The art of cellular communication: tunneling nanotubes bridge the divide

The ability of cells to receive, process, and respond to information is essential for a variety of biological processes. This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms. In the latter, most cells do not exist as independent units, but are organized into specialized tissues. Within these functional assemblies, cells communicate with each other in different ways to coordinate physiological processes. Recently, a new type of cell-to-cell communication was discovered, based on de novo formation of membranous nanotubes between cells. These F-actin-rich structures, referred to as tunneling nanotubes (TNT), were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components. The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity. At the same time it emerged that the direct transfer of cargo between cells is a common functional property, suggesting a general role of TNT-like structures in selective, long-range cell-to-cell communication. Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling, it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes

Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes

Background: Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. Methodology and Principal Findings: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca2+ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. Conclusions and Significance: Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic

Corneal Alternations Induced by Topical Application of Benzalkonium Chloride in Rabbit

Benzalkonium chloride (BAC) is the most common preservative in ophthalmic preparations. Here, we investigated the corneal alternations in rabbits following exposure to BAC. Twenty-four adult male New Zealand albino rabbits were randomly divided into three groups. BAC at 0.01%, 0.05%, or 0.1% was applied twice daily to one eye each of rabbits for 4 days. The contralateral untreated eyes were used as control. Aqueous tear production and fluorescein staining scores of BAC-treated eyes were compared with those of controls. The structure of the central cornea was examined by in vivo confocal microscopy. Expression of mucin-5 subtype AC (MUC5AC) in conjunctiva was detected by immunostainig on cryosections. Corneal barrier function was assessed in terms of permeability to carboxy fluorescein (CF). The distribution and expression of ZO-1, a known marker of tight junction, and reorganization of the perijunctional actomyosin ring (PAMR) were examined by immunofluorescence analysis. Although there were no significant differences between control and BAC-treated eyes in Schirmer scores, corneal fluorescein scores and the number of conjunctival MUC5AC staining cells, in vivo confocal microscopy revealed significant epithelial and stromal defects in all BAC-treated corneas. Moreover, BAC at 0.1% resulted in significant increases in central corneal thickness and endothelial CF permeability, compared with those in control eyes, and endothelial cell damage with dislocation of ZO-1 and disruption of PAMR. Topical application of BAC can quickly impair the whole cornea without occurrence of dry eye. A high concentration of BAC breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR and remodeling of apical junctional complex in vivo

Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Femtosecond linear dichroism of DNA-intercalating chromophores: Solvation and charge separation dynamics of [Ru(phen)₂dppz]²⁺ systems

The DNA-intercalating chromophore [Ru(phen)(2)dppz](2+) has unique photophysical properties, the most striking of which is the "light-switch" characteristic when binding to DNA. As a dimer, it acts as a molecular staple for DNA, exhibiting a remarkable double-intercalating topology. Herein, we report femtosecond dynamics of the monomeric and the covalently linked dimeric chromophores, both free in aqueous solution and complexed with DNA, Transient absorption and linear dichroism show the electronic relaxation to the lowest metal-to-ligand charge-transfer (CT) state, and subpicosecond kinetics have been observed for this chromophore for what is, to our knowledge, the first time. We observe two distinct relaxation processes in aqueous solution with time constants of 700 fs and 4 ps. Interestingly, these two time constants are very similar to those observed for the reorientational modes of bulk water. The 700-fs process involves a major dichroism change. We relate these observations to the change in charge distribution and to the time scales involved in solvation of the CT state. Slower processes, with lifetimes of approximate to 7 and 37 ps, were observed for both monomer and dimer when hound to DNA, Such a difference can be ascribed to the change of the structural and electronic relaxation experienced in the DNA intercalation pocket. Finally, the recombination lifetime of the final metal-to-ligand CT state to the ground state, which is a key in the light-switch process, is found in aqueous solution to be sensitive to structural modification, ranging from 260 ps for [Ru(phen)(2)dppz](2+) and 360 ps for the monomer chromophore derivative to 2.0 ns for the dimer. This large change reflects the direct role of solvation in the light-switch process

Recognition of synthetic polyanionic ligands underlies “spontaneous” reactivity of Vγ1γδTCRs

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1‐containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self‐ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate‐like αβ T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long‐standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation‐independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.ISSN:0741-5400ISSN:1938-367