Prion aggregates transfer through tunneling nanotubes in endocytic vesicles

Abstract

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrP^Sc. The intercellular transfer of PrP^Sc is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrP^Sc can move between cells. Here we investigate further the details of this mechanism and show that PrP^Sc travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs.     

Prion aggregates transfer through tunneling nanotubes in endocytic vesicles

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrP^Sc. The intercellular transfer of PrP^Sc is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrP^Sc can move between cells. Here we investigate further the details of this mechanism and show that PrP^Sc travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs.     

Exosomes Are Unlikely Involved in Intercellular Nef Transfer

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81^low/TSG101^low exosomes, but not in detergent-soluble AChE-/CD81^high/TSG101^high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81^low/TSG101^low exosomes and AChE-/CD81^high/TSG101^high exosomes.     

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca²⁺-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP₃ and Ca²⁺ that initiate the propagation of the Ca²⁺-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca²⁺-wave propagation are provided by gap junction channels through the direct transfer of IP₃ and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca²⁺-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca²⁺-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca²⁺-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.     

Tumour promoter-mediated reversible inhibition of cell-cell communication (electrical coupling) : Relationship with phorbol ester binding and de novo macromolecule synthesis

A tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), reversibly inhibits the onset and maintenance of cell-cell communication measured by electrophysiological method. We have now studied the mechanism by which TPA inhibits communication of human cells (FL) in culture. Using [³H]phorbol-12,13-dibutyrate ([³H]PDBu), we found a class of specific, high-affinity, saturable binding sites in intact FL cells; they have a dissociation constant of 15.4 nM, and at saturation about 3 × 10⁵ PDBu molecules were bound to each cell. The binding of [³H]PDBu to FL cells was inhibited by TPA, phorbol-12-13-didecanoate and mezerein, whereas phorbol and 4α-phorbol-12-13-didecanoate had no effect. There is a close correlation between the ability of the former compounds to inhibit [³H]PDBu binding and their capacity to inhibit cell-cell communication. When FL cells are dispersed with EDTA and plated onto a culture dish, they start to couple electrically within 2 h; such cell coupling was not affected by the presence of cycloheximide or actinomycin D. TPA inhibits the formation of electrical cell coupling as well as its maintenance, even in the presence of cycloheximide; the recovery of cell-cell communication after the removal of TPA was not significantly affected by the addition of cycloheximide or actinomycin D. Taken together, these results suggest that TPA-mediated reversible inhibition of intercellular communication is mediated by specific binding of TPA to cellular receptors and that macromolecular synthesis is not necessary.     

Intercellular Sodium Regulates Repolarization in Cardiac Tissue with Sodium Channel Gain of Function

In cardiac myocytes, action potentials are initiated by an influx of sodium (Na⁺) ions via voltage-gated Na⁺ channels. Na⁺ channel gain of function (GOF), arising in both inherited conditions associated with mutation in the gene encoding the Na⁺ channel and acquired conditions associated with heart failure, ischemia, and atrial fibrillation, enhance Na⁺ influx, generating a late Na⁺ current that prolongs action potential duration (APD) and triggering proarrhythmic early afterdepolarizations (EADs). Recent studies have shown that Na⁺ channels are highly clustered at the myocyte intercalated disk, facilitating formation of Na⁺ nanodomains in the intercellular cleft between cells. Simulations from our group have recently predicted that narrowing the width of the intercellular cleft can suppress APD prolongation and EADs in the presence of Na⁺ channel mutations because of increased intercellular cleft Na⁺ ion depletion. In this study, we investigate the effects of modulating multiple extracellular spaces, specifically the intercellular cleft and bulk interstitial space, in a novel computational model and experimentally via osmotic agents albumin, dextran 70, and mannitol. We perform optical mapping and transmission electron microscopy in a drug-induced (sea anemone toxin, ATXII) Na⁺ channel GOF isolated heart model and modulate extracellular spaces via osmotic agents. Single-cell patch-clamp experiments confirmed that the osmotic agents individually do not enhance late Na⁺ current. Both experiments and simulations are consistent with the conclusion that intercellular cleft narrowing or expansion regulates APD prolongation; in contrast, modulating the bulk interstitial space has negligible effects on repolarization. Thus, we predict that intercellular cleft Na⁺ nanodomain formation and collapse critically regulates cardiac repolarization in the setting of Na⁺ channel GOF.     

Evidence for Distinct Aggregation Factors and Receptors in Cells

SINCE the original observations by Wilson¹ that dissociated sponge cells could reassociate in vitro, cell aggregation (or reaggregation) has been widely used as an operational criterion for the study of intercellular adhesion². The introduction of rotation-mediated methods to promote cell aggregation^3,4 led to the possibility of obtaining reproducible quantitative data. In these methods, suspensions of dissociated single cells are shaken under defined conditions of speed and temperature and cell aggregation is measured by either the size of aggregates or the number of single cells. The aggregation of dissociated cells from sponges⁵, chick and mouse embryos⁴ and tissue culture cells⁶ has been investigated with this method. Cells maintained in vitro seemed particularly suitable for studying mechanisms of cell aggregation as they represent a histotypically homogeneous population.     

Reversible Inhibition of Gap Junctional Intercellular Communication, Synchronous Contraction, and Synchronism of Intracellular Ca Fluctuation in Cultured Neonatal Rat Cardiac Myocytes by Heptanol

We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca²⁺ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca²⁺ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca²⁺ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca²⁺ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca²⁺ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca²⁺ fluctuations, which facilitate synchronized contraction of cardiac myocytes.     

Mitochondria modulate the spatio-temporal properties of intra- and intercellular Ca signals in cochlear supporting cells

In the cochlea, cell damage triggers intercellular Ca²⁺ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca²⁺ waves are thought to convey information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt), and yet little is known about their role during the propagation of such intercellular Ca²⁺ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell [Ca²⁺]_cyt signals that are triggered by ATP or by hair cell damage. ATP application (0.1–50 μM) caused a dose dependent increase in [Ca²⁺]_cyt which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca²⁺ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, enhanced the peak amplitude and duration of ATP-induced [Ca²⁺]_cyt transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca²⁺ waves was significantly increased. Thus, mitochondria function as spatial Ca²⁺ buffers during agonist-evoked [Ca²⁺]_cyt signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca²⁺ waves.     

A locally-induced increase in intracellular Ca propagates cell-to-cell in the presence of plasma membrane Ca ATPase inhibitors in non-excitable cells

Intercellular Ca²⁺ waves are commonly observed in many cell types. In non-excitable cells, intercellular Ca²⁺ waves are mediated by gap junctional diffusion of a Ca²⁺ mobilizing messenger such as IP₃. Since Ca²⁺ is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca²⁺ to intercellular Ca²⁺ waves is limited. Here, we report that in the presence of plasma membrane Ca²⁺ ATPase inhibitors, locally-released Ca²⁺ from the flash-photolysis of caged-Ca²⁺ appeared to induce further Ca²⁺ release and were propagated from one cell to another, indicating that Ca²⁺ was self-amplified to mediate intercellular Ca²⁺ waves. Our findings support the notion that non-excitable cells can establish a highly excitable medium to communicate local responses with distant cells.     

Effects of thymidine analogs upon growth control in cultured hormone-dependent ray ovary cells.

Growth-promoting alpha-globulin (GPAG) has a strong binding capacity for ³H-thymidine, ³H-uridine, ³H-lysine, and ³²PO₄? and causes an enormous increase in the rate of RNA, DNA, and protein synthesis in metazoan cells. GPAG is taken into the cell by pinocytosis in S and G 2 periods of the cell cycle. Pinosomes travel rapidly towards the perinuclear region before the bound precursors are used in macromolecule biosynthesis. As the GPAG is needed for an actively proliferating culture of metazoan cells in vitro, it is concluded that pinocytosis of GPAG is the controlling factor in deciding whether a cell will continue to grow or will go to the resting state and that the formation of DNA, RNA, and protein may be controlled by the availability of usable precursors in the site of macromolecule biosynthesis. The sources of GPAG in metazoan organism have not been identified but it is reasonable to believe that cell division is regulated primarily by this metazoan factor on an intercellular level.     

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system^{1, 2}; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis³ have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions⁴. Radiation pressure was first observed and applied to optical tweezer systems in 1970^{5, 6}, and was first used to control biological specimens in 1987⁷. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena^8-13.We describe a method¹⁴ that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals^{15, 16} (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.     

Hydrogen peroxide induces microvilli on human retinal pigment epithelial cells in culture.

We have found that hydrogen peroxide (10^-4 - 10^-2 M) rapidly induces microvilli on separate cells and confluent sheets of human retinal pigment epithelium in culture. t-butyl hydroperoxide and sodium arsenite do not induce microvilli. A role for hydrogen peroxide as an intercellular messenger has previously been proposed in the inflammatory response, in which hydrogen peroxide from phagocytes may signal to vascular endothelial cells. Our observations thus provide a second example of the induction of what may be a physiological response by this potentially toxic agent. In the retina, hydrogen peroxide released from illuminated photoreceptors may elongate the microvilli which extend into the spaces between them. Increased numbers of microvilli and their protrusion further into the photoreceptor layer may enhance various interactions between the two cell types, including the antioxidant functions of the epithelium.     

E-Cadherin–dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27KIP1

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell–cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell– cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell–cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27^kip1 and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin–neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin–dependent growth inhibition, we engineered E-cadherin–positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin–neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.     

Involvement of the neuregulins and their receptors in cardiac and neural development

The neuregulin gene encodes a series of polypeptide growth factors that can influence the growth state of target vertebrate cells in culture. Recently, three studies have explored the in vivo function of the neuregulin signaling system in mice by disrupting the genes encoding the neuregulin ligand⁽¹⁾ and two of its receptors, ErbB2⁽²⁾ and ErbB4⁽³⁾. Each of the genes is essential for development, and aberrations in cardiac and neural development are particularly prominent in mutant embryos. The observed defects, together with the localization of expression of the neuregulin signaling components within these tissues, highlight a paracrine mechanism for neuregulin-mediated intercellular communication.     

Application of freeze-etching to the study of myogenesis in cell culture

A method is described for in situ freeze-etching of cells grown in monolayer culture, with preservation of delicate surface structures such as filopodia. The close intercellular contact which is a prerequisite for fusion of myogenic cells appears to depend on and be mediated by filopodial contacts: cells grown in standard fusion-permitting medium show frequent filopodial contacts, whereas cells in medium depleted of Ca² ions by the addition of EGTA lack filopodia and fail to fuse.     

Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/CCdh1 Complex

Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C^Cdh1 complex, but not by the APC/C^Cdc20 complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.     

Unloading of intercellular tension induces the directional translocation of PKCα

The migration of endothelial cells (ECs) is closely associated with a Ca²⁺-dependent protein, protein kinase Cα (PKCα). The disruption of intercellular adhesion by single-cell wounding has been shown to induce the directional translocation of PKCα. We hypothesized that this translocation of PKCα is induced by mechanical stress, such as unloading of intercellular tension, or by intercellular communication, such as gap junction-mediated and paracrine signaling. In the current study, we found that the disruption of intercellular adhesion induced the directional translocation of PKCα even when gap junction-mediated and paracrine signaling were inhibited. Conversely, it did not occur when the mechanosensitive channel was inhibited. In addition, the strain field of substrate attributable to the disruption of intercellular adhesion tended to be larger at the areas corresponding with PKCα translocation. Recently, we found that a direct mechanical stimulus induced the accumulation of PKCα at the stimulus area, involving Ca ²⁺ influx from extracellular space. These results indicated that the unloading of intercellular tension induced directional translocation of PKCα, which required Ca ²⁺ influx from extracellular space. The results of this study indicate the involvement of PKCα in the Ca ²⁺ signaling pathway in response to mechanical stress in ECs.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by Connexin43

Abstract

Intercellular Ca^{2 +} wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca^{2 +} wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca^{2 +} waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca^{2 +} wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca^{2 +} waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca^{2 +} waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.