The inducing capacity of the presumptive endoderm of Xenopus laevis studied by transfilter experiments

Summary The inducing capacity of the vegetal hemisphere of early amphibian blastulae was studied by placing a Nucleopore filter (pore size 0.4 m) between isolated presumptive endoderm and animal (ectodermal) caps. The inducing effect was shown to traverse the Nucleopore membrane. The reacting ectoderm differentiated into mainly ventral mesodermal derivatives. Expiants consisting of five animal caps also formed dorsal mesodermal and neural structures. Those results together with data published elsewhere suggest that, in addition to a vegetalizing factor, different mesodermal factors must be taken into consideration for the induction of either the ventral or the dorsal mesodermal derivatives. The neural structures are thought to be induced by the primarily induced dorsal mesodermal tissue. Electron microscopic (TEM) examination did not reveal any cell processes in the pores of the filter. The results indicate that transmissible factors rather than signals via cytoplasmic contacts or gap junctions are responsible for the mesodermal induction of ectodermal cells. The data support the view that in normogenesis the mesoderm is determined by the transfer of inducing factors from vegetal blastomeres to cells of the marginal zone (presumptive mesodermal cells).     

Periaxonal surface calcium binding and distribution of charge on the faces of squid axon potassium channel molecules

Summary The calcium binding constant associated with external surface charge in a position to influence the voltage sensing charges for potassium channel gating appears to be 30 molar^–1, a value much larger than previously thought and in approximate agreement with that found for artificial membranes composed of the lipid brain phosphatidylserene. Fixed charge on the periaxonal membrane surface is distributed in such a way that much larger charges occur at a distance of at least 8 angstroms from the channel pore openings. The separation between the ion pathway and the channel gating charge appears to be greater than or equal to 8 angstroms. Periaxonal surface charge which is in a position to determine the surface potential for gating has a magnitude greater than or equal to one (negative) electronic charge per 182 square angstrom before calcium binding, which is reduced to –e/625 Å in a normal divalent ionic environment. With the normal divalent ionic composition of seawater the surface potential at a position to influence the gating voltage sensor is –15 millivolts relative to the bulk external potential. The external surface potential is –3 mV at the pore mouth. There appears to be a negligible amount of fixed charge on the axoplasmic surface in the vicinity of the ion channel opening. Further, our results confirm earlier measurements that have given a negligible amount of axoplasmic surface fixed charge whose field components would be in a position to influence the channel gating charges.     

Electrostatic properties of cells estimated by absorption and fluorescence spectroscopy

A colloid titration method was used to determine the surface charge of cells of a human colon adenocarcinoma cell line WiDr; 6.2±0.8×10⁸ charges per cell were found. The apparent surface charge density was calculated using the cell surface area estimated by a Coulter counter. Alternatively, the lower limit of the cell surface area was estimated by visible microscopy. The same procedure was applied for human skin fibroblasts, resulting in the value 9.4±1.1×10⁸ charges per cell. This is significantly higher (p<0.05) than that of WiDr cells, presumably because of the different size of the cells. According to the estimations using the Coulter counter, the median diameter was higher in the case of skin fibroblasts. Fluorimetric titration of the fluorescent probe U-6 was used to estimate the interfacial potential of the WiDr cells. A shift of the titration curve of the U-6 probe toward higher pH values compared to that in pure buffer solutions was found in the presence of the WiDr cells. From the displacement of the midpoints of the titration curves, the interfacial potential of the WiDr cells was found to be about−35.8 mV. Incubation of the cells at two different pH values (7.4 and 6.8) did not result in any significant modification of the electrostatic properties of the cells under the experimental conditions of the present study. Electron microscopy revealed a distinct difference in the surface morphology of the WiDr cells compared to human skin fibroblasts. Numerous microvilli present on the surface of WiDr cells indicated marked uncertainties in cell surface area estimations. This gives large uncertainties in the real surface charge densities of cells.     

Role of Membrane-bound, Fixed-charge Changes in Phytochrome-mediated Mung Bean Root Tip Adherence Phenomenon

The movement of cells and cell fragments in an electric field provided a means for determining the nature of cellular surface charges. We found that changes in ionic strength and particularly changes in Ca²⁺ and H⁺ in the bathing medium cause changes in the surface charges on the root cap cells in the absence of red light. Red light-induced charge changes are demonstrable only on root cap cells and are reversible with far red light. By osmotically separating the membrane from the wall, we demonstrated that both light-induced and ionically mediated charge changes are associated with the cell membrane and not the cell wall.     

Influence of age on transmembrane potential and cell surface charge of human peripheral blood lymphocytes estimated with fluorescent probes.

Transmembrane potential (TMP) and surface charge (CSC) of human peripheral blood lymphocytes were estimated in relation to donor's age. Cyanine dye DiOC(6) and fluorescein--coupled poly-L-ornithine EPLO were used as representative fluorescent probes for microfluorimetric determination of relative TMP and CSC values. Significant decrease in cyanine fluorescence was observed in lymphocytes of old people, which was interpreted as relative depolarization of these cells. No such difference was found when surface charges of young and old cells were compared, although some brightly fluorescent, i.e. highly charged cells appeared among the latter.     

Conduction through the inward rectifier potassium channel, Kir2.1, is increased by negatively charged extracellular residues

Ion channel conductance can be influenced by electrostatic effects originating from fixed "surface" charges that are remote from the selectivity filter. To explore whether surface charges contribute to the conductance properties of Kir2.1 channels, unitary conductance was measured in cell-attached recordings of Chinese hamster ovary (CHO) cells transfected with Kir2.1 channels over a range of K+ activities (4.6-293.5 mM) using single-channel measurements as well as nonstationary fluctuation analysis for low K+ activities. K+ ion concentrations were shown to equilibrate across the cell membrane in our studies using the voltage-sensitive dye DiBAC4(5). The dependence of gamma on the K+ activity (a(K)) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as a(K) approaches 0 mM, suggesting electrostatic surface charge effects. Following the addition of 100 mM N-methyl-D-glucamine (NMG+), a nonpermeant, nonblocking cation or following pretreatment with 50 mM trimethyloxonium (TMO), a carboxylic acid esterifying agent, the gamma-a(K) relationship did not show nonzero intercepts, suggesting the presence of surface charges formed by glutamate or aspartate residues. Consistent with surface charges in Kir2.1 channels, the rates of current decay induced by Ba2+ block were slowed with the addition of NMG or TMO. Using a molecular model of Kir2.1 channels, three candidate negatively charged residues were identified near the extracellular mouth of the pore and mutated to cysteine (E125C, D152C, and E153C). E153C channels, but not E125C or D152C channels, showed hyperbolic gamma-a(K) relationships going through the origin. Moreover, the addition of MTSES to restore the negative charges in E53C channels reestablished wild-type conductance properties. Our results demonstrate that E153 contributes to the conductance properties of Kir2.1 channels by acting as a surface charge.     

Quantum dots as a sensor for quantitative visualization of surface charges on single living cells with nano-scale resolution

We developed a technique using quantum dot (QD) as a sensor for quantitative visualization of the surface charge on biological cells with nano-scale resolution. The QD system was designed and synthesized using amino modified CdSe/ZnS nanoparticles. In a specially designed buffer solution, they are positively charged and can homogeneously disperse in the aqueous environment to label all the negative charges on the surfaces of living cells. Using a wide-field optical sectioning microscopy to achieve 2D/3D imaging of the QD-labeled cells, we determined the charge densities of different kinds of cells from normal to mutant ones. The information about the surface charge distribution is significant in evaluating the structure, function, biological behavior and even malignant transformation of cells.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Formation of the Neural Plate and the Mesoderm in Normally Developing Embryos of Xenopus laevis

Normally developing embryos of Xenopus were fixed at various stages between the blastula and early tail bud stage, and their serial sections were examined. The marginal belt of the blastula was characterized by abundance of cells with RNA-rich peripheral cytoplasm called mesoplasm. At the early gastrula stage, the marginal belt was folded into two layers giving rise to mesodermal material and marginal ectoderm. During gastrulation, the mesodermal material, which consisted of RNA-rich cells, spread to enclose the blastocoel and the endoderm, and a large part of it was shifted to the dorsal side of the embryo. It gradually established the mesodermal layer. The notochord was formed on the dorsal lip of the blastopore by involution, separately from preformed mesodermal material. The RNA-rich cells in the marginal ectoderm became columnar, forming a broad belt in the marginal zone. This belt was deformed and shifted to the dorsal side during gastrulation, eventually establishing the neural plate showing quantitative differentiation along the head-tail axis. Possible mechanisms involved in the formation of the neural plate and mesoderm were discussed with reference to the organizer and the mesoplasm.     

Characterization of amalgam: a member of the immunoglobulin superfamily from Drosophila

The immunoglobulin superfamily is a diverse group of proteins that are involved in various aspects of cell surface recognition. Here, we report the characterization of amalgam (ama), a gene in the Antennapedia complex (ANT-C) of D. melanogaster that exhibits amino acid similarity to vertebrate neural cell adhesion molecules and other members of the immunoglobulin superfamily. The putative 333 amino acid ama protein consists of a signal sequence, three immunoglobulin-like domains, and a short slightly hydrophobic carboxy-terminal region. Antibodies against the ama protein reveal that it accumulates on the surface of various mesodermal and neural cells during embryogenesis. The function of this protein remains elusive, as no mutations have been recovered for ama during saturation EMS mutagenesis of this chromosomal region.     

Anionic sites on the envelope of Salmonella typhimurium mapped with cationized ferritin

Binding of either ferritin (F) or cationized ferritin (CF) was employed to indicate the surface charge of the envelope of mainly two Salmonella typhimurium strains (395 MR10, a Rd-mutant, and LT2-M1, a UDP-galactose-4-epimerase-less mutant). Lowering the pH from 7 to 4 decreased binding of CF, but increased binding of F. At low concentrations, the distribution of CF on S. typhimurium 395 MR10 was in general random, with individual ferritin molecules often forming clusters of two or three particles. At ionic strengths of 0.25M NaCl, ferritin produced distinctive, larger clusters at relatively few sites (10-50/cell). Addition of galactose to cultures of growing S. typhimurium, LT2-M1 reduced the binding of CF in 1-10 min, and numerous ferritin-free areas became visible. Possibly this is caused by a pluri-focal reduction in the negative cell surface charge that was generated at the multiple sites of export of new, smooth-type lipopolysaccharide, which either exhibits lesser charge or masks a preexisting surface charge. Dividing cells may show unequal charges on the prospective daughter cells, and the difference in the capacity for ferritin adsorption of both daughter cells is sharply separated at the division site.     

Biochemical properties of viral envelopes of insect baculoviruses and their role in infectivity

The enveloped virions of a nuclear polyhedrosis virus (NPV) and those of a granulosis virus (GV) of the armyworm, Pseudaletia unipuncta, were isolated and purified from their inclusion bodies. The enveloped virion of NPV contained a large amount of phosphatidyl choline which was not detected in that of GV. The total electric charges distributed on the surface of the envelopes of NPV and GV were negative in neutral and alkaline solutions. Although there was little difference in charges between NPV and GV, the charge was less negative in NPV than in GV. When the negative charges were neutralized by cationic detergents, the NPV infectivity was enhanced.     

Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development

Background

In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/Principal Findings

Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1^−/− mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/Significance

This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.     

Clonal analysis of mesoderm induction in Xenopus laevis

Acidic fibroblast growth factor (aFGF) has been used to induce mesoderm from single animal pole cells of midblastula stage Xenopus embryos. The cells are individually cultured in a completely defined medium and are able to differentiate as small clones in a high proportion of cases. FGF-treated cells can give rise to several mesodermal cell types, while untreated cells show only epidermal or neural differentiation. Mesodermal differentiation can occur in clones of as few as eight cells, indicating that any additional cell-cell interactions required for mesodermal differentiation can be met by the medium used.     

Granulysin crystal structure and a structure-derived lytic mechanism

Our crystal structure of granulysin suggests a mechanism for lysis of bacterial membranes by granulysin, a 74-residue basic protein from human cytolytic T lymphocyte and natural killer cells. We determined the initial crystal structure of selenomethionyl granulysin by MAD phasing at 2A resolution. We present the structure model refined using native diffraction data to 0.96A resolution. The five-helical bundle of granulysin resembles other "saposin folds" (such as NK-lysin). Positive charges distribute in a ring around the granulysin molecule, and one face has net positive charge. Sulfate ions bind near the segment of the molecule identified as most membrane-lytic and of highest hydrophobic moment. The ion locations may indicate granulysin's orientation of initial approach towards the membrane. The crystal packing reveals one way to pack a sheet of granulysin molecules at the cell surface for a concerted lysis effort. The energy of binding granulysin charges to the bacterial membrane could drive the subsequent lytic processes. The loosely packed core facilitates a hinge or scissors motion towards exposure of hydrophobic surface that we propose tunnels the granulysin into the fracturing target membrane.     

Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis

During amphibian development, non-canonical Wnt signals regulate the polarity of intercalating dorsal mesoderm cells during convergent extension. Cells of the overlying posterior neural ectoderm engage in similar morphogenetic cell movements. Important differences have been discerned in the cell behaviors associated with neural and mesodermal cell intercalation, raising the possibility that different mechanisms may control intercalations in these two tissues. In this report, targeted expression of mutants of Xenopus Dishevelled (Xdsh) to neural or mesodermal tissues elicited different defects that were consistent with inhibition of either neural or mesodermal convergent extension. Expression of mutant Xdsh also inhibited elongation of neural tissues in vitro in Keller sandwich explants and in vivo in neural plate grafts. Targeted expression of other Wnt signaling antagonists also inhibited neural convergent extension in whole embryos. In situ hybridization indicated that these defects were not due to changes in cell fate. Examination of embryonic phenotypes after inhibition of convergent extension in different tissues reveals a primary role for mesodermal convergent extension in axial elongation, and a role for neural convergent extension as an equalizing force to produce a straight axis. This study demonstrates that non-canonical Wnt signaling is a common mechanism controlling convergent extension in two very different tissues in the Xenopus embryo and may reflect a general conservation of control mechanisms in vertebrate convergent extension.     

Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation

Vertebrate neural induction requires inhibition of bone morphogenetic protein (BMP) signaling in the ectoderm. However, whether inhibition of BMP signaling is sufficient to induce neural tissues in vivo remains controversial. Here we have addressed why inhibition of BMP/Smad1 signaling does not induce neural markers efficiently in Xenopus ventral ectoderm, and show that suppression of both Smad1 and Smad2 signals is sufficient to induce neural markers. Manipulations that inhibit both Smad1 and Smad2 pathways, including a truncated type IIB activin receptor, Smad7 and Ski, induce early neural markers and inhibit epidermal genes in ventral ectoderm; and co-expression of BMP inhibitors with a truncated activin/nodal-specific type IB activin receptor leads to efficient neural induction. Conversely, stimulation of Smad2 signaling in the neural plate at gastrula stages results in inhibition of neural markers, disruption of the neural tube and reduction of head structures, with conversion of neural to neural crest and mesodermal fates. The ability of activated Smad2 to block neural induction declines by the end of gastrulation. Our results indicate that prospective neural cells are poised to respond to Smad2 and Smad1 signals to adopt mesodermal and non-neural ectodermal fates even at gastrula stages, after the conventionally assigned end of mesodermal competence, so that continued suppression of both mesoderm- and epidermis-inducing Smad signals leads to efficient neural induction.     

Differentiation of primary embryonic neuroblasts in purified neural cell cultures from Drosophila

Embryonic neuroblasts of Drosophila are undifferentiated precursor cells that give rise to the central nervous system. Centrifugal elutriation has been employed to fractionate embryonic cells on the basis of size. A fraction of large cells was found to be greatly enriched for neuroblasts, whereas mesodermal precursor cells were completely excluded. This allowed a second step of purification, based upon adhesion to glass, to provide virtually pure cultures of neural cells. The cells in these cultures had the properties of neurons of the Drosophila CNS: They gave rise to ganglion-like clusters from which neurites extended on the culture substrate, and they expressed the enzyme, acetylcholinesterase, and the cell surface antigens recognized by antisera raised against horseradish peroxidase. The production of large-scale neuronal cell cultures will be useful for immunological and molecular studies of neural cell differentiation.