Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey

The modern exploration of endothelial cell biology is a largely interdisciplinary exercise. Cell biological, physiological, and more recently molecular biology approaches were used to study the pathways and the organelles involved in transcytosis of macromolecules in endothelial cell (EC). Here we discuss mainly the cell biological findings that revealed that EC have the attributes to fulfill the transport function. They are polarized cells, heterogeneous, and, thus, structurally and functionally adapted to the vascular bed in which they reside. The structural heterogeneity involves the number and distribution of plasmalemmal vesicles (caveolae), their generated channels, and the organization of intercellular junctions. The closely related functional heterogeneity comprises the degree of permeability for plasma molecules that vary as a function of organ. The EC are endowed with the cellular machinery to perform (1) endocytosis, that is to take up plasma proteins and the molecules they carry to be used for themselves (cholesterol-carrying low density lipoproteins, fatty acid carrying albumin, iron carrying transferrin, etc.), and (2) transcytosis, which implies to transport plasma proteins to the subjacent cells and tissues. The possible pathways for transport of molecules are transcellular, via caveolae and channels, and paracellular via intercellular junctions. Most of the results obtained, so far, indicate that transcytosis of albumin, low-density lipoproteins, metaloproteases, and insulin, is performed by cargo-vesicles and their generated channels. The paracellular pathway can be used for water and ions; in postcapillary venules, at the level of which approximately 30% of junctions are open to a space of 6 nm, small molecules may take this route. Recent data obtained by molecular biology techniques revealed that caveolae are endowed with the molecular machinery for fusion/fission, docking, and movement across cells. Moreover, the various and numerous molecules that have been detected in the caveolae membrane and the different functions assumed by this differentiated microdomain strengthen the postulate that there are at least two or more types of vesicles molecularly tailored for the local physiological requirements.     

Gap-junction quantification in biological tissues: freeze-fracture replicas versus thin sections

The relative efficiency of freeze-fracture replicas versus thin sections for the visualization and quantification of gap junctions in biological tissues has been evaluated. Both methods may underestimate gap-junction number—thin sections for reasons of tissue resolution and freeze-fracture replicas due to the mechanics of the fracturing process. Freeze-fracture misses gap junctions in regions of plasma membrane which are highly contoured, such as the overlapping basal cell processes of Drosophila imaginal wing discs and the interdigitating lateral membrane plications of intercalated discs in cardiac tissue. If the missed gap junctions are relatively large, as they are in both of these examples, freeze-fracture significantly underestimates the total gap-junctional area. Thin sections may miss small gap junctions, but in tissues which contain a range of gap-junction sizes the lost junctions constitute a relatively small fraction of the total junctional area. In neoplastic imaginal wing discs, thin sections were as efficient as freeze-fracture replicas in identifying even the smallest gap junctions. Although freeze-fracture may be the better technique for the qualitative and quantitative documentation of small gap junctions in tissues with relatively flat to gently contoured plasma membranes, and thin sections may be the superior method for gap-junction quantification in tissues containing a range of gap-junctional sizes and highly contoured cellular processes, the data suggest that a combination of the two approaches should be utilized whenever possible.     

Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model

Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction—the cardiac gap junction—is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart. Freeze-fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision-related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasicrystalline arrangement, not a ‘random’ arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap-junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap-junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131–142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three-dimensional precision mapping of gap junctions through thick slices of cardiac tissue. By exploiting the serial optical sectioning ability of the confocal microscope, we have succeeded in (1) elucidating the organization of gap junctions at the intercalated disc, (2) establishing temporal and spatial patterns of gap-junctional protein expression in embryogenesis that correlate with functional differentiation in subsets of cardiac cells, and (3) demonstrating abnormalities of gap-junction distribution and quantity that may contribute to the genesis of arrhythmias in ischaemic heart disease.     

