were compared with those obtained for the same plasma pools by centrifugal ultrafiltration.     

Directionality of wingless protein transport influences epidermal patterning in the Drosophila embryo.

Active endocytotic processes are required for the normal distribution of Wingless (Wg) protein across the epidermal cells of each embryonic segment. To assess the functional consequences of this broad Wg distribution, we have devised a means of perturbing endocytosis in spatially restricted domains within the embryo. We have constructed a transgene expressing a dominant negative form of shibire (shi), the fly dynamin homologue. When this transgene is expressed using the GAL4-UAS system, we find that Wg protein distribution within the domain of transgene expression is limited and that Wg-dependent epidermal patterning events surrounding the domain of expression are disrupted in a directional fashion. Our results indicate that Wg transport in an anterior direction generates the normal expanse of naked cuticle within the segment and that movement of Wg in a posterior direction specifies diverse denticle cell fates in the anterior portion of the adjacent segment. Furthermore, we have discovered that interfering with posterior movement of Wg rescues the excessive naked cuticle specification observed in naked (nkd) mutant embryos. We propose that the nkd segment polarity phenotype results from unregulated posterior transport of Wg protein and therefore that wild-type Nkd function may contribute to the control of Wg movement within the epidermal cells of the segment.