Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut

Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.     

Marked differences in the swainsonine inhibition of rat liver lysosomal alpha-D-mannosidase, rat liver Golgi mannosidase II, and jack bean alpha-D-mannosidase

Swainsonine, a plant toxin, strongly inhibits certain alpha-D-mannosidases but has no effect on others [D. R. P. Tulsiani, T. M. Harris, and O. Touster (1982) J. Biol. Chem. 257, 7936-7939]. The reversible inhibition of jack bean and lysosomal alpha-D-mannosidases has previously been suggested to be similar in nature but quite complex. Specific differences in the action of swainsonine on these two enzymes and on Golgi mannosidase II are reported. (a) The inhibition of the jack bean mannosidase, but not rat liver lysosomal alpha-D-mannosidase or Golgi mannosidase II, is increased by preincubation with the alkaloid. (b) The inhibition of the jack bean and lysosomal enzymes, but not mannosidase II, is competitive at inhibitor concentrations of less than or equal to 0.5 microM. (c) The inhibition of jack bean alpha-mannosidase is largely irreversible, its very limited reversibility being partially dependent upon the swainsonine concentration used and on the time of preincubation with the inhibitor. On the other hand, the inhibition of lysosomal alpha-mannosidase is largely reversible, as shown by dilution experiments and by the use of [3H]swainsonine. Golgi mannosidase II shows intermediate reversibility, the results indicating two modes of binding; one rapid and irreversible, the other much slower and reversible.     

Retrotransposition and cell-to-cell transfer of foamy viruses

A remarkable feature of the prototype foamy virus (PFV) replication pathway has been reported to consist of the ability to retrotranspose intracellularly with high efficiency (M. Heinkelein, T. Pietschmann, G. Jármy, M. Dressler, H. Imrich, J. Thurow, D. Lindemann, M. Bock, A. Moebes, J. Roy, O. Herchenr?der, and A. Rethwilm, EMBO J. 19:3436-3345, 2000). PFV intracellular retrotransposition (IRT) was reported to be enhanced by coexpression of fusion-defective envelope protein. To investigate the possibility of cell-to-cell transfer of PFV genomes, which could mimic IRT, we performed cocultivation experiments with cells transfected with an IRT-competent and marker gene-expressing PFV vector together with cells expressing a different marker and measured cells positive for both markers. The findings corroborated the initial report on IRT of Env-deficient PFV. Furthermore, they indicated that viral cores that have incorporated fusion-deficient Env can be transferred from cell to cell in a cell type-specific manor. One possible explanation consists of a minor alternative cleavage site in Env that can be used to expose the fusion peptide of the Env transmembrane protein, which appears to be required for virus uptake.     

Transfer of Haemophilus influenzae chromosomal genes by cell-to-cell contact

A low-frequency exchange of chromosomal markers was observed in matings of Haemophilus influenzae. Transfer did not appear to be due to classical transformation or to be plasmid mediated, and chromosomal gene transfer differed in several respects from plasmid transfer by conjugation.     

Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal alpha-D-mannosidase, decreases Golgi alpha-D-mannosidase II, and increases the plasma levels of several acid hydrolases

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D.R.P. Tulsiani, T.M. Harris, and O. Touster, (1982) J. Biol. Chem. 257, 7936-7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal alpha-D-mannosidase and are probably a consequence of abnormal processing of glycoproteins.     

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.     