Moving in the right direction-nanoimaging in cancer cell motility and metastasis

Although genetic and protein manipulations have been the cornerstone for the study and understanding of biological processes for many decades, complimentary nanoscale observations have only more recently been achieved in the live-imaging mode. It is at the nano measurement level that events such as protein-protein interactions, enzymatic conversions, and single-molecule stochastic behavior take place. Therefore, nanoscale observations allow us to reinterpret knowledge from large-scale or bulk techniques and gain new insight into molecular events that has cellular, tissue, and organismal phenotypic manifestations. This review identifies pertinent questions relating to the sensing and directional component of cancer cell chemotaxis and discusses the platforms that provide insight into the molecular events related to cell motility. The study of cell motility at the molecular imaging level often necessitates the use of devices such as microinjection, microfluidics, in vivo/intravital and in vitro chemotaxis assays, as well as fluorescence methods like uncaging and FRET. The micro- and nanofabricated devices that facilitate these techniques and their incorporation to specialized microscopes such as the multiphoton, AFM, and TIR-FM, for high-resolution imaging comprise the nanoplatforms used to explore the mechanisms of carcinogenesis. In real-time observations, within a milieu of physiological protein concentrations, true states of dynamic and kinetic fluxes can be monitored.     

AFM与SNOM表征抗原抗体分子的分布与特异性作用

分析免疫系统中抗原抗体分子的分布及其特异性作用对研究生命体生理机能、疾病的诊断与治疗等方面有着重要的作用。随着免疫学研究从细胞水平向分子水平的深入,高分辨和高灵敏度仪器得到更重要和广泛地应用。简单介绍了原子力显微镜(AFM)和扫描近场光学显微镜(SNOM)的特点,对近几年AFM和SNOM原位探测细胞表面分子的研究进行了分析总结,同时着重阐述了AFM和SNOM在抗原抗体分子之间特异性作用、抗体分子结构以及细胞表面抗原抗体的分布等方面的应用。     

Involvement of the adrenal glands and testis in gap junction formation via testosterone within the male rat anterior pituitary gland

We investigated the influence of testicular and adrenal androgens on the presence of gap junctions between folliculo‐stellate cells in the anterior pituitary glands of 60‐day‐old Wistar‐Imamichi strain male rats. The animals were separated into six groups: Group A served as the controls and had free access to a normal diet and water, Group B was given a normal diet and 0.9% NaCl for their drinking water as the controls of adrenalectomized groups, Group C was castrated, Group D was adrenalectomized, Group E was both castrated and adrenalectomized, and Group F was also both castrated and adrenalectomized. In addition, the animals of Group F were administered a dose of testosterone that is known to produce high physiological levels of the hormones in plasma. Five rats from each group were sacrificed 1, 2, 3, 4, 5, 6, and 7 days after their respective operation, and the anterior pituitary glands were removed and prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicle profile. Simultaneous removal of adrenal glands with castration resulted in a significantly decrease in the number of gap junctions, whereas the administration of testosterone to these rats compensated for this change. These observations indicate that the preservation of gap junctions between folliculo‐stellate cells is mainly dependent on androgens from both the testes and adrenal glands in adult male rats. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Are there gap junctions between chief (glomus,type I) cells in the carotid body chemoreceptor? A review

Since the dye- and electronic couplings between the carotid body chief cells have been demonstrated, the detection and localization of the gap junctions in the carotid body is crucial to understanding the functional mechanism of chemoreception. However, conventional electron microscopy has been unsuccessful in unquestionably detecting ultrastructural features equivalent to the gap junctions, such as close (2 nm in width) membrane appositions in ultrathin sections and aggregations of intramembranous particles in freeze-fracture replicas of the carotid body. We previously reported using a modified electron microscopic study by chemically fixed and subsequent rapid freezing and freeze-substitution method a number of close membrane appositions comparable to the gap junctions. However, we later found that the freeze-substitution also induces numerous close apposition of the membrane in sites where the gap junctions are not known to occur, indicating that the modified electron microscopy by freeze-substitution is not always confirmative in the detection of the gap junction. With regard to the molecular evidence for the gap junction in the carotid body, there have so far been few data on the immunohistochemical demonstration on connexin 32 and 43 in cultured chief cells, but not in the in situ cells.     

Molecular ultrastructure of the urothelial surface: Insights from a combination of various microscopic techniques

The urothelium forms the blood–urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno‐TEM), and freeze‐fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno‐FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues. Microsc. Res. Tech. 77:896–901, 2014. © 2014 Wiley Periodicals, Inc.     

