Effect of temperature on the occluding junctions of monolayers of epithelioid cells (MDCK)

Summary In previous works it was demonstrated that the monolayer of MDCK cells behaves as a leaky epithelium where the electrical resistance across reflects the sealing capacity of the occluding junction. In the present work we study whether this sealing capacity can be modified by temperature and whether this is accompanied by changes in the structure of the occluding junction. Monolayers were prepared on disks of nylon cloth coated with collagen and mounted as a flat sheet between two Lucite chambers. The changes in resistance elicited by temperature were large (306% between 3 and 37°C), fast (less than 2 sec), and reversible. An Arrhenius plot of conductance versus the inverse of temperature shows a broken curve (between 22 and 31°C), and the activation energies calculated (3.2 and 4.0 kcal·mol^–1) fall within the expected values for processes of simple diffusion. The morphology of the occuluding the number of evaluated in freeze-fracture replicas by counting the number of strands and the width of the band occupied by the junction every 133 nm. In spite of the change by 306% of the electrical resistance and the phase transition, we were unable to detect any appreciable modification of the morphology of the occluding junction. Since the freeze-fracture replicas also show a density of intramembrane particles (IMP) different in the apical from that in the basolateral regions of the plasma membrane, as well as differences between faceE and faceP, we also investigated whether this is modified by temperature. Cold increases the population of IMP, but does not affect their polarization with the incubation time it takes to elicit changes in electrical resistance.     

Cellular distributions of the ERM proteins in MDCK epithelial cells: regulation by growth and cytoskeletal integrity

The highly homologous ERM (ezrin/radixin/moesin) proteins, molecular cross-linkers which connect the cell membrane with the underlying cytoskeleton, have molecular weights of 81, 80 and 78 kDa respectively. We present data which shows significant variation in the molecular weight and presence of multiple forms of ERM proteins in different cell lines, such that specific antibodies to each protein are essential for unambiguous detection. Biochemical fractionation of MDCK cells demonstrates that although the individual ERM fractionation patterns are unaltered by cell density, the multiple forms of moesin each associate with different subcellular fractions. Since ERM proteins can exist in dormant or active conformations corresponding to their phosphorylation state, we propose that the partitioning of ERM proteins between subcellular compartments may depend on their activation status. In addition, we show that when the co-localization between ezrin and F-actin is disrupted by cytochalasin D, MDCK cells undergo a dramatic morphology change during which long, branching, ezrin-rich protrusions are formed. Consistent with other workers, our data suggest that maintenance of ezrin:F-actin interactions are required for the maintenance of normal cellular morphology.     

Arginine-vasopressin-induced structural alterations in MDCK cells

Alterations in the structural organization of MDCK cells under the effect of arginine-vasopressin (AVP) have been studied using electron and fluorescent microscopy methods. Electron microscopy has confirmed that the MDCK cells in the monolayer have structurally different apical and basolateral surfaces separated by well-formed zones of intercellular contacts. AVP has been proven to bind specifically to receptors on the basolateral cell surface and be internalized from the cell surface after 10–15 min. AVP produces fragmentation of the Golgi apparatus and swelling in its cisternae due to the appearance of an osmotic water flow across the monolayer. The significant depolymerization of the cell’s actin cytoskeleton has been revealed under effect of AVP or forskolin (an adenylyl cyclase activator). The functional role and regulatory mechanisms of the described structural alterations are discussed.     

Tight junction formation in cultured epithelial cells (MDCK)

Summary Synthesis and assembly of tight junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca²⁺, 20 hr later, while monolayers with Ca²⁺ have fully developed junctions that confer an electrical resistance across of 346±51 cm², those without Ca²⁺ have a negligible resistance. If at this time Ca²⁺ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cycloheximide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Golgi compartment. Upon addition of Ca²⁺ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 min, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca²⁺ is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25°C. Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains.     

应用Steedman's wax切片法观察植物细胞微管骨架

微管骨架在植物发育过程中起重要作用.由于植物细胞的特殊性,与动物细胞相比植物微管骨架的研究遇到更多的困难.简略地介绍了曾被国内外学者应用的植物微管骨架的各种研究方法及其局限性.Steedman's wax是一种多脂蜡.它熔点低(35～37℃),具有与石蜡相同的切片性质,能够切成不同厚度的连续切片,适合深埋于器官内部的组织或细胞的免疫细胞化学研究.介绍了应用Steedman's wax切片法观察植物细胞微管骨架的一般程序和方法以及经过作者检验且切实可行的一些技术改进.     

Fetal bovine serum and other sera used in tissue culture increase epithelial permeability

Summary Fetal bovine serum (FBS) or heat-inactivated FBS (56° C for 30 min, HFBS) caused a dose-dependent decrease in the transepithelial electrical resistance of an epithelial monolayer (MDCK). A saturating concentration of HFBS (30%) caused an average fall of 25 ± 2% within 60 min. Upon removal of HFBS, the resistance returned to its starting value within 1 h. Flux studies with [³H]mannitol demonstrate that the fall in resistance is due to an increased permeability of the tight junctions. Thirty percent heat inactivated sera from goat, newborn calf, calf, bovine, and horse caused falls ranging from 26 to 47%. In contrast with the basolateral preference of human and bovine adult sera, fetal bovine and newborn calf sera elicit this response primarily by interacting with the apical surface of the epithelium. HFBS-treated monolayers show a significant increase in the condensation of F-actin at points where ≥3 cells meet. These results demonstrate that FBS and other sera used as nutritional supplements can increase the permeability of the tight junctions of cultured epithelial cells.     

Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.     

一种改良的肌细胞骨架染色方法

为了观察肌细胞骨架,对传统考马斯亮蓝染色法进行改良,并与免疫荧光染色法进行了比较。培养的血管平滑肌细胞先用多聚甲醛预固定后再进行考马斯亮蓝染色,可使细胞骨架非常清晰的显色,解决了传统考马斯亮蓝染色易使肌细胞变形、脱片的问题,其效果与免疫荧光染色相近。因此,多聚甲醛预固定．考马斯亮蓝染色法是一种适于肌细胞骨架染色的简便方法。     

The Large GTPase Mx1 Is Involved in Apical Transport in MDCK Cells

In epithelial cells apical proteins are transported by specific transport carriers to the correct membrane domain. The composition of these carriers is heterogeneous and comprises components such as motor proteins, annexins, lectins, Rab GTPases and cargo molecules. Here, we provide biochemical and fluorescence microscopic data to show that the dynamin‐related large GTPase Mx1 is a component of post‐Golgi vesicles carrying the neurotrophin receptor p75^NTR. Moreover, siRNA‐mediated depletion of Mx1 significantly decreased the transport efficiency of apical proteins in MDCK cells. In conclusion, Mx1 plays a crucial role in the delivery of cargo molecules to the apical membrane of epithelial cells.      

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

Bovine Brain Endothelial Cells Express Tight Junctions and Monoamine Oxidase Activity in Long-Term Culture

The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.     

Electrical Conductance of Cell-to-Cell Junctions and the Cytoskeleton of Plant Cells

In the submerged trichomes of floating-moss (Salvinia auriculataAubl.) and the roots of the higher water plant Trianea bogotensisKarst., the dependence of the electrical resistance of intercellular junctions on the presence of the agents that destroy microfilaments (cytochalasin B) and microtubules (colchicine) was investigated using the microelectrode technique. The resistance of the junctions (R _c) was estimated taking into account the input resistance and the coefficient of intercellular electrical communication. Should the cells be connected via symplast, R _cwill describe the resistance of plasmodesmata. Cytochalasin B (3–30 g/ml) reversibly changed R _cduring the first minutes after application. The extent of the change depended on the concentration of the inhibitor; its character of action depended on the initial strength of intercellular communication. When the initial conductance of the contact was high, cytochalasin B elevated the resistance; when it was low, the inhibitor decreased it. In all the experiments, cytochalasin B reduced the input resistance (R _i) that suggests the dependence of plasma membrane resistance on actin cytoskeleton. The effect of colchicine (0.1–1.0 mM) on R _iand R _cwas observed only when the cellular membrane was hyperpolarized or after a prolonged action of the inhibitor (for about 0.5 h). It was concluded that the electrical conductance of plasmodesmata and plasma membrane depended on the state of actin cytoskeleton. A complex and probably mediated interaction of microtubules with the processes affecting these characteristics of the cells was suggested.     

Junctions between interstitial cells of the renal medulla: A freeze-fracture study

Summary Cell junctions between interstitial cells of the renal medulla were studied in freeze-fracture replicas of kidneys from rat, rabbit, hamster and the tree-shrew Tupaia belangeri. In all species studied a composite type of intercellular junction was found comprising elements of tight junctions and irregular gap junctions of highly variable size and shape. The number of these junctions increased towards the tip of the papilla.Our findings suggest that the composite junctions observed play a role in the maintainance of the ladder-like arrangement of the interstitial cells in the inner zone. The existence of irregular gap junctions raises the possibility that the functions of the interstitial cells are coordinated, especially during alterations of the functional state of the kidney.Supported by the Deutsche Forschungsgemeinschaft     

The expression of essential components for human influenza virus internalisation in Vero and MDCK cells

MDCK and Vero cell lines have been used as substrates for influenza virus replication. However, Vero cells produced lower influenza virus titer yield compared to MDCK. Influenza virus needs molecules for internalisation of the virus into the host cell, such as influenza virus receptor and clathrin. Human influenza receptor is usually a membrane protein containing Sia(α2,6) Gal, which is added into the protein in the golgi apparatus by α2,6 sialyltransferase (SIAT1). Light clathrin A (LCA), light clathrin B (LCB) and heavy clathrin (HC) are the main components needed for virus endocytosis. Therefore, it is necessary to compare the expression of SIAT1 and clathrin in Vero and MDCK cells. This study is reporting the expression of SIAT1 and clathrin observed in both cells with respect to the levels of (1) RNA by using RT-PCR, (2) protein by using dot blot analysis and confocal microscope. The results showed that Vero and MDCK cells expressed both SIAT1 and clathrin proteins, and the expression of SIAT1 in MDCK was higher compared to Vero cells. On the other hand, the expressions of LCA, LCB and HC protein in MDCK cells were not significantly different to Vero cells. This result showed that the inability of Vero cells to internalize H1N1 influenza virus was possibly due to the lack of transmembrane protein receptor which contained Sia(α2,6) Gal.     

Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells

Summary When MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25mm), taurine (19 mm) and glycine (9 mm). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na⁺ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mm for nine different amino acids in Na⁺-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K⁺ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.     

Effect of cell-substratum interaction on hemicyst formation by MDCK cells

Summary On impermeable substrate MDCK cells, a cell line derived from normal dog kidney, forms a confluent monolayer that is studded with numerous hemicysts. Previous studies with this cell line suggest that these hemicysts develop as a result of active fluid accumulation between cell sheet and substratum. However, the formation of hemicysts as a multifocal phenomenon is still unexplained. The results presented here show that the hemicysts are not only expressions of active transport of solutes and water, but also of cell-substratum interaction. The increase in number and size of the hemicyst produced by dbcAMP may be explained by a decrease in the adhesive strength to substrata produced by this compound. Moreover, when the strength of the cell-substratum adhesion was increased the number of hemicysts was reduced or abolished. On the contrary, when this strength was reduced, larger hemicysts occurred, covering practically all the area available for growth. Results from cinematographic time lapse studies, showing that 90% of the area of the monolayer is able to produce hemicysts, also suggest that hemicyst formation as a multifocal phenomenon is more an expression of local variations in cell-substratum interaction than of regional changes in transepithelial active transport.     

Thyroid Hormones Reorganize the Cytoskeleton of Glial Cells Through Gfap Phosphorylation and Rhoa-Dependent Mechanisms

Thyroid hormones (3,5,3′-triiodo-l-thyronine, T₃; 3,5,3′,5′-l-tetraiodothyronine, T₄; TH) play crucial roles in the growth and differentiation of the central nervous system. In this study, we investigated the actions of TH on proliferation, viability, cell morphology, in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and actin reorganization in C6 glioma cells. We first observe that long-term exposure to TH stimulates cell proliferation without induce cell death. We also demonstrate that after 3, 6, 12, 18, and 24 h treatment with TH, C6 cells and cortical astrocytes show a process-bearing shape. Furthermore, immunocytochemistry with anti-actin and anti-GFAP antibodies reveals that TH induces reorganization of actin and GFAP cytoskeleton. We also observe an increased in vitro ³²P incorporation into GFAP recovered into the high-salt Triton insoluble cytoskeletal fraction after 3 and 24 h exposure to 5×10⁻⁸ and 10⁻⁶ M T₃, and only after 24 h exposure to 10⁻⁹ M T₄. These results show a T₃ action on the phosphorylating system associated to GFAP and suggest a T₃-independent effect of T₄ on this cytoskeletal protein. In addition, C6 cells and astrocytes treated with lysophosphatidic acid, an upstream activator of the RhoA GTPase pathway, totally prevented the morphological alterations induced by TH, indicating that this effect could be mediated by the RhoA signaling pathway. Considering that IF network can be regulated by phosphorylation leading to reorganization of IF filamentous structure and that alterations of the microfilament organization may have important implications in glial functions, the effects of TH on glial cell cytoskeleton could be implicated in essential neural events such as brain development.     

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.