Virus-like particle formation in Drosophila melanogaster germ cells suggests a complex translational regulation of the retrotransposon cycle and new mechanisms inhibiting transposition.

Transposition of 1731, a Drosophila melanogaster LTR retrotransposon, was investigated in reproductive organs by RNA, protein and VLP distribution during its life cycle. We detected 1731 transcription in oogonia but not in spermatogonia; in all cells during oogenesis but only in primary spermatocytes; and in ovarian cytoplasm but both in nuclei and cytoplasm of primary spermatocytes. By confocal scanning, we showed that whereas Gag protein appeared in all cytoplasms during oogenesis, in testes Gag detection began in late premeiotic primary spermatocytes and increased in elongating spermatids suggesting distinct mechanisms of 1731 transcription and translation regulation. By electron microscopy, we did not detect 1731 VLPs in ovaries, suggesting a complex post-translational control blocking VLP assembly and transposition. Interestingly, in testes we discovered VLP aggregates in cystic cytoplasm of maturing partially individualized spermatids. In testes, we observed two delays in 1731 product expressions, suggesting a complex temporal control mechanism. Transcriptional/translational delay may be determined by accumulation of 1731 RNAs in primary spermatocyte nuclei. Translational/VLP assembly delay may be determined by post-transductional mechanisms controlling +1 frameshift and Pol-protein degradation. Our results indicated two differential mechanisms inhibiting 1731 transposition in Drosophila melanogaster ovaries and testes. In addition, we proposed a new mechanism for transposition control at the cell cycle level.

Asymmetrical segregation of chromosomes with a normal metaphase/anaphase checkpoint in polyploid megakaryocytes.

During differentiation, megakaryocytes increase ploidy through a process called endomitosis, whose mechanisms remain unknown. As it corresponds to abortive mitosis at anaphase and is associated with a multipolar spindle, investigation of chromosome segregation may help to better understand this cell-cycle abnormality. To examine this variation, a new method was developed to combine primed in situ labeling to label centromeres of one chromosome category and immunostaining of tubulin. Human megakaryocytes were obtained from normal bone marrow culture. By confocal microscopy, this study demonstrates an asymmetrical distribution of chromosomes (1 or 7) either between the spindle poles at anaphase stage of endomitosis and between the different lobes of interphase megakaryocyte nuclei. The metaphase/anaphase checkpoint appears normal on the evidence that under nocodazole treatment megakaryocytes progressively accumulate in pseudo-metaphase, without spontaneous escape from this blockage. Immunostaining of p55CDC/hCDC20 with similar kinetochore localization and dynamics as during normal mitosis confirms this result. HCdh1 was also expressed in megakaryocytes, and its main target, cyclin B1, was normally degraded at anaphase, suggesting that the hCdh1-anaphase-promoting complex checkpoint was also functional. This study found the explanation for these unexpected results of an asymmetrical segregation coupled to normal checkpoints by careful analysis of multipolar endomitotic spindles: whereas each aster is connected to more than one other aster, one chromosome may segregate symmetrically between 2 spindle poles and still show asymmetrical segregation when the entire complex spindle is considered.

Type I protein kinase a is localized to interphase microtubules and strongly associated with the mitotic spindle.

We show here that type I protein kinase A is localized to microtubules during the entire cell cycle in epithelial (hepatoma, cervical carcinoma) and nonepithelial (myoblast) cell lines. The association of the type Ialpha regulatory subunit is very strong in all phases of mitosis, from prophase to cytokinesis. In interphase, the association appears weaker, reflecting perhaps a more dynamic molecular interaction. This regulatory subunit appears to recruit catalytic subunits as the latter are also associated with microtubules. BW1J hepatoma cells, stably transfected with either wild-type or mutant Ialpha regulatory subunit, are enriched in aberrant mitoses with multipolar spindles and in mono- or multinucleated giant cells. This suggests that type I protein kinase A could have a role in centrosome duplication and/or segregation, sister chromatid separation, or cytokinesis.

H-2M molecules, like MHC class II molecules, are targeted to parasitophorous vacuoles of Leishmania-infected macrophages and internalized by amastigotes of L. amazonensis and L. mexicana.