Nature of cell-to-cell transfer of auxin in polar transport

Summary By application of agar blocks (side blocks) against the inner and outer epidermis of maize (Zea mays L.) coleoptiles whose cuticle has been abraded it is found that radioactive auxin in the polar transport stream exchanges rapidly with the tissue's free space and therefore does not move confined within the symplast. Polar transport of IAA is demonstrable in Avena coleoptile segments plasmolyzed in 0.5 and 0.7 M mannitol, in which most of the plasmodesmatal connections between successive cells in the polar transport pathway appear to have been broken. We conclude that during polar transport IAA probably moves from cell to cell by crossing the plasmalemmas and the free space between successive cells, rather than via plasmodesmata. Auxin in the polar transport stream exchanges rapidly with side blocks by a cyanide-and azide-insensitive, presumably passive, process. A similarly passive uptake takes place into the cells from an external donor. NPA almost completely inhibits efflux from the polar transport stream even though it does not inhibit uptake; its inhibition of efflux is completely reversed by azide or cyanide. These findings are compatible either with the traditional model of polar transport as passive uptake combined with an active basal efflux pump for IAA, or with the model of purely passive polar transport driven by pH and/or potential differences across the plasma membrane, provided certain ad hoc assumptions are made about the characteristics of the IAA anion carrier that would be operating in either model.Abbreviations IAA indoleacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Acid alpha-D-mannosidase of human leukocytes in the norm and during chronic myeloid leukemia

The activity and properties of acid alpha-mannosidase were studied in normal granulocytes and in two types of myeloid cells from patients with chronic myeloid leukemia. The activity of the enzyme in leukemic cells was 2-fold higher than that in normal granulocytes and in morphologically matured myeloid cells. Two latter types of cells did not differ in alpha-mannosidase activity. Kinetic properties, thermo- and pH stability of alpha-mannosidase from normal and leukemic cells were similar. alpha-mannosidase in leukemic and normal cells existed in two forms (A and B), which were easily separated on DEAE-cellulose column. These two forms differed in molecular mass (300 and 290 kD, respectively) and in the degree of sialylation. The quantitative ratios of A and B forms in normal and leukemic cells were different. In normal granulocytes and in mature cells from patients this ratio was 0.60 and 0.67, respectively. In leukemic cells the ratio was found to be 1.31. Thus, in leukemic cells form A of alpha-mannosidase predominanted, whereas in normal cells the predominance of form B was observed. It was suggested therefore that in leukemic cells the enhanced synthesis of alpha-mannosidase occurred in parallel with the accumulation of the B form. This accumulation was assumed as the cause of enhanced activity of the enzyme in immature leukemic cells.     

The effect of cell-to-cell contact on the surface morphology of Chinese hamster ovary cells

In the previous report (Porter et al., in this issue) morphological changes in Chinese hamster ovary (CHO) cells during the cell cycle were described. In this report we describe the role of intercellular contact on these changes. We find that intercellular contact is required for cells to exhibit the morphologies Porter et al. described for S and G₂. When cells are synchronized by mitotic selection and plated onto cover slips at very low density such that no intercellular contact occurs, the cells remain in a G₁ configuration (rounded and highly blebbed through G₁, S, and G₂). This G₁ morphology is also observed in nonsynchronized log phase cells plated at low densities and allowed to grow for several generations. The addition of conditioned medium from confluent cultures does not induce low density cells to change morphology during the cell cycle. These results indicate that extensive intercellular contact is required for the complete expression of the morphological changes associated with the cell cycle (as described by Porter et al.). It is concluded that although classic contact inhibition of movement and of growth may be absent in this transformed cell line, some contact-dependent response persists.     

Multiploid inheritance of HIV-1 during cell-to-cell infection

During cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), many viral particles can be simultaneously transferred from infected to uninfected CD4 T cells through structures called virological synapses (VS). Here we directly examine how cell-free and cell-to-cell infections differ from infections initiated with cell-free virus in the number of genetic copies that are transmitted from one generation to the next, i.e., the genetic inheritance. Following exposure to HIV-1-expressing cells, we show that target cells with high viral uptake are much more likely to become infected. Using T cells that coexpress distinct fluorescent HIV-1 variants, we show that multiple copies of HIV-1 can be cotransmitted across a single VS. In contrast to cell-free HIV-1 infection, which titrates with Poisson statistics, the titration of cell-associated HIV-1 to low rates of overall infection generates a constant fraction of the newly infected cells that are cofluorescent. Triple infection was also readily detected when cells expressing three fluorescent viruses were used as donor cells. A computational model and a statistical model are presented to estimate the degree to which cofluorescence underestimates coinfection frequency. Lastly, direct detection of HIV-1 proviruses using fluorescence in situ hybridization confirmed that significantly more HIV-1 DNA copies are found in primary T cells infected with cell-associated virus than in those infected with cell-free virus. Together, the data suggest that multiploid inheritance is common during cell-to-cell HIV-1 infection. From this study, we suggest that cell-to-cell infection may explain the high copy numbers of proviruses found in infected cells in vivo and may provide a mechanism through which HIV preserves sequence heterogeneity in viral quasispecies through genetic complementation.     

Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H(+)-ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.     

Cobalt-ion chelate affinity chromatography for the purification of brain neutral alpha-D-mannosidase and its separation from acid alpha-D-mannosidase.

A method is described for the purification of neutral alpha-D-mannosidase and its separation from acid alpha-D-mannosidase from monkey brain by utilizing Co2+ chelate affinity chromatography. The neutral enzyme, which selectively bound to the metal-ion chelate column, was elutable by Tris at pH 7.5 and gave over 80-fold purification in a single step with 100% recovery.     

Motility,linear arrangement and cell-to-cell contact of myogenic cells prior to fusion

Summary Time-lapse cinematography elucidates the genesis of a uniform and approximately linearly arranged myogenic cell aggregate, stemming from two larger cell groups. The ultimate aggregate is created by continuous movement of one cell group toward the other. Following this motion, the angle between the cell groups is reduced as they approach each other.Different patterns of cell motility can be recognized. Some cells move in a preferred direction in relation to the aggregate as a whole, whereas others alter their direction of movement.The myogenic cells are aligned end-to-end and side-by-side. The latter is often accomplished in the following manner: two cells in end-to-end contact form as crescent-shaped free space with their polar extensions; a neighboring spindle-shaped cell then settles in this space. An arrangement of cells such that their greatest cytoplasmic widths lie at the same level can also be seen. During the recording period, two cells in one of the groups were replicating. One of them realized karyo- and cytokinesis in approximately 80min. The daughter cells moved apart in opposite directions, but never lost contact to the aggregate. This observation shows that contact between presumptive myoblasts and myoblasts is established.This research was supported by a grant from the Deutsche Forschungsgemeinschaft (Ba 689/1)     

Tracking and quantitation of fluorescent HIV during cell-to-cell transmission

The green fluorescent protein (GFP) is a powerful genetic marking tool that has enabled virologists to monitor and track viral proteins during HIV infection. Expression-optimized Gag-GFP constructs have been used to study virus-like particle (VLP) assembly and localization in cell types that are easily transfected. The development of HIV-1 variants carrying GFP within the context of the viral genome has facilitated the study of infection and has been particularly useful in monitoring the transfer of virus between cells following virological synapse formation. HIV Gag-iGFP, a viral clone that contains GFP inserted between the matrix (MA) and capsid (CA) domains of Gag, is the first replication competent molecular clone that generates fluorescent infectious particles. Here, we discuss some methods that exploit HIV Gag-iGFP to quantify cell-to-cell transmission of virus by flow cytometry and to track the proteins during assembly and transmission using live-cell imaging.     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

Induction of lysosomal enzymes in contact inhibited 3T3 cells

The pattern of changing activities of three lysosomal enzymes, N-acetyl-β-D-glucosaminidase, aryl sulfatase, and DNase II, and that of DNA polymerase was followed in homogenates of 3T3 cells during the logarithmic phase of growth and in stationary cultures. The change in activities of the polymerase and the lysosomal enzymes is antiparallel. DNA polymerase exhibits highest activity in growing cultures, and shows a three-fold decline of the specific activity in stationary cultures. The lysosomal enzymes show a very marked increase in their specific activity after density saturation is reached, which can be prevented by the addition of cycloheximide. Colcemid added to logarithmically growing cultures also causes an increase in the specific activities of lysosomal enzymes.     

CCK-58 is the only detectable endocrine form of cholecystokinin in rat

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radiolabeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 +/- 0.5 and 17 +/- 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin.