Multiple Adjacent Cells Research Articles

NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer

Adoptive cellular immunotherapy as a promising and alternative cancer therapy platform is critical for future clinical applications. Natural killer (NK) cells have attracted attention as an important type of innate immune regulatory cells that can rapidly kill multiple adjacent cancer cells. However, these cells are significantly less effective in treating solid tumors than in treating hematological tumors. Herein, we report the synthesis of a Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobe labeled with NK-92 cells that can be used for adoptive cellular immunotherapy, photodynamic therapy and dual-modality imaging-based in vivo fate tracking. The labeled NK-92 cells specifically target the tumor cells, which increases the amount of cancer cell apoptosis in vitro. Furthermore, the in vivo results indicate that the labeled NK-92 cells can be used for tumor magnetic resonance imaging and fluorescence imaging, adoptive cellular immunotherapy, and photodynamic therapy after tail vein injection. These data show that the developed multifunctional nanostructure is a promising platform for efficient innate immunotherapy, photodynamic treatment and noninvasive therapeutic evaluation of breast cancer.

Projecting RNA measurements onto single cell atlases to extract cell type-specific expression profiles using scProjection

Multi-modal single cell RNA assays capture RNA content as well as other data modalities, such as spatial cell position or the electrophysiological properties of cells. Compared to dedicated scRNA-seq assays however, they may unintentionally capture RNA from multiple adjacent cells, exhibit lower RNA sequencing depth compared to scRNA-seq, or lack genome-wide RNA measurements. We present scProjection, a method for mapping individual multi-modal RNA measurements to deeply sequenced scRNA-seq atlases to extract cell type-specific, single cell gene expression profiles. We demonstrate several use cases of scProjection, including identifying spatial motifs from spatial transcriptome assays, distinguishing RNA contributions from neighboring cells in both spatial and multi-modal single cell assays, and imputing expression measurements of un-measured genes from gene markers. scProjection therefore combines the advantages of both multi-modal and scRNA-seq assays to yield precise multi-modal measurements of single cells.

Dynamic conversion of cell sorting patterns in aggregates of embryonic stem cells with differential adhesive affinity

BackgroundMammalian early development comprises the proliferation, differentiation, and self-assembly of the embryonic cells. The classic experiment undertaken by Townes and Holtfreter demonstrated the ability of dissociated embryonic cells to sort and self-organize spontaneously into the original tissue patterns. Here, we further explored the principles and mechanisms underlying the phenomenon of spontaneous tissue organization by studying aggregation and sorting of mouse embryonic stem (ES) cells with differential adhesive affinity in culture.ResultsAs observed previously, in aggregates of wild-type and E-cadherin-deficient ES cells, the cell assemblies exhibited an initial sorting pattern showing wild-type cells engulfed by less adhesive E-cadherin-deficient ES cells, which fits the pattern predicted by the differential adhesive hypothesis proposed by Malcom Steinberg. However, in further study of more mature cell aggregates, the initial sorting pattern reversed, with the highly adhesive wild-type ES cells forming an outer shell enveloping the less adhesive E-cadherin-deficient cells, contradicting Steinberg’s sorting principle. The outer wild-type cells of the more mature aggregates did not differentiate into endoderm, which is known to be able to sort to the exterior from previous studies. In contrast to the naive aggregates, the mature aggregates presented polarized, highly adhesive cells at the outer layer. The surface polarity was observed as an actin cap contiguously spanning across the apical surface of multiple adjacent cells, though independent of the formation of tight junctions.ConclusionsOur experimental findings suggest that the force of differential adhesive affinity can be overcome by even subtle polarity generated from strong bilateral ligation of highly adhesive cells in determining cell sorting patterns.

Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas.

Single-cell RNA sequencing (scRNA-seq) enables the systematic identification of cell populations in a tissue, but characterizing their spatial organization remains challenging. We combine a microarray-based spatial transcriptomics method that reveals spatial patterns of gene expression using an array of spots, each capturing the transcriptomes of multiple adjacent cells, with scRNA-Seq generated from the same sample. To annotate the precise cellular composition of distinct tissue regions, we introduce a method for multimodal intersection analysis. Applying multimodal intersection analysis to primary pancreatic tumors, we find that subpopulations of ductal cells, macrophages, dendritic cells and cancer cells have spatially restricted enrichments, as well as distinct coenrichments with other cell types. Furthermore, we identify colocalization of inflammatory fibroblasts and cancer cells expressing a stress-response gene module. Our approach for mapping the architecture of scRNA-seq-defined subpopulations can be applied to reveal the interactions inherent to complex tissues.

NK cells for cancer immunotherapy.

Natural killer (NK) cells can swiftly kill multiple adjacent cells if these show surface markers associated with oncogenic transformation. This property, which is unique among immune cells, and their capacity to enhance antibody and T cell responses support a role for NK cells as anticancer agents. Although tumours may develop several mechanisms to resist attacks from endogenous NK cells, ex vivo activation, expansion and genetic modification of NK cells can greatly increