Wholemount histofluorescence of catecholamine-containing neurones in a hemipteran brain

Whole brains from Rhodnius prolixus treated with glyoxylic acid display catecholamine histofluorescence in eleven bilaterally distributed clusters of neurones, and in three circumscribed neuropile regions. This simple method offers resolution of some features not detected in studies on sectioned insect tissue; cephalic neurosecretory cells in chronically starved animals are histofluorescent and diverse catecholamine-handling fibres converge within the brain of this insect.     

Viral DNA in transformed cells: II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome

³²P-labeled adenovirus 2 DNA was treated with restricting endonuclease from Escherichia coli strain RY-13 (Yoshimori, 1972) (EcoRI) or restricting endonuclease from Hemophilus parainfluenzae (Hpa I) and the resulting fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete adenovirus 2 DNA were measured in the presence of DNA extracted from nine lines of adenovirus 2-transformed rat cells and from control cells. Six of the transformed cell lines contained viral DNA sequences homologous to two of the seven Hpa I⁴ fragments and to part of one of the six EcoRI fragments. From the order of the fragments formed by EcoRI and Hpa I on the adenovirus 2 map we conclude that these cell lines contain only the segment of viral DNA that stretches from the left-hand end to a point about 14% along the viral genome. Thus, any viral function expressed in transformed cells must be coded by this small section of viral DNA. The three remaining lines of adenovirus 2-transformed rat cells are more complicated and contain not only the sequences from the left-hand end of the viral DNA, but also other segments of the viral genome. However, no adenovirus 2-transformed rat cell contained DNA sequences homologous to the complete viral genome.     

Lysosomal enzyme inactivation associated with defects in post-translational modification during development in Dictyostelium discoideum

The developmental accumulation of lysosomal alpha-mannosidase-1 activity in Dictyostelium discoideum is controlled at the level of de novo enzyme precursor biosynthesis. Aggregation-deficient mutants are defective with regard to the accumulation of alpha-mannosidase-1 activity beyond 8-16 h of development. We used enzyme-specific monoclonal antibodies to show that the activity defect in aggregation-deficient strains is not due to a lack of alpha-mannosidase-1-precursor synthesis or processing, or to preferential degradation of the mature enzyme protein. Instead, the defect is a result of enzyme inactivation: cells of aggregation-deficient strains contain significant amounts of inactive alpha-mannosidase-1 protein late in development. The alpha-mannosidase-1 inactivation phenotype is associated with a more general defect in lysosomal enzyme modification. A change in the post-translational modification system occurs during normal slime-mold development, as shown by differences in enzyme isoelectric point, antigenicity, and thermolability. We found that this change in modification does not occur in mutant strains blocked early in development. We propose a model in which pleiotropic mutations in early aggregation-essential genes can indirectly affect the accumulation of alpha-mannosidase-1 activity by preventing the expression of a developmentally controlled change in the post-translational modification system, a change which is required for the stability of several lysosomal enzymes late in development.     

Quantification of the nonspecific intercellular transfer of fluorescent molecules between labeled and unlabeled rat thymocytes

Fluorescein isothiocyanate has been used to label normal or tumor cells in order to study their migration in vivo. This requires that any transfer of fluorescence to neighbouring cells be carefully ruled out. The aim of the present report is to demonstrate the possibility of a transfer of fluorescein or fluorescein-bound molecules between untreated and labeled cells. When normal rat thymocytes were co-incubated with labeled cells (about 5 X 10(6) fluorescein molecules/cell) under continuous agitation or exposed to supernatants of these labeled cells, they bound an average of 10(4) fluorescein molecules. When the incubation was done on cell pellets after centrifugation, this transfer was increased tenfold. Hence, intercellular molecular exchange may occur in the absence of any specific interaction.     

α-Mannosidase-1 mutants of Dictyostelium dkoideum: Early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.     

Cytokinetic activities in a human cell line: the midbody and intercellular bridge

The midbody and intercellular bridge of D-98S cells were studied by time lapse cinemicrography. Before separation interconnected daughter cells tended to move in opposition to each other, maintaining tension across the bridge and, in some cases, lengthening the bridge. Cell separation was preceded by a reduction in width of the bridge, on one or both sides of the midbody, to a cytoplasmic strand less than 0-5 μ wide, which was then stretched and broken by movements of the daughter cells, completing cytokinesis. Wave-like movements were observed to pass along longer intercellular bridges from the midbody to the daughter cells. This activity was judged to be involved with the retraction of cytoplasm from the bridge into the daughter cells. Wave activity was disrupted by the drug cytochalasin B.     

Actin localization in male germ cell intercellular bridges in the rat and ground squirrel and disruption of bridges by cytochalasin D

Filaments about 6-7 nm in diameter were seen associated with germ cell intercellular bridges in detergent-permeabilized cells treated with tannic acid. Approximately 40-50 filaments were present subjacent to the bridge density. Filaments encircled the bridge channel in a manner similar to contractile ring actin filaments of dividing cells. NBD-phallacidin and myosin S-1 subfragments were employed to demonstrate that the filaments observed at intercellular bridges are actin. Intratesticular injection of a single dose of cytochalasin D, a specific inhibitor of actin filaments, caused certain intercellular bridges of spermatids to open within 3 hr after injection, leading to the production of symplasts. During bridge opening, remnants of bridge densities were gradually incorporated into the lateral aspect of the plasma membrane of the symplast. Thus actin, present in bridge structures, appeared to participate in maintaining certain intercellular bridges. A model of intercellular bridge structure is presented.     

A quantitative method for determining polarization of neutrophil adhesion molecules associated with ischemia reperfusion

Ischemia-reperfusion-induced neutrophil adhesion to endothelium is CD18-dependent, but information regarding polarity of CD18 adhesion molecules remains speculative. This study evaluated neutrophil adhesion using an in vitro cell adhesion assay and introduces a quantitative method of measuring CD18 membrane distribution using confocal microscopy. Neutrophils from normal animals were isolated from whole blood and incubated with plasma from rat gracilis muscle flaps with no ischemia and reperfusion (nonischemic control, n = 10) or 4 hours of ischemia and 90 minutes of reperfusion (ischemia/reperfusion, n = 10), on coverslips pretreated with and without (phosphate-buffered saline) soluble intercellular adhesion molecules. Coverslips without intercellular adhesion molecules represented a negative control (intercellular adhesion molecules were required for adhesion). Percent adherence to intercellular adhesion molecules was expressed as a ratio of adherent cells/total cells. CD18 polarization was assessed by staining neutrophils with fluorescein isothiocyanate-labeled anti-CD11b, followed by confocal microscopy and Z-stack analysis. Membrane-associated CD18 was expressed as fluorescence intensity units in three equal areas of the cell membrane. Capping was defined as twice as much fluorescence in 33 percent of the cell membrane as in the remaining 67 percent. Neutrophils exposed to ischemia and reperfusion plasma showed a significant increase in adhesion (0.8 +/- 0.1 percent versus 16.7 +/- 2.2 percent, p < 0.001) and CD18 polarization (6.2 +/- 1.7 percent versus 43.9 +/- 12.2 percent, p = 0.0206) compared with controls. This article describes an in vitro assay that reliably reproduces the neutrophil adhesion phenomenon associated with ischemia-reperfusion injury. Results from confocal microscopy allowed for quantitative estimation of membrane-associated receptor polarization.     

Highly permeable intercellular junctions in normal and transformed fibroblast cultures]

Intercellular junctions permeable to ions and fluorescein Na were studied with the aid of intracellular microelectrodes in the cultures of normal and transformed mouse-embryo and hamster-embryo fibroblast-like cells. Normal cells were effectively coupled by the highly permeable junctions. In cultures of 7 types of transformed cells, 3 types of coupling were detected: effective, decreased, and fully reduced couplings. The degree of uncoupling was not correlated with morphological patterns of malignization and tumorogeneity of transformed cultures. The decrease of permeability of intercellular junctions to ions and small molecules is concluded not to be necessary for malignization.     

Terminal phase of cytokinesis in D-98S cells

The events leading to the completion of cytokinesis after the formation of the midbody and intercellular bridge in D-98S cells were studied with light and electron microscopy. Pairs of daughter cells corresponding to different stages of cytokineses, as determined previously form time lapse films, were selected from embedded monolayers for serial sectioning. Separation of daughter cells is preceded by the reduction in diameter of the intercellular bridge from 1-1.5 μm to approx. 0.2 μm. Two processes contribute to this reduction: (a) The intercellular bridge becomes gradually thinner after telophase; a progressive breakdown of midbody structures accompanies this change; and (b) the more significant contribution to reduction in bridge diameter occurs through the localized constriction of a segment of the intercellular bridge.. The microtubules within the constricted portion of the bridge are forced closer together, and some microtubules disappear as this narrowing progresses. The plasma membrane over the narrowed segments is thrown into a series of wavelike ripples. Separation of daughter cells is achieved through movements of the cells which stretch and break the diameter-reduced bridge. The midbody is discarded after separation and begins to deteriorate. Occasional pairs of daughter cells were found in which incomplete karyokineses resulted in their nuclei being connected by a strand of nuclear material traversing the bridge and midbody. Such cells do not complete cytokinesis but merge together several hours after telophase. This merging of daughter cells coincides with the nearly complete breakdown of the midbody.     

Conversion of midbodies into germ cell intercellular bridges

Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP) and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7 and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell-specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.     

Highly permeable contacts between epithelial cells in culture]

Cultured epithelial cells producing monolayered sheets were used to study intercellular contacts permeable to sodium fluorescein. Cells in dense cultures were more capable of forming permeable junctions than cells of sparse cultures. In addition, the standard microelectrode technique revealed some differences in cellular membrane potentials in dense and sparse cultures. A possible correlation is discussed between intercellular contact formation and other features of cells depending on cell culture density.     

Mechanistic insights into the physiological functions of cell adhesion proteins using single molecule force spectroscopy

Intercellular adhesion molecules play an important role in regulating several cellular processes such as a proliferation, migration and differentiation. They also play an important role in regulating solute diffusion across monolayers of cells. The adhesion characteristics of several intercellular adhesion molecules have been studied using various biochemical assays. However, the advent of single molecule force spectroscopy as a powerful tool to analyze the kinetics and strength of protein interactions has provided us with an opportunity to investigate these interactions at the level of a single molecule. The study of interactions involving intercellular adhesion molecules has gained importance because of the fact that qualitative and quantitative changes in these proteins are associated with several disease processes. In this review, we focus on the basic principles, data acquisition and analysis in single molecule force spectroscopy experiments. Furthermore, we discuss the correlation between results obtained using single molecule force experiments and the physiological functions of the proteins in the context of intercellular adhesion molecules. Finally, we summarize some of the diseases associated with changes in intercellular adhesion molecules.     

Translocation in the staminal hairs ofSetcreasea purpurea

Summary Investigations into plant intercellular communication were initiated through an examination of plasmodesmata and cell-to-cell passage of molecular probes in the staminal hairs ofSetcreasea purpurea. Plasmodesmata connecting staminal hair cells of small buds are filled with an electron-opaque homogenous material. To examine the permeation selectivity of plasmodesmata, molecular probes made up of fluorescein isothiocyanate (FITC) complexed with amino acids and peptides were injected into the staminal hair cells and the spread of these fluorescent molecules through the symplast, was monitored. Molecules composed of FITC complexed to single amino acids with polar and aliphatic R groups travel rapidly, while those which include peptides travel slowly. Dye molecules composed of an amino acid with an aromatic side group do not pass from cell to cell at all. It is hypothesized that the material occluding the plasmodesmata constitutes the diffusion barrier, by presenting a hydrophilic environment which allows passage of molecules with maximum molecular weights of 700–800 daltons, but which retains those with aromatic side groups.     

Observations on populations of the horseshoe crabLimulus (=Xiphosura) polyphemus

1. Marked populations of Limulus (=Xiphosura) polyphemus reveal that in Cold Spring Harbor, New York, they consisted of 10,000–18,000 adults in 1957 and 1961. The sex ratio in 1957 was about 4 males: 1 female. Pairs may remain attached for as long as 9 days. An undisturbed female may lay as many as 12,000 eggs in one nest.
2. The Cold Spring Harbor populations appear to be rather sedentary: none of the 1,000 animals marked on the north edge of the sandspit in 1961 were detected in the outer harbor either at Laurel Hollow Beach or the peninsula adjacent to the Cold Spring Harbor Yacht Club 500–800 meters from the tagging site (see Fig. 1), nor were they found in the small beach adjacent to the Biological Laboratory in the inner harbor. Similarly, none of the 300 animals marked at this last site were found at the north edge of the sandspit.
3. The phenotype of the compound eye varies from black to pigmentless. Samples observed in Cold Spring Harbor and in the Marine Biological Laboratory, Woods Hole, Massachusetts (separated by Long Island Sound and a distance of 150 miles) differ in the frequency of the various phenotypes scored, but the mode of inheritance of eye color remains obscure.
4. The available evidence indicates Limulus has considerable phenotypic variation in regard to body size, eye color, and other characters believed to be inherited, with the result that demes or physiological races are created. It is argued that the belief that this organism is stable and has not changed since the Triassic 200 million years ago has foundation only in regard to the pattern of the body of Limulus, but not in regard to its genotype. Limulus does not seem to be different from other organisms for which considerable genetic evidence is available, and thus the statement that DNA is fairly stable and has remained so for 200 million years is open to question.

     

Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis

Cytokinesis is the final step of cell division and leads to the physical separation of the daughter cells. After the ingression of a cleavage membrane furrow that pinches the mother cell, future daughter cells spend much of the cytokinesis phase connected by an intercellular bridge. Rab proteins are major regulators of intracellular transport in eukaryotes, and here, we report an essential role for human Rab35 in both the stability of the bridge and its final abscission. We find that Rab35, whose function in membrane traffic was unknown, is localized to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway. Consistent with a key requirement for Rab35-regulated recycling during cell division, inhibition of Rab35 function leads to the accumulation of endocytic markers on numerous cytoplasmic vacuoles in cells that failed cytokinesis. Moreover, Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid and the septin SEPT2. We propose that the Rab35-regulated pathway plays an essential role during the terminal steps of cytokinesis by controlling septin and PIP2 subcellular distribution during cell division.     

Electrically mediated protein movement inDrosophila follicles

Summary Distribution of rhodamine-conjugated lysozyme injected into the sixteen-cell syncytium comprising the germ-line portion of theDrosophila follicle is shown to be affected by charge. Positive molecules are able to migrate through intercellular bridges from the oocyte to the nurse cells, but are unable to migrate detectably from nurse cells to the oocyte. Their negatively charged counterparts can move from the nurse cells to the oocyte, but are unable to traverse the intercellular bridges in the counter direction. This charge-dependent movement of molecules is accompanied by an electrical potential difference, focused across the nurse cell-oocyte bridges, which makes the nurse cells negatively charged to the oocyte. The addition of insect hemolymph to the physiological salt solution in which the experiments were performed resulted in only a small increase in the transmembrane resistance, but enhanced the potential difference between oocyte and nurse cells from 0.2±0.3 (SE) mV (nurse cells negative) to 2.3±0.45 (SE) mV (nurse cells negative). Supported by NSF Grant # DB-18617     

Mechanical interactions between dendritic cells and T cells correlate with T cell responsiveness

Ag recognition is achieved through the communication across intercellular contacts between T cells and APCs such as dendritic cells (DC). Despite remarkable progress in delineating detailed molecular components at the intercellular contacts, little is known about the functional roles of physical cross-junctional adhesion between T and DC in shaping T cell responses. In addition, the mechanisms underlying sensitivity and specificity of Ag discrimination by T cells at intercellular contacts remain to be elucidated. In this study, we use single-cell force spectroscopy to probe the mechanical interactions between DC and T cells in response to stimulation with a panel of altered peptide ligands. The results show that intercellular interactions of DC-T cell conjugates exhibited different ranges of interaction forces in peptide-dependent manners that match the ability of the peptides to activate T cells. Elevated calcium mobilization and IL-2 secretion by T cells were only promoted in response to antigenic peptides that induce strong interaction forces, suggesting that mechanically stable DC-T cell contacts are crucial for driving T cell activation. Strong interactions were not solely dependent on cell-surface molecules such as TCRs and the adhesion molecule LFA-1, but were also controlled by cytoskeletal dynamics and the integrity of membrane lipid rafts. These data provide novel mechanical insights into the effect of Ag affinity on intercellular contacts that align with T cell responsiveness.     

Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules

The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.