Maruša Lokar

Different Types of Cell-to-Cell Connections Mediated by Nanotubular Structures

Communication between cells is crucial for proper functioning of multicellular organisms. The recently discovered membranous tubes, named tunneling nanotubes, that directly bridge neighboring cells may offer a very specific and effective way of intercellular communication. Our experiments on RT4 and T24 urothelial cell lines show that nanotubes that bridge neighboring cells can be divided into two types. The nanotubes of type I are shorter and more dynamic than those of type II, and they contain actin filaments. They are formed when cells explore their surroundings to make contact with another cell. The nanotubes of type II are longer and more stable than type I, and they have cytokeratin filaments. They are formed when two already connected cells start to move apart. On the nanotubes of both types, small vesicles were found as an integral part of the nanotubes (that is, dilatations of the nanotubes). The dilatations of type II nanotubes do not move along the nanotubes, whereas the nanotubes of type I frequently have dilatations (gondolas) that move along the nanotubes in both directions. A possible model of formation and mechanical stability of nanotubes that bridge two neighboring cells is discussed.

Mechanisms for the formation of membranous nanostructures in cell-to-cell communication

Cells interact by exchanging material and information. Two methods of cell-to-cell communication are by means of microvesicles and by means of nanotubes. Both microvesicles and nanotubes derive from the cell membrane and are able to transport the contents of the inner solution. In this review, we describe two physical mechanisms involved in the formation of microvesicles and nanotubes: curvature-mediated lateral redistribution of membrane components with the formation of membrane nanodomains; and plasmamediated attractive forces between membranes. These mechanisms are clinically relevant since they can be affected by drugs. In particular, the underlying mechanism of heparin’s role as an anticoagulant and tumor suppressor is the suppression of microvesicluation due to plasma-mediated attractive interaction between membranes.

Binding of plasma proteins to titanium dioxide nanotubes with different diameters

Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices.

The role of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes

Intercellular membrane nanotubes (ICNs) are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant) urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are discussed.

Temperature and cholera toxin B are factors that influence formation of membrane nanotubes in RT4 and T24 urothelial cancer cell lines.

The growth of membrane nanotubes is crucial for intercellular communication in both normal development and pathological conditions. Therefore, identifying factors that influence their stability and formation are important for both basic research and in development of potential treatments of pathological states. Here we investigate the effect of cholera toxin B (CTB) and temperature on two pathological model systems: urothelial cell line RT4, as a model system of a benign tumor, and urothelial cell line T24, as a model system of a metastatic tumor. In particular, the number of intercellular membrane nanotubes (ICNs; ie, membrane nanotubes that bridge neighboring cells) was counted. In comparison with RT4 cells, we reveal a significantly higher number in the density of ICNs in T24 cells not derived from RT4 without treatments (P = 0.005), after 20 minutes at room temperature (P = 0.0007), and following CTB treatment (P = 0.000025). The binding of CTB to GM1–lipid complexes in membrane exvaginations or tips of membrane nanotubes may reduce the positive spontaneous (intrinsic) curvature of GM1–lipid complexes, which may lead to lipid mediated attractive interactions between CTB–GM1–lipid complexes, their aggregation and consequent formation of enlarged spherical tips of nanotubes. The binding of CTB to GM1 molecules in the outer membrane leaflet of membrane exvaginations and tips of membrane nanotubes may also increase the area difference between the two leaflets and in this way facilitate the growth of membrane nanotubes.

Protruding membrane nanotubes: attachment of tubular protrusions to adjacent cells by several anchoring junctions

Membrane nanotubes are a morphologically versatile group of membrane structures (some resembling filopodia), usually connecting two closely positioned cells. In this article, we set morphological criteria that distinguish the membrane nanotubes from filopodia, as there is no specific molecular marker known to date that unequivocally differentiates between filopodia and protruding nanotubes. Membrane nanotubes have been extensively studied from the morphological point of view and the transport that can be conducted through them, but little is known about the way they connect to the adjacent cell. Our results show that the nanotubes may connect to a neighboring cell by anchoring junctions. Among cell adhesion proteins, N-cadherin, β-catenin, nectin-2, afadin and the desmosomal protein desmoplakin-2 were immune-labeled. We found that N-cadherin and β-catenin are concentrated in nanotubes, while the concentrations of other junction-involved proteins are not increased in these structures. On the basis of data from transmission electron microscopy, we propose a model of the nanotube attachment where the connection of nanotubes is stabilized by several anchoring junctions, most likely adherens junctions that are formed when the nanotube is sliding along the target cell membrane.

Agglutination of like-charged red blood cells induced by binding of β2-glycoprotein I to outer cell surface

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

Chapter 3 Membrane Nanotubes in Urothelial Cell Line T24

Abstract Membrane nanotubes (also referred as tunnelling nanotubes—TNTs, nanotubules, cytonemes), that directly connect separated neighboring cells, may offer a very specific and effective way of intercellular transport and communication. Our experiments on T24 cell line show that TNTs can be divided into two types with respect to their biochemical and biophysical characteristics and the nature of their formation. As type I were characterized the nanotubes which are shorter, more dynamic and contain actin filaments. These structures remain stabile even if underlying actin cytoskeleton is disintegrated by cytochalasin D. The nanotubes of type II are much longer, appear more stable and contain cytokeratin filaments. In both types microtubules can be found, but this type of cytoskeleton is present in only a small fraction of the TNTs. On the nanotubes of both types small vesicular dilatations were found as an integral part of the nanotubes (i.e., dilatations of the nanotubes, gondolas). Vesicular dilatations of type I nanotubes move along the nanotubes in both directions, while the vesicular dilatations of type II nanotubes do not move along the nanotubes. Both TNTs by themselves and the transporting gondolas were proposed to be involved in intercellular communication and transport. The possible mechanisms of stabilization of membrane nanotubular protrusions and TNTs are also discussed.

The Effect of in vitro Anticoagulant Disodium Citrate on Beta-2-glycoprotein I - Induced Coalescence of Giant Phospholipid Vesicles

In order to elucidate the mechanisms of blood coagulation the complex interactions between phospholipid membranes, serum protein beta-2-glycoprotein I (β2GPI), antiphospholipid antibodies (aPL) and disodium citrate were studied by observing collective interactions between giant phospholipid vesicles (GPVs) in the sugar solution. GPVs composed of palmitoyl-oleoyl-sn-glycero-3-phosphocholine (POPC), tetraoleoyl cardiolipin and cholesterol were obtained by the electroformation method and observed under the phase contrast microscope. β2GPI or aPL acted as mediators inducing the coalescence of the vesicles. The strength of the adhesion between the coalesced vesicles was dependent on the content of cardiolipin and the species of the mediator. The addition of disodium citrate to the coalesced GPVs solution caused disintegration of the complexes of coalesced vesicles. The extend of the disintegration between coalesced vesicles was interpreted to be connected to the strength of the adhesion between GPVs. It was found that the disintegration of the GPV complexes was more pronounced in the system where the vesicles coalesced due to the presence of antiphospholipid antibodies compared to the system where the vesicles coalesced due to the presence of β2GPI. The effect of the disintegration of the coalesced GPVs was more pronounced for smaller vesicles which originated in the budding of the the membrane of larger GPVs.