Rearrangements of the Actin Cytoskeleton and E-Cadherin–Based Adherens Junctions Caused by Neoplasic Transformation Change Cell–Cell Interactions

E-cadherin–mediated cell–cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell–cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin–based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell–cell interactions, preventing contact paralysis after the establishment of cell–cell contact and promoting dynamic cell–cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells

Characteristics and in vitro response of thin hydroxyapatite-titania films produced by plasma electrolytic oxidation of Ti alloys in electrolytes with particle additions

The enhancement of the biological properties of Ti by surface doping with hydroxyapatite (HA) is of great significance, especially for orthodontic applications. This study addressed the effects of HA particle size in the electrolyte suspension on the characteristics and biological properties of thin titania-based coatings produced on Ti–6Al–4V alloy by plasma electrolytic oxidation (PEO). Detailed morphological investigation of the coatings formed by a single-stage PEO process with two-step control of the electrical parameters was performed using the Minkowski functionals approach. The surface chemistry was studied by glow discharge optical emission spectroscopy and Fourier transform infrared spectroscopy, whereas mechanical properties were evaluated using scratch tests. The biological assessment included in vitro evaluation of the coating bioactivity in simulated body fluid (SBF) as well as studies of spreading, proliferation and osteoblastic differentiation of MC3T3-E1 cells. The results demonstrated that both HA micro- and nanoparticles were successfully incorporated in the coatings but had different effects on their surface morphology and elemental distributions. The micro-particles formed an irregular surface morphology featuring interpenetrated networks of fine pores and coating material, whereas the nanoparticles penetrated deeper into the coating matrix which retained major morphological features of the porous TiO2 coating. All coatings suffered cohesive failure in scratch tests, but no adhesive failure was observed; moreover doping with HA increased the coating scratch resistance. In vitro tests in SBF revealed enhanced bioactivity of both HA-doped PEO coatings; furthermore, the cell proliferation/morphometric tests showed their good biocompatibility. Fluorescence microscopy revealed a well-organised actin cytoskeleton and focal adhesions in MC3T3-E1 cells cultivated on these substrates. The cell alkaline phosphatase activity in the presence of ascorbic acid and β-glycerophosphate was significantly increased, especially in HA nanoparticle-doped coatings

РЕОРГАНИЗАЦИЯ МЕЖКЛЕТОЧНЫХ АДГЕЗИОННЫХ КОНТАКТОВ И ПОЯВЛЕНИЕ МИГРАЦИОННОЙ АКТИВНОСТИ У КЛЕТОК MCF-7-SNAI1 ПРИ ИНДУКЦИИ ЭПИТЕЛИАЛЬНО-МЕЗЕНХИМАЛЬНОГО ПЕРЕХОДА

Using DIC and confocal microscopy, changes in morphology, migratory characteristics and adherence junctions (AJs) were analyzed in the mammary carcinoma cell line MCF-7-SNAI1  after activation of the EMT transcription factor SNAI1. Western Blot analysis showed that  after removal of tetracycline from the cell culture medium expression of SNAI1 reached its  peak in 24 hours and then plateaued for 7 days. During the 7 days the cells continued to  express E-cadherin; however, tangential AJs typical for cells with stable cell-cell adhesion,  changed into radial AJs. The radial AJs continued to accumulate E-cadherin during 24‑72  hours after tetracycline removal. As a result of SNAI1 activation, the cells underwent  epithelial-mesenchymal transition (EMT) and became migratory. On a two-dimensional  substrate, cells exhibited both individual and collective migration. As the tetracycline  washout period progressed, the fraction of the cells capable of migrating through migration chamber membranes increased; on the contrary, cells’ ability to invade an epithelial  monolayer decreased. These results demonstrate that retaining a hybrid epithelial/mesenchymal  phenotype and accumulation of E-cadherin in AJs during early stages of EMT do not impede  disruption of stable cell-cell adhesion and cells’ acquisition of migratory activity.С помощью DIC-микроскопии и конфокальной микроскопии были проанализированы изменения морфологии,  миграционных характеристик и межклеточных адгезионных контактов в культуре клеток рака молочной железы MCF-7-SNAI1 при активации экспрессии транскрипционного фактора ЭМП – SNAI1. Как показал Вестерн-блот  анализ, экспрессия SNAI1 достигала максимальных значений через 24 часа после переноса клеток в среду без  тетрациклина и поддерживалась на этом уровне в течение семи дней. В клетках в течение семи дней  сохранялась экспрессия Е-кадхерина, при этом тангенциальные межклеточные адгезионные контакты,  характерные для клеток со стабильной межклеточной адгезией, замещались радиальными контактами. В  радиальных контактах в течение 24–72 часов отмывки от тетрациклина продолжалась аккумуляция Е- кадхерина. В результате активации SNAI1 клетки вступали в ЭМП и приобретали миграционную активность. На  двумерном субстрате клетки мигрировали как индивидуально, так и коллективно. С увеличением  продолжительности отмывки от тетрациклина повышался процент клеток, мигрировавших через поры в  миграционных камерах, способность клеток инвазировать эпителиальный монослой, напротив, снижалась.  Полученные данные свидетельствуют о том, что сохранение гибридного эпителиально-мезенхимального  фенотипа и аккумуляция Е-кадхерина в межклеточных адгезионных контактах на ранних этапах ЭМП не  препятствуют разрушению стабильной межклеточной адгезии и приобретению клетками миграционной активности

Comparison of Different Approaches to Surface Functionalization of Biodegradable Polycaprolactone Scaffolds

Due to their good mechanical stability compared to gelatin, collagen or polyethylene glycol nanofibers and slow degradation rate, biodegradable poly-epsilon-caprolactone (PCL) nanofibers are promising material as scaffolds for bone and soft-tissue engineering. Here, PCL nanofibers were prepared by the electrospinning method and then subjected to surface functionalization aimed at improving their biocompatibility and bioactivity. For surface modification, two approaches were used: (i) COOH-containing polymer was deposited on the PCL surface using atmospheric pressure plasma copolymerization of CO2 and C2H4, and (ii) PCL nanofibers were coated with multifunctional bioactive nanostructured TiCaPCON film by magnetron sputtering of TiC-CaO-Ti3POx target. To evaluate bone regeneration ability in vitro, the surface-modified PCL nanofibers were immersed in simulated body fluid (SBF, 1x) for 21 days. The results obtained indicate different osteoblastic and epithelial cell response depending on the modification method. The TiCaPCON-coated PCL nanofibers exhibited enhanced adhesion and proliferation of MC3T3-E1 cells, promoted the formation of Ca-based mineralized layer in SBF and, therefore, can be considered as promising material for bone tissue regeneration. The PCL-COOH nanofibers demonstrated improved adhesion and proliferation of IAR-2 cells, which shows their high potential for skin reparation and wound dressing

Роль Е-кадхерина в неопластической эволюции эпителиальных клеток

Interaction of the extracellular domains of the transmembrane proteins cadherins provides cell-cell adhesion. For many years epithelial E- cadherin was regarded as a tumor suppressor and was used as a prognostic marker in cancer. Suppression of E-cadherin expression was observed in many carcinomas. During recent years, the tumor suppressor function of E-cadherin is being reconsidered. It has been shown that ductal breast carcinomas, colorectal carcinomas, oral cavity carcinomas, and squamous cell carcinomas of the head and neck can retain E-cadherin expression. Immunohistochemical staining with a panel of monoclonal antibodies revealed membrane localization of E-cadherin in many tumors. It was shown that transformed epithelial cells in vitro form dynamic adherens junctions that are essential for the effective collective migration of these cells.Трансмембранные белки кадхерины обеспечивают межклеточную адгезию через взаимодействие внеклеточных доменов. На протяжении многих лет эпителиальный Е-кадхерин считался опухолевым супрессором и рассматривался в качестве прогностического маркера у онкологических больных. Угнетение экспрессии Е-кадхерина наблюдали во многих карциномах. В последние несколько лет пересматриваются представления о супрессирующей оли Е-кадхерина. Показано, что протоковые карциномы молочной железы, карциномы толстой кишки, карциномы полости рта, плоскоклеточные карциномы головы и шеи могут сохранять экспрессию Е-кадхерина. При иммуногистохимическом окрашивании с использованием панели моноклональных антител во многих опухолях была выявлена мембранная локализация Е-кадхерина. В трансформированных эпителиоцитах in vitro были обнаружены динамичные межклеточные адгезионные контакты, образованные Е-кадхерином, которые важны для эффективной коллективной миграции клеток

Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations

Aims: Recent immunohistochemical studies observed the loss of plakoglobin (PG) from the intercalated disc (ID) as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting a final common pathway for this disease. However, the underlying molecular processes are poorly understood. Methods and results: We have identified novel mutations in the desmosomal cadherin desmocollin 2 (DSC2 R203C, L229X, T275M, and G371fsX378). The two missense mutations (DSC2 R203C and T275M) have been functionally characterized, together with a previously reported frameshift variant (DSC2 A897fsX900), to examine their pathogenic potential towards PG's functions at the ID. The three mutant proteins were transiently expressed in various cellular systems and assayed for expression, processing, localization, and binding to other desmosomal components in comparison to wild-type DSC2a protein. The two missense mutations showed defects in proteolytic cleavage, a process which is required for the functional activation of mature cadherins. In both cases, this is thought to cause a reduction of functional DSC2 at the desmosomes in cardiac cells. In contrast, the frameshift variant was incorporated into cardiac desmosomes; however, it showed reduced binding to PG. Conclusion: Despite different modes of action, for all three variants, the reduced ability to provide a ligand for PG at the desmosomes was observed. This is in agreement with the reduced intensity of PG at these structures observed in ARVC patients

Cadherin exits the junction by switching its adhesive bond

Intercellular traction forces or lateral alignment of cadherin molecules can influence adherens junction dynamics by altering the cadherin dimerization interface

Different concepts for creating antibacterial yet biocompatible surfaces: Adding bactericidal element, grafting therapeutic agent through COOH plasma polymer and their combination

Antibacterial coatings have become a rapidly developing field of research, strongly stimulated by the increasing urgency of identifying alternatives to the traditional administration of antibiotics. Such coatings can be deposited onto implants and other medical devices and prevent the inflammations caused by hospital-acquired infections. Nevertheless, the design of antibacterial yet biocompatible and bioactive surfaces is a challenge that biological community has faced for many years but the "materials of dream" have not yet been developed. In this work, the biocompatible yet antibacterial multi-layered films were prepared by a combination of magnetron sputtering (TiCaPCON film), ion implantation (Ag-doped TiCaPCON film), plasma polymerization (COOH layer), and the final immobilization of gentamicin (GM) and heparin (Hepa) molecules. The layer chemistry was thoroughly investigated by means of FTIR and X-ray photoelectron spectroscopies. It was found that the immobilization of therapeutic components occurs throughout the entire thickness of the plasma-deposited COOH layer. The influence of each type of bactericide (Ag+ ions, GM, and Hepa) on antibacterial activity and cell proliferation was analyzed. Our films were cytocompatible and demonstrated superior bactericidal efficiency toward antibioticresistant bacterial E. coli K261 strain. Increased toxicity while using the combination of Ag nanoparticles and COOH plasma polymer is discussed

A Novel Pathway of TEF Regulation Mediated by MicroRNA-125b Contributes to the Control of Actin Distribution and Cell Shape in Fibroblasts

BACKGROUND: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF. PRINCIPAL FINDINGS: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts

α5β1 Integrin-Mediated Adhesion to Fibronectin Is Required for Axis Elongation and Somitogenesis in Mice

The arginine-glycine-aspartate (RGD) motif in fibronectin (FN) represents the major binding site for α5β1 and αvβ3 integrins. Mice lacking a functional RGD motif in FN (FNRGE/RGE) or α5 integrin develop identical phenotypes characterized by embryonic lethality and a severely shortened posterior trunk with kinked neural tubes. Here we show that the FNRGE/RGE embryos arrest both segmentation and axis elongation. The arrest is evident at about E9.0, corresponding to a stage when gastrulation ceases and the tail bud-derived presomitic mesoderm (PSM) induces α5 integrin expression and assumes axis elongation. At this stage cells of the posterior part of the PSM in wild type embryos are tightly coordinated, express somitic oscillator and cyclic genes required for segmentation, and form a tapered tail bud that extends caudally. In contrast, the posterior PSM cells in FNRGE/RGE embryos lost their tight associations, formed a blunt tail bud unable to extend the body axis, failed to induce the synchronised expression of Notch1 and cyclic genes and cease the formation of new somites. Mechanistically, the interaction of PSM cells with the RGD motif of FN is required for dynamic formation of lamellipodia allowing motility and cell-cell contact formation, as these processes fail when wild type PSM cells are seeded into a FN matrix derived from FNRGE/RGE fibroblasts. Thus, α5β1-mediated adhesion to FN in the PSM regulates the dynamics of membrane protrusions and cell-to-cell communication essential for elongation and segmentation of the body axis