In their amastigote stage, Leishmania are obligatory intracellular parasites of mammalian macrophages, residing and multiplying within phagolysosomal compartments called parasitophorous vacuoles (PV). These organelles have properties similar to those described for the MHC class II compartments of antigen-presenting cells, sites where peptide-class II molecule complexes are formed before their expression at the cell surface. After infection with Leishmania amazonensis or L. mexicana, endocytosis and degradation of class II molecules by intracellular amastigotes have also been described, suggesting that these parasites have evolved mechanisms to escape the potentially hazardous antigen-presentation process. To determine whether these events extend to other molecules of the antigen-presentation machinery, we have now studied the fate of the MHC molecule H-2M in mouse macrophages infected with Leishmania amastigotes. At least for certain class II alleles, H-2M is an essential cofactor, which catalyses the release of the invariant chain-derived CLIP peptide from the peptide-binding groove of class II molecules and facilitates the binding of antigenic peptides. H-2M was detected in PV of mouse macrophages infected with various Leishmania species including L. amazonensis, L. mexicana, L. major and L. donovani. PV thus contain all the molecules required for the formation of peptide-class II molecule complexes and especially of complexes with parasite peptides. The present data indicate, however, that if this process occurs, it does not lead to a clear increase of SDS-stable compact (alpha)(beta) dimers of class II. In PV that contained L. amazonensis or L. mexicana, both class II and H-2M molecules often colocalized at the level where amastigotes bind to the PV membrane, suggesting that these molecules are physically associated, directly or indirectly, and possibly interact with parasite components. Furthermore, as class II molecules, H-2M molecules were internalized by amastigotes of these Leishmania species and reached parasite compartments that also contained class II molecules. Immunostaining of H-2M within parasites was increased by treatment of infected macrophages with the cysteine protease inhibitors Z-Phe-AlaCHN2 or Z-Phe-PheCHN2 or by incubation of the parasites with the same inhibitors before infection. These data thus support the idea that amastigotes of certain Leishmania species capture and degrade some of the molecules required for antigen presentation. To examine whether endocytosis of class II molecules by the parasites occurs through interactions with parasite components involving their peptide-binding groove, we made use of the fact that a large fraction of the class II molecules of H-2M(alpha) knock-out H-2(b) mice are occupied by the peptide CLIP and are unable to bind other peptides. We found that, in Leishmania-infected macrophages of these mutant mice, class II-CLIP complexes reached PV and were internalized by amastigotes. These results thus prove that endocytosis of class II molecules by amastigotes (1) is H-2M-independent and (2) does not necessarily involve the peptide-binding pocket of these molecules. Altogether, these data are compatible with an endocytic mechanism based on general properties shared by classical and non-classical class II molecules.

Indications for GABA-immunoreactive axo-axonic contacts on the intraspinal arborization of a Ib fiber in cat: a confocal microscope study.

Confocal microscopy was used to detect GABA-immunoreactive axo-axonic appositions, indicating possible synaptic contacts, on Ib fiber terminals in the lumbosacral spinal cord. A Ib fiber from posterior biceps-semitendinosus muscles was labeled by intra-axonal ejection of tetramethylrhodamine dextran (red), and serial sections of S1-L7 spinal cord segments were processed for GABA immunocytochemistry revealed by fluorescein isothiocynate (green). Appositions between GABA-immunoreactive structures and the labeled fiber appeared as yellow spots because of the presence of both fluorochromes in small volumes (0.3 * 0.3 * 0.5 micrometer(3)) of tissue. These spots were identified as probable axo-axonic contacts when: (1) they were observed in two to four serial confocal planes, indicating that they did not occur by chance; and (2) their sizes, shapes, and locations were similar to those of axo-axonic contacts found on Ia terminals, known to bear presynaptic boutons, and resembled the axo-axonic synapses described in electron microscope studies of Ib boutons in Clarke's column. A total of 59 presumed axo-axonic contacts was observed on two Ib collaterals, representing an estimated 20% of the total complement. In a three-dimensional reconstruction of one collateral, they were mostly located in terminal positions, and some branches bore more contacts than others. Such differential distribution could not result from chance appositions between GABAergic structures and Ib arborization and further supported the identification of axo-axonic contacts. Segmental Ib collaterals bear axo-axonic synapses that might ensure differential funneling of information toward different targets.

Alteration of the actin-based cytoskeleton by rabies virus.

