Razporejanje mikrotubulov v celicah V-79 po delovanju citohalazina B

Background. The aim of this work was to study the configuration of the microtubules in the cytochalasin B treated V-79 cells in connection to the cell shape and to see whether there are any similarities to the phenomena taking place in phospholipid vesicles. Subjects and methods. An experiment wasperformed where cytochalasin B was added to the V-79 cells (lung fibroblasts of Chinese hamster). Results. The cell shape changed from an elongated one into the shape with a profile resembling the Greek letter phi. The microtubules were found to be organized into a rod within the symmetry axis of the cell. Conclusion. As similar shapes were previously observed also in the phospholipid vesicles with entrapped microtubule rods, our results support the hypothesis that similar physical mechanisms may pertain in simple systems as well as in living cells.Izhodišča. V tej raziskavi smo proučevali razporeditev mikrotubulov celicah V-79 po delovanju citohalazina B. Poleg njihove oblike smo poskušali ugotoviti, ali je ta pojav v čemer koli podoben dogajanjem v fosfolipidnih mehurčkih. Material in metode. Opravili smo preiskus s citohalazinom B, ki smoga dodali celicam V 79 (pljučni fibroblasti kitajskega hrčka). Rezultati. Oblika celice se je spremenila iz podolgovate v obliko, katere profil je podoben grški črki fi. Mikrotubuli so se uredili v palico v simetrijski osi celice. Zaključek. Ker so podobne oblike pred tem opazili tudi pri fosfolipidnih mehurčkih, ki so vsebovali paličasto strukturo iz mikrotubulov, prikazani rezultati podpirajo hipotezo, da so podobni fizikalni mehanizmi prisotni v preprostih sistemih kot tudi v živih celicah

Colocalization of Serum Amyloid A with Microtubules in Human Coronary Artery Endothelial Cells

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency

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

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

Comparative lipidomic study of urothelial cancer models: association with urothelial cancer cell invasiveness

A joint NMR/LC-MS approach allows to establish significant differences in the lipidoma of invasive urothelial carcinoma cells (T24) with respect to noninvasive urothelial cells (RT4)

Post - prandial rise of microvesicles in peripheral blood of healthy human donors

<p>Abstract</p> <p>Background</p> <p>Microvesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood.</p> <p>Aim</p> <p>To study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations.</p> <p>Results</p> <p>The average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01) with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C) and average high density lipoprotein cholesterol concentration (HDL-C) all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12) and a 6% decrease in blood glucose (p < 0.01, P = 0.74). The post - prandial number of microvesicles negatively correlated with the post - fasting total cholesterol concentration (r = - 0.46, p = 0.035) while the difference in the number of microvesicles in the isolates between post - prandial and post - fasting states negatively correlated with the respective difference in blood glucose concentration (r = - 0.39, p = 0.05).</p> <p>Conclusions</p> <p>In a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.</p

Morphological alterations of T24 cells on flat and nanotubular TiO2 surfaces

Aim To investigate morphological alterations of malignant cancer cells (T24) of urothelial origin seeded on flat titanium (Ti) and nanotubular titanium dioxide (TiO2) nanostructures. Methods Using anodization method, TiO2 surfaces composed of vertically aligned nanotubes of 50-100 nm diameters were produced. The flat Ti surface was used as a reference. The alteration in the morphology of cancer cells was evaluated using scanning electron microscopy (SEM). A computational model, based on the theory of membrane elasticity, was constructed to shed light on the biophysical mechanisms responsible for the observed changes in the contact area of adhesion. Results Large diameter TiO2 nanotubes exhibited a significantly smaller contact area of adhesion (P < 0.0001) and had more membrane protrusions (eg, microvilli and intercellular membrane nanotubes) than on flat Ti surface. Numerical membrane dynamics simulations revealed that the low adhesion energy per unit area would hinder the cell spreading on the large diameter TiO2 nanotubular surface, thus explaining the small contact area. Conclusion The reduction in the cell contact area in the case of large diameter TiO2 nanotube surface, which does not enable formation of the large enough number of the focal adhesion points, prevents spreading of urothelial cells

Poly-L-lysine as an effective and safe desquamation inducer of urinary bladder epithelium

Induced desquamation of urinary bladder epithelial cells, also called urothelial cells, is frequently used in studies of bladder epithelial regeneration and also in treating recurrent bacterial cystitis. Positively charged polymer chitosan is known to cause large-scale desquamation of terminally differentiated urothelial cells called umbrella cells. Aiming to compare the desquamation ability of another polycation poly-L-lysine, we studied the effect of this polymer on the functional and structural integrity of the urothelium in ex vivo and in vivo experiments. The urothelium was analyzed by measuring transepithelial electrical resistance, and the structural changes of its luminal surface were analyzed with scanning electron microscopy. The results revealed a selective and concentration-dependent desquamation effect of poly-L-lysine on superficial urothelial cells followed by quick regeneration of the urothelium, which functionally and structurally recovers in 2 to 3 h after poly-L-lysine-induced injury. Poly-L-lysine was thus proven to be a promising polymer to be used when desquamation of urothelial cells is required in basic and potentially clinical studies