A combined optical and atomic force microscope for live cell investigations

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.     

ACTH and adrenocortical gap junctions

Since the initial identification of gap junctions in the adrenal gland, it has been proposed that a system involving direct cell-cell communication might be involved in adrenal cortical functions. Gap junction channels do, in fact, provide pathways for direct intercellular exchange of small molecules (<1,000 Da), many of which have the potential to influence a wide range of cellular activities. Gap junctions are composed of proteins called connexin which, in the adrenal cortex, have proven to be remarkably consistent in both type and zonal distribution with connexin 43 (Cx43) as the predominant component in mammalian adrenal glands thus far evaluated. Only the inner two zones of the cortex (zonae fasciculata and reticularis) exhibit significant amounts of Cx43 and functional coupling. Adrenocorticotropin (ACTH) has been shown to increase Cx43 protein in vivo and in vitro, and a strong correlation has been noted between the presence of gap junctions and certain adrenal cortical functions, especially steroidogenic capacity and cell proliferation. This review summarizes evidence of the Cx43 expression in adrenal cortical cells and the likely role of Cx43 in steroidogenesis and cell proliferation. It is concluded that control of gap junction expression in the adrenal gland is hormonally dependent and is functionally linked to adrenal gland zonation.     

The fracture-flip technique reveals new structural features of the Escherichia coli cell wall

With few exceptions, all bacteria possess a wall which protects them and controls their communication with the environment. In Gram-negative bacteria the cell wall exhibits a complex and unique multilayered organization. We have applied a modification of the freeze-fracture technique known as 'fracture-flip' to visualize the real surfaces of the different wall layers in a Gram-negative bacterium, Escherichia coli . In combination with treatments to weaken the interlayer connections, this technique has provided new insights into the structure of the bacterial wall. Large areas of an intermediate layer (most probably the peptidoglycan-containing matrix) have been visualized for the first time between the plasma membrane and the outer membrane of the wall. Extensive regions corresponding to the cytoplasmic face of the plasma membrane have also been obtained. These images provide new three-dimensional views of the bacterial cell wall and provide the structural framework for the analysis of the molecular relationships between the different cell wall components.     

Atomic force microscopy for ultrafiltration membrane imaging

Atomic force microscopy (AFM) can reveal nanometer-scale structure of samples without the sample preparation techniques that involve dehydration. This is particularly important for hydrophilic organic materials. An asymmetric polysulfone ultrafiltration membrane (molecular weight cutoff rated at 10 kg/mol) was imaged by AFM. Sample mounting methods tried include cyanoacrylate, double-sided tape, and paraffin. Wax and tape bonding did not lead to usable images. Cyanoacrylate bonding resulted in images that appear to show 2.8° 10⁹ pores/m² approximately 3 nm in diameter, creating a porosity of 2%. This is consistent with estimates of molecular sizes for 10 kg/mol proteins, but not with the results of other AFM studies of similar membranes. The discrepancies can be explained largely by differences in sample preparation techniques.     

Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope

Atomic force microscopy has been used to visualize nano‐scale structures of various cellular components and to characterize mechanical properties of biomolecules. In spite of its ability to measure non‐fixed samples in liquid, the application of AFM for living cell manipulation has been hampered by the lack of knowledge of the mechanical properties of living cells. In this study, we successfully combine AFM imaging and force measurement to characterize the mechanical properties of the plasma membrane and the nuclear envelope of living HeLa cells in a culture medium. We examine cantilevers with different physical properties (spring constant, tip angle and length) to find out the one suitable for living cell imaging and manipulation. Our results of elasticity measurement revealed that both the plasma membrane and the nuclear envelope are soft enough to absorb a large deformation by the AFM probe. The penetrations of the plasma membrane and the nuclear envelope were possible when the probe indents the cell membranes far down close to a hard glass surface. These results provide useful information to the development of single‐cell manipulation techniques.     

Sample preparation procedures for biological atomic force microscopy

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.     

Vascular wall: potential target for the physicochemical effects of cis-unsaturated free fatty acids

Diets rich in monounsaturated cis-FFA (cis FFA) are associated with a significant reduction of cardiovascular risk. Although several different mechanisms have been proposed to explain this protective effect, the biochemical processes involved have not been fully elucidated. It has been shown that upon their incorporation into the plasma membrane, cis FFA induce a marked perturbation of the lipid domains, altering membrane fluidity as well as lipid-lipid and lipid-protein interactions in the bilayer plane. During the last few years, several lines of evidence have shown that these perturbations disrupt the activity of several membrane proteins and enzymatic systems. As a result, several critical transmembrane signaling systems, including the Ins(1,4,5)P(3)/DAG/[Ca(2+)](i), the cAMP/PKA, and the voltage-operated Ca(2+) influx are strongly inhibited by cis FFA in different experimental models. Furthermore, this inhibition is associated with alterations in the timing of the cell cycle as well as in the final steps of the secretory pathway. We propose that this complex set of biological actions exerted by cis FFA at the plasma membrane may contribute to explain the protective roles that these molecules appear to exert on the vascular wall.     

Improved structural detail in freeze-fracture replicas: high-angle shadowing of gap junctions cooled below -170 degrees C and protected by liquid nitrogen-cooled shrouds.

In conventional freeze-fracture replicas, precise complementarity of membrane faces is seldom achieved. In a model system frequently used to evaluate replica quality, vertebrate gap junctions are usually visualized as patches of 8-10 nm P-face intramembrane particles separated by 1-2 nm spaces, while E-face images are represented by 4-6 nm conical pits separated by 5-7 nm wide membrane ridges. However, that disparity in sizes of particles versus pits, as well as the disparity in the widths of the spaces separating particles versus pits, suggests that a significant reduction in complementarity of membrane faces has occurred. In this investigation, a JEOL JFD-9000 freeze-etch machine was modified so that fracturing and replication could be performed at temperatures much colder than commonly employed. With the addition of cryopumps to improve overall vacuum and the installation of optically tight LN2-cooled shrouds surrounding the specimen and the knife, water vapor contamination arising from all sources within the vacuum chamber was reduced substantially, allowing replicas to be made at temperatures down to -185 degrees C. With the specimen at these much colder temperatures, water vapor released by the heat of cleaving was also reduced significantly, providing additional improvement in replica quality. In addition, with higher shadowing angles (greater than 60 degrees) and with the specimen at a much lower temperature, the grain size of the platinum film was noticeably reduced, thereby improving resolution at the molecular level. Under these improved conditions, replicas of rat liver gap junctions revealed that many of the P-face IMPs were tubes 6-7 nm in diameter, but that other IMPs had been stretched and distorted by the fracturing process. More important, however, these high resolution replicas revealed that the replicas of the E-face pits represented three-dimensional molecular casts of the transmembrane proteins comprising the connexon hexamer. This means that before they were replicated, the E-face pits faithfully maintained the shape that the IMPs had before fracturing. These more detailed images revealed a new structure in the center of each E-face pit: a 2-3 nm "peg" that may represent the frozen aqueous matrix of the connexon ion channel that remained after elastic extraction of the surrounding six connexin molecules. Thus, high-angle shadowing at very low specimen temperature under virtually non-contaminating conditions has revealed a new level of detail for membrane structure in freeze-fracture replicas.     

TectoRNA and 'kissing-loop' RNA: atomic force microscopy of self-assembling RNA structures

RNA molecules have been much less studied by atomic force microscopy (AFM) than have DNA molecules. In this paper, AFM imaging is presented for two different RNA molecules able to self‐assemble into complex supramolecular architectures. The first one is a molecular dimer of a 230‐nt RNA fragment coming from the RNA genome of a murine leukaemia virus. The monomeric RNA fragment, which appears by AFM as an elongated structure with a mean aspect ratio of 1.4, assembles into a dimer of elongated structures through the formation of a ‘kissing‐loop’ RNA interaction. The second one is a large supramolecular fibre formed of artificial self‐assembling RNA molecular units called tectoRNA. The fibre lengths by AFM suggest that there are 50–70 tectoRNA units per fibre. Some methods and limitations are presented for measuring molecular volumes from AFM images.     

The blood-testis barrier and Sertoli cell junctions: structural considerations.

In this review, a few well-established axioms have been challenged while others were viewed from a new perspective. The extensive literature on the blood-testis barrier has been scrutinized to help probe its mechanics and hopefully to promote understanding of the constant adaptation of the barrier function to germ cell development. Our principal conclusions are as follows: (1) Although the barrier zonule is topographically located at the base of the seminiferous epithelium it actually encircles the apex of the Sertoli cell. Consequently the long irregular processes specialized in holding and shaping the developing germ cells should be considered as apical appendages analogous to microvilli. (2) The development of the barrier zonule does not coincide with the appearance of a particular class of germ cells. (3) The barrier compartmentalizes the epithelium into only two cellular compartments: basal and lumenal. (4) Although the blood-testis barrier does sequester germ cells usually considered antigenic, immunoregulator factors other than the physical barrier seem to be involved in preventing autoimmune orchitis. (5) Structurally, a Sertoli cell junctional complex is composed of occluding, gap, close, and adhering junctions. The Sertoli cell membrane segments facing germ cells are presumably included in the continuum of the Sertoli cell junctional complex that extends all over the lateral and apical Sertoli cell membranes. (6) The modulation (i.e., formation and dismantling) of the junctions in a baso-apical direction is characteristic of the seminiferous epithelium and may be dictated by germ cell differentiation. The formation of tubulobulbar complexes and the following internalization of junction vesicles conceivably represent sequential steps of a single intricate junction elimination process that involves junction membrane segments from different cell types as part of a continual cell membrane recycling system. (7) The preferential association of junctional particles with one or the other fracture-face reflect a response to various stimuli including seasonal breeding. Changes in the affinity of the particles are generally coincidental with cytoskeletal changes. However, changes in the cytoskeleton are not necessarily accompanied by permeability changes. The number of strands seems to reflect neither the junctional permeability nor the transepithelial resistance. The diverse orientation of the strands seems to be related to the plasticity of the Sertoli cell occluding zonule. (8) Cooperation between all constituents (Sertoli cells, myoid cells, cell substratum, and germ cells) of the epithelium seems essential for the barrier zonule to function in synchrony with the germ cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epithelial cell polarity as reflected in enterocytes

Absorptive cells are the main cells present in the intestinal epithelium. The plasma membrane of these tall columnar cells reflects their high degree of polarization, by dividing into apical and basolateral domains with different compositions. The most characteristic structure of these cells consists of closely packed apical microvilli with the same height, looking like a brush, which is why they were named the brush border. The concentrated pattern of some apical markers observed in a restricted brush border domain shows that mature enterocytes are hyperpolarized epithelial cells: the filamentous brush border glycocalyx is anchored at the top of the microvilli and the annexin XIII is concentrated in the lower three fourths. Many studies have been carried out on the biosynthesis and intracellular pathway of domain markers. The results show clearly that the basolateral markers take a direct pathway from the trans-Golgi network to the basolateral membrane. However, the two apical pathways, one direct and one indirect pathway via the basolateral membrane, are used, depending on the apical protein involved. Efficient protein sorting and addressing are essential to the establishment and maintenance of cell polarity, on which the integrity of the epithelial barrier depends.     

Integration of non-linear cellular mechanisms regulating microvascular perfusion

It is becoming increasingly evident that interactions between the different cell types present in the vessel wall and the physical forces that result from blood flow are highly complex. This short article will review evidence that irregular fluctuations in vascular resistance are generated by non-linearity in the control mechanisms intrinsic to the smooth muscle cell and can be classified as chaotic. Non-linear systems theory has provided insights into the mechanisms involved at the cellular level by allowing the identification of dominant control variables and the construction of one-dimensional iterative maps to model vascular dynamics. Experiments with novel peptide inhibitors of gap junctions have shown that the coordination of aggregate responses depends on direct intercellular communication. The sensitivity of chaotic trajectories to perturbation may nevertheless generate a high degree of variability in the response to pharmacological interventions and altered perfusion conditions.