We investigated the effect of rabies virus infection on the actin cytoskeleton using various techniques. Confocal microscopic examination of rabies virus-infected neuroblastoma cells at late stages of infection revealed a dramatic decrease in F-actin staining. The results of a fluorimetric assay with pyrenylated actin indicated that purified rabies virus nucleocapsid has no direct action on the kinetics of actin polymerization and only a weak effect on the final extent of polymerization. Video-microscopy experiments with purified components showed that rabies virus nucleocapsid inhibits the actin-bundling effect induced by dephospho-synapsin I, a neuron-specific protein which is known to exert a control on the actin-based cytoskeleton. Thus, the observed decrease in F-actin staining in infected cells might be ascribed to an indirect action of rabies nucleocapsid on the effects of actin-binding proteins such as synapsin I.

Distribution of endosomal, lysosomal, and major histocompatability complex markers in a monocytic cell line infected with Chlamydia psittaci.

The intracellular fate of Chlamydia psittaci during infection of a monocytic cell line, THP1, was characterized. Cytochalasin D inhibited phagocytosis of latex beads but had no effect on infection by C. psittaci, and vacuoles expressed the transferrin receptor, suggesting accessibility to the endocytic pathway. Early Chlamydia-containing vacuoles expressed major histocompatibility complex (MHC) class I molecules, and most vacuoles fused with host cell lysosomes, since they expressed LAMP-1 and had acidic pHs. In cells prestimulated with gamma interferon, vacuoles also expressed MHC class II molecules, suggesting that the monocytes might effectively process Chlamydia-derived antigens for presentation by MHC class I and class II molecules.

Heparin-mediated inhibition of Chlamydia psittaci adherence to HeLa cells.

The adherence of human strains of Chlamydia trachomatis has been recently shown to be inhibitable by heparin and heparitinase, leading to the proposal that Chlamydia binding to host cells may be mediated by a glycosaminoglycan (GAG)-dependent mechanism. We here describe the adherence of the guinea-pig pathogen, Chlamydia psittaci GPIC, to HeLa cells, which was measured by cytofluorometry with chlamydiae whose DNA was fluorescently labelled. Adherence could be inhibited by heat or trypsin pretreatment of the bacteria, and binding was much faster at 37 degrees C (reaching a plateau within 1 h) than 4 degrees C. Little binding remained when host cells were pre-fixed with paraformaldehyde, suggesting that host cell receptor mobility may be required for effective adherence. Visualization by confocal microscopy confirmed that the bacteria were at or near the host cell surface during the entire time-course of these experiments. Adherence increased as a function of pH between pH 6 and pH 8.0-8.5. Both adherence and infection of HeLa cells could be inhibited with heparin when the adherence step was performed at 4 degrees C, but only infection was inhibited when the adherence step was performed at 37 degrees C, even though heparitinase could block adherence at either 4 degrees C or 37 degrees C. Even at 4 degrees C, heparin-mediated inhibition was significantly lower at pH 8 than pH 7.4, suggesting that GAG-independent mechanisms may play a role in the higher adherence observed at basic pH. These results therefore demonstrate that a GAG-dependent adherence step may be operative in C. psittaci, and raise the possibility that other adherence mechanisms may also contribute to binding by this chlamydial strain. Furthermore, they suggest that there may not be a strict correlation between C. psittaci adherence and the ability to cause productive infections.

Accumulation in fetal muscle and localization to the neuromuscular junction of cAMP-dependent protein kinase A regulatory and catalytic subunits RI alpha and C alpha.

Using probes specific for cAMP-dependent protein kinase, we have analyzed by in situ hybridization the patterns of expression of regulatory and catalytic subunits in mouse embryos and in adult muscle. RI alpha transcripts are distributed in muscle fibers exactly as acetylcholinesterase, showing that this RNA is localized at the neuromuscular junction. The transcript levels increase upon denervation of the muscle, but the RNA remains localized, indicating a regulation pattern similar to that of the epsilon subunit of nicotinic acetylcholine receptor. RI alpha transcripts have accumulated in the muscle by day 12 of mouse embryogenesis, and localization is established by day 14, at about the time of formation of junctions. This localization is maintained throughout development and in the adult. Immunocytochemical analysis has demonstrated that RI alpha protein is also localized. In addition, RI alpha recruits C alpha protein to the junction, providing at this site the potential for local responsiveness to cAMP. PKA could be implicated in the establishment and/or maintenance of the unique pattern of gene expression occurring at the junction, or in the modulation of synaptic activity via protein phosphorylation. Embryonic skeletal muscle shows a high level of C alpha transcripts and protein throughout the fiber; the transcripts are already present by day 12 of embryogenesis, and their elevated level is maintained only through fetal life. In the adult, the C alpha hybridization signal of muscle is weak and homogeneous.

Immunology and the confocal microscope.

The techniques of classical epifluorescence microscopy are already widely used by the immunological community to detect antigens at the cellular level. Coupled with the use of specific inhibitors that affect diverse intracellular events, these techniques have provided valuable information on the mechanisms involved in antigen presentation. The same biological samples can now be examined by confocal microscopy, which has a higher resolution than conventional microscopy and allows one to analyse quantitatively single cross-sections of the sample. The confocal microscope is therefore especially well-suited for studies on intracellular membrane traffic, cell-to-cell interactions, and the distribution of particular antigens and their co-localization with other intracellular markers. This review describes the technique of confocal microscopy and the goals of sample preparation, along with several detailed protocols for fixing and permeabilizing cells and mounting them on microscope slides. Representative examples are cited from studies on the endocytosis of surface receptors, the distribution of adhesion and major histocompatibility complex (MHC) molecules, and the interaction of an intracellular parasite with MHC molecules of the host cell.

Internalization and biological effects of serum albumin in the breast cancer MCF-7 and MDA-MB 231 cells.

We show that albumin is internalized by human breast cancer cells (MDA-MB 231 and MCF-7) in culture by using confocal laser scanning microscopy. Moreover, albumin has an effect on the level of radioactivity incorporated when the cells are incubated with radioactive estradiol, and it is necessary to observe the mitogenic effect of estradiol towards the MCF-7 cells. This finding opens some possibilities regarding the internalization mechanisms and fate (degradation, recycling) of albumin as well as the role played by this protein in the intracellular metabolism of estradiol and in the intra-extracellular traffic of estradiol and its metabolites.

Intracellular Leishmania amazonensis amastigotes internalize and degrade MHC class II molecules of their host cells.

In their amastigote stage, Leishmania live in mammalian macrophages within parasitophorous vacuoles (PV), organelles of phagolysosomal origin that, in macrophages activated with IFN-gamma, contain major histocompatibility complex (MHC) class II molecules apparently devoid of invariant chains. We have now studied the fate of PV-associated class II molecules in mouse bone marrow-derived macrophages infected with L. amazonensis amastigotes using immunocytochemical and biochemical approaches. We have found that at least a part of these class II molecules was internalized by amastigotes and reached structures very often located in their posterior poles. This process was much more obvious if infected macrophages were incubated with protease inhibitors like antipain, chymostatin, Z-Phe-AlaCHN2 and Z-Phe-PheCHN2, or if amastigotes were pre-treated with the irreversible cysteine protease inhibitor Z-Phe-AlaCHN2 before infection, clearly indicating that amastigotes also degraded the internalized class II molecules. Study of infected macrophage cryosections by immuno-electron microscopy allowed the identification of the class II-positive structures in amastigotes as the lysosome-like organelles known as megasomes. Other PV membrane components like the prelysosomal/lysosomal glycoproteins Igp110, Igp120 and macrosialin could not be detected in megasomes of amastigotes even after treatment of macrophages with protease inhibitors, suggesting the involvement of some specific mechanism(s) for the internalization of class II molecules. Interestingly, after treatment of infected macrophages with various protease inhibitors (antipain, leupeptin, E-64, Z-Phe-AlaCHN2, Z-Phe-PheCHN2), PV membrane as well as megasomes of amastigotes become positive for invariant chains. A quantitative analysis of amastigote-associated class II molecules based on enzyme immunoassays showed that: (a) amastigotes extracted from macrophages treated with both IFN-gamma and antipain or Z-Phe-AlaCHN2 contained a much greater amount of class II than amastigotes extracted from macrophages treated with IFN-gamma alone; (b) class II molecules associated with the former were mainly intracellular and, at least some of them, were complexed with invariant chains or fragments of invariant chains; (c) amastigotes pre-incubated with Z-Phe-AlaCHN2 before infection accumulated a greater amount of intracellular class II than amastigotes pre-incubated without inhibitor, clearly indicating that the blockade of parasite cysteine proteases was sufficient to enhance the pool of these molecules within megasomes. On the whole, these data are consistent with the idea that class II molecules reaching PV are newly synthesized and still complexed with intact invariant chains or with partially degraded invariant chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Targeting of Listeria monocytogenes ActA protein to the plasma membrane as a tool to dissect both actin-based cell morphogenesis and ActA function.

Actin assembly on the surface of Listeria monocytogenes in the cytoplasm of infected cells provides a model to study actin-based motility and changes in cell shape. We have shown previously that the ActA protein, exposed on the bacterial surface, is required for polarized nucleation of actin filaments. To investigate whether plasma membrane-associated ActA can control the organization of microfilaments and cell shape, variants of ActA, in which the bacterial membrane signal had been replaced by a plasma membrane anchor sequence, were produced in mammalian cells. While both cytoplasmic and membrane-bound forms of ActA increased the F-actin content, only membrane-associated ActA caused the formation of plasma membrane extensions. This finding suggests that ActA acts as an actin filament nucleator and shows that permanent association with the inner face of the plasma membrane is required for changes in cell shape. Based on the observation that the amino-terminal segment of ActA and the remaining portion which includes the proline-rich repeats cause distinct phenotypic modifications in transfected cells, we propose a model in which two functional domains of ActA cooperate in the nucleation and dynamic turnover of actin filaments. The present approach is a new model system to dissect the mechanism of action of ActA and to further investigate interactions of the plasma membrane and the actin cytoskeleton during dynamic changes of cell shape.

Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains.

Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and IL15 have common beta and gamma chains, while those for IL2, 4, 7, and 9 have a common gamma chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the alpha, beta, and gamma chains had different intracellular localizations after being endocytosed together. The alpha chain was always in transferrin-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the beta and gamma chains were excluded from transferrin-positive organelles and did not colocalize with alpha. Furthermore, beta could be found in rab7-positive vesicles. These differences suggest that the alpha chain recycles to the plasma membrane, while the beta and gamma chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The beta and gamma chains are very short-lived polypeptides since their half-life on the surface is only approximately 1 h, whereas alpha is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.

Leishmania donovani-infected macrophages: characterization of the parasitophorous vacuole and potential role of this organelle in antigen presentation.

Leishmania donovani amastigotes, the etiological agents of visceral leishmaniasis, are obligate intracellular parasites residing in membrane-bound compartments of macrophages called parasitophorous vacuoles (PV). The study of these organelles is of paramount importance to understanding how these parasites resist the microbicidal mechanisms of macrophages and how they escape the immune response of their hosts. Confocal microscopy of mouse bone marrow-derived macrophages infected with L. donovani amastigotes and stained for various prelysosomal/lysosomal markers and for major histocompatibility complex (MHC) molecules was used to define PV with respect to the endocytic compartments of the host cells and to address the issue of their potential role in antigen processing and presentation. Forty-eight hours after infection, many PV contained cathepsins B, D, H and L and they were all surrounded by a membrane enriched for the lysosomal glycoprotein lgp120/lamp 1 but apparently devoid of the cation-independent mannose 6-phosphate receptor, a membrane protein generally absent from the lysosomes. These data suggested that PV acquire within 48 hours the characteristics of a lysosomal compartment. However, both macrosialin and the GTP-binding protein rab7p (specific markers of the prelysosomal compartment) were found to be highly expressed in/on PV membrane. Thus, at this stage, PV appear to exhibit both lysosomal and prelysosomal features. Infected macrophages activated with IFN-gamma before or after infection showed PV strongly stained for MHC class II molecules but not for MHC class I molecules. This suggests that, if infected macrophages can act as antigen-presenting cells for class I-restricted CD8+ T lymphocytes, Leishmania antigens must exit the PV. MHC class II molecules reached the PV progressively, indicating that they were not plasma membrane-bound molecules trapped during internalization of the parasites. The redistribution of class II observed in infected cells did not alter their quantitative expression on the plasma membrane at least during the first 48 hours following the phagocytosis of the parasites. The invariant chains, which are transiently associated with class II molecules during their intracellular transport and which mask their peptide-binding sites, did not reach PV or were rapidly degraded in these sites, suggesting that PV-associated class II are able to bind peptides. This last assumption is strengthened by the fact that class II located in PV could bind conformational antibodies that preferentially recognize class II with tightly associated peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of MHC class I and of MHC class II molecules in macrophages infected with Leishmania amazonensis.

