Evaluation of VP22 spread in tissue culture

We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.     

Intercellular adhesions

Summary The submicroscopic morphology of freeze-etched cellular adhesions in rat hepatocytes is reported. In contrast to the reticulated tight junctions which line the bile capillaries, the junctional complexes of the described type appear as ovally or irregularly shaped patches. They are randomly distributed over the cell surface with the exception of the villous biliary and sinusoidal surface. In tangential fractures hepatocellular adhesions reveal one single layer of densely packed globular particles measuring 85–95 Å in diameter. These particles are interpreted as a product of both the plasma membranes involved in the complex formation. They also are supposed to be identical with the subunits demonstrated by Benedetti and Emmelot (1968) in liver cell membranes and more recently by Brightman and Reese (1969) in the gap junctions of vertebrate brain cells. Based on the findings, a hypothetical model of the adhesional complex-architecture is presented. The model may well explain the special task of this type of cellular adhesion which is to permit the ion flow from one cell to the other.Supported by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung, Nr. 4131.     

Intercellular adhesion

Summary A quantitative procedure for determining the early kinetics of cell aggregation (adhesion) is described. The cells used for this study were obtained by dissociation of 8-day-old embryonic chicken neural retina with crude trypsin. The method is based on determining the decrease in single cells in an aggregating population with the Coulter electronic particle counter. A variety of experiments show that the method is reproducible and capable of detecting relatively small changes in the rate of aggregation. Using a number of criteria, the loss of single cells from the population with increasing time of incubation was shown to result from the formation of aggregates, and not from other phenomena such as cell death or changes in cell permeability. The intercellular adhesions formed under these conditions were stable to mechanical shear and to ethylenediaminetetraacetate, and were partially resistant to crude trypsin. The logarithm₁₀ of the number of single cells in the population was found to be directly related to the time of incubation. The slope of the resultant straight lines could be used as a measure of the rate of aggregation. No lag in aggregation was demonstrable under the standard assay conditions. the rate was affected by the initial cell density, speed of rotation during aggregation, temperature, and by Ca²⁺ and Mg²⁺. It was not affected by inhibitors of protein synthesis, metabolic inhibitors, ATP, ADP, cyclic-AMP, or horse serum at 37 °C. The quantitative method for determining the initial rate of adhesion should be applicable to studies on the chemistry of this process.Contribution No. 557 from the McCollum-Pratt Institute.     

Intercellular Alignments of the PlantCytoskeleton

   apd: 3 July 2001     

Intercellular junctions in myriapods

Tissue from the intestinal tract of myriapods, including millipedes, centipedes and pauropods were examined in tracer-impregnated sections and freeze-fracture replicas. The foregut and hindgut of all three classes exhibit pleated septate junctions; these display undulating intercellular ribbons in thin sections. In replicas they show discrete intramembranous particle (IMP) arrays aligned in rows in parallel; with one another. The tissues of the hindgut also possess scalariform junctions, characterized by cross-striated intercellular clefts in sections and IMP-enriched membranes in replicas. Gap junctions occur in all groups, but they are atypical in replicas in that their component IMPs do not always fracture onto the E face, as is characteristic of other arthropods; some IMPs cleave to the P face and others to the E face. The midgut of these organisms exhibits smooth septate junctions with conventional straight septal ribbons and occasional interseptal columns. However the intramembranous appearance in replicas is variable, particularly in centipedes, in that the rows of IMPs in chemically-unfixed propanecryofixed tissues, are prominent and adhere preferentially to the E face, with complementary P face grooves, while in fixed tissues the IMPs are much less distinct and fracture to either P face or E face. They tend not to protrude far beyond the mid-plane of the membrane bilayer and lie in rows which commonly take on the form of a network. Individual rows of the network sometimes curve to run beside a second row, over a short distance, before bending away into another part of the network. The aligned particle rows, which are much more prominent in millipedes, where they frequently lie in close parallel appositions, do not fuse into ridges as often occurs in insect tissues. The myriapod junctions, therefore, are of the same general kind as are found in the gut tract of other arthropod groups, but differ with respect to the subtleties of their intramembranous organization and disposition.     

Intercellular communication and carcinogenesis

Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.     

Intercellular channels in animals

Gap junctions are considered to serve a similar function in all multicellular animals (Metazoa). Two unrelated protein families are involved in this function: connexins, which are found only in chordates, and pannexins, which are present in the genomes of both chordates and invertebrates. Recent sequence data from different organisms show important exceptions to this simplified scheme. It looks as if Chordate lancelet has only pannexins and no connexins in its genome. New data indicate that some metazoans have neither connexins nor pannexins and use other unidentified proteins to form gap junctions.     

Intercellular lipid in oilseeds

Summary Electron microscopic observations of three oilseeds, zucchini, yucca, and okra, have revealed a substance occurring in the intercellular spaces in the embryos of these seeds. Extraction methods and histochemical tests for light microscopy have characterized the material as lipid. Disappearance of the intercellular lipid during seed germination suggests that it may be utilized by the germinating seed.     

Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus

In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.     

Intercellular Transport of Macromolecules in Nitella

We injected three different fluorescein isothiocyanate (FITC)-labeledproteins, two different FITC-labeled dextrans and the photoproteinaequorin (the molecular weight of each being more than 20 kDa)into internodal cells of Nitella. All macromolecules with molecularweights equal to or less than 45 kDa moved from the injectedcell to the neighboring nodal and internodal cells within 24h after injection. The injected aequorin emitted light in theadjacent internodal cell upon a transient increase in the cytoplasmicconcentration of Ca²⁺, an indication that the aequorin retainedits function after transport between cells. (Received November 2, 1991; Accepted March 7, 1992)     

Intercellular NOR-AG variability in man

Summary A new modification of the Ag I technique has been developed using human cultured blood lymphocytes, which involves ultra-violet irradiation of chromosome preparations during incubation in AgNO₃. This technique enables detection in a short incubation time all NORs capable of being stained with silver. A peculiar morphological change in Ag-stainable NORs during the incubation is described, which can be used as a criterion of the completion of Ag staining. With the refined Ag-staining procedure, acrocentric marker chromosomes were studied which showed one or two satellite stalks within the same individual. Ag staining was highly coincident with this variability.     

Intercellular Deformation in Compressed Organs

Intercellular deformations, caused by increasing levels of compressionapplied by a pressure chamber to an organ covered with a plasticsealant and evaluated according to the internal atmosphere removalrate, were observed in carrots (Daucus carota L. sativa), potatoes(Solanum tuberosum L.) and sweet-potatoes (Ipomea batatas L.Lam). The maximum internal gas volume removed in these kineticassays was close to the intercellular air volume (V_g) measuredby the pycnometric method. Presumably a compression larger thanthe average organ turgor was required to remove all V_g and abovethis point the cells should become completely flattened againsteach other. The intercellular deformation caused by a compressingload, observed by constant pressure volumetry, induced a reductionin the endogenous O₂ concentration at the stressed area, accordingto polarographic measurements. Cellular deformations and eventualV_g flooding caused by water movement from the symplasm to theapoplasm of externally compressed organs were distinct fromthe usual pressure chamber assays, where all cells are exposedto homogeneous gas pressurization, without the development offorces to cause large cellular deformation and intercellularflooding. These gas transport restrictions were suggested aspotential causes for post harvest deterioration in fragile commoditiessubjected to compression.Copyright 1995, 1999 Academic Press Carrot, compression, Daucus carota, gas volumetry, Ipomea batatas, oxygen, porosity, potato, pressure chamber, Solanum tuberosum, stress, sweet-potato, turgor, suction     

Intercellular junctions in nerve-free hydra

Summary Epithelial cells of nerve-free hydra contain septate and gap junctions. In thin sections the gap junctions are characterized by a gap of 3–4 nm. Freeze-fracture demonstrates the presence of septate junctions and two further types of structures: (i) the E-type or inverted gap junctions with particles in an en plaque conformation appearing as a raised plateau on the E-face or as a depression on the P-face; (ii) structures morphologically similar to gap junctions in rat liver, containing particles on the P-face and corresponding pits on the E-face, both having hexagonal packing with a lattice constant of 8 nm. We propose that these structures are also gap junctions.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.     

植物转录因子的胞间运动

植物体的组织和器官由多细胞组成,细胞之间的通信对植物体的生长发育必不可少。转录因子作为一类特殊的蛋白质分子不仅在转录水平上参与植物生长发育的调控,而且新近研究发现,转录因子的胞间运动是细胞之间通信方式之一,具有重要的功能。对转录因子胞间运动的发现过程、转录因子胞间运动的机制及其通道进行了论述。转录因子的胞间运动有基于扩散作用的非目标性转运和具有目标性的主动转运两种模式。转录因子胞间运动具有明显的组织特异性和方向性。分析了影响转录因子胞间运动的因素,讨论了转录因子胞间运动的功能以及转录因子胞间运动所参与的植物生长发育及形态建成的调控。     

Intercellular communication and tissue growth

Summary Epithelial cells of normal rat (adult) liver and hamster embryo in tissue culture communicate through membrane junctions: the membrane regions of cell contact are highly ion-permeable. Cancerous counterparts of these cells, cells from Morris' and Reuber's liver tumors and from x-ray-transformed embryo cultures, do not communicate under the same experimental conditions. These cells also fail to communicate with contiguous normal cells. Cancerous fibroblastic cells from a variety of tissues, including cells transformed by virus, x-radiation and chemicals, communicate as well as their normal counterparts; this is so for long- and short-term cell cultures. Communication in some fibroblastic cells is sensitive to components of blood serum: normal and transformed hamster embryo fibroblasts, which communicate when cultured in medium containing fetal calf serum, appear to lose communication in medium containing calf serum; the converse holds for hamster (adult) fibroblasts and 3T3 cells.The preceding papers of this series appeared in the Journal of Cell Biology.Trainee of the National Institutes of Health, National Cancer Institute, Grant CA 05011.