Macrophages, being apparently the only cells that in vivo allow the growth of the intracellular pathogen Leishmania, are likely candidates to present antigens to Leishmania-specific CD4+ and CD8+ T lymphocytes, known to be involved in the resolution or in the development of lesions induced by these parasites, and recognizing processed antigens bound to MHC class I and MHC class II molecules, respectively. In the present study, we analysed by confocal microscopy and by immunoelectron microscopy the subcellular distribution of both MHC class I and class II molecules in mouse (Balb/c and C57BL/6 strains) bone marrow-derived macrophages infected for 12 to 48 hours with Leishmania amazonensis amastigotes and activated with gamma interferon to determine the intracellular sites where Leishmania antigens and MHC molecules meet and can possibly interact. Double labelings with anti-MHC molecule antibodies and with either propidium iodide or an anti-amastigote antibody allowed localization of MHC molecules with regard to the endocytic compartments housing Leishmania amastigotes, organelles known as the parasitophorous vacuoles (PV) and which most likely contain the highest concentration of parasite antigens in the host cell. Both uninfected and infected macrophages from Balb/c mice expressed the MHC class I molecules H-2Kd and H-2Dd on their cell surface but no significant amount of these molecules could be detected in the PV, which indicates that, if infected macrophages play a role in the induction of Leishmania-specific CD8+ T lymphocytes, PV are probably not loading compartments for MHC class I molecules. In contrast, MHC class II molecules were found to be associated with the PV membranes as shown previously with microscopic techniques at lower resolution (Antoine et al. Infect. Immun. 59, 764-775, 1991). In addition, we show here that, 48 hours after infection of Balb/c macrophages, in about 90% of PV containing MHC class II molecules, the latter were mainly or solely localized at the attachment zone of amastigotes to PV membranes. This peculiar distribution, especially well demonstrated using confocal microscopy, was confirmed by subcellular fluorescence cytometry of infected macrophages stained for the MHC class II molecules. The following data agree with the idea that PV-associated MHC class II molecules establish specific interactions with plasma membrane components of amastigotes. First, the polarized localization of class II appeared specific to these molecules, since the distribution of the lysosomal glycoproteins Igp110 and Igp120, of the macrosialin (a macrophage-specific marker of endocytic compartments) and of the GTP-binding protein rab7p, shown here as being PV membrane components, was homogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

Polarized distribution of Listeria monocytogenes surface protein ActA at the site of directional actin assembly.

The facultative intracellular pathogen Listeria monocytogenes can infect host tissues by using directional actin assembly to propel itself from one cell into another. The movement is generated by continuous actin assembly from one end of the bacterium into a tail, which is left behind in the cytoplasm. Bacterial actin assembly requires expression of the bacterial gene actA. We have used immunocytochemistry to show that the actA gene product, ActA, is distributed asymmetrically on the bacterial surface: it is not expressed at one pole and is increasingly concentrated towards the other. This polarized distribution of ActA was linked to bacterial division: ActA protein was not, or only faintly, expressed at the pole that had been formed during the previous division. On intracellular bacteria ActA was expressed at the site of actin assembly, suggesting that ActA may be involved in actin filament nucleation off the bacterial surface. We predict that the asymmetrical distribution of this protein is required for the ability of intracellular Listeria to move in the direction of the non-ActA expressing pole.

Both the alpha and beta chains of high-affinity interleukin 2 receptors are located in intracellular vesicles when their ligand is endocytosed.

The growth factor interleukin 2 (IL2) binds to high and low-affinity receptors (Kd approximately 10-100 pM and 10 nM, respectively) present on activated T lymphocytes. High-affinity receptors are composed of two non-convalently linked polypeptides, alpha and beta of 55 and 70 kDa. These two polypeptides do not share any sequence homology, but each of them, in the absence of the other, binds IL2: alpha with a Kd approximately 10 nM and beta with a Kd approximately 1 nM. When these two chains are associated in lymphocytes, they form high-affinity receptors that mediate IL2 endocytosis and degradation, and transduce IL2 signaling. On cells that physiologically express IL2 receptors, such as activated T lymphocytes, both high and low affinity-receptors are present simultaneously on the cell surface, and low-affinity receptors (alpha without beta) are, in most instances, more abundant by a factor 5 to 10 than high-affinity receptors (alpha associated to beta). Low-affinity receptors bind IL2 but do not induce its internalization and signaling. The physiological role of the complexity of this receptor system is not fully understood. In the present study, we have investigated directly the fate of the high-affinity receptors when the ligand is endocytosed. By confocal microscopy, using two monoclonal antibodies specific for alpha and for beta, respectively, we show that each of these two polypeptides is located in intracellular endocytic compartments. Therefore, when the alpha chain is part of high-affinity receptors, it is endocytosed, as opposed to when it is part of low-affinity receptors and is not endocytosed.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene.

Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5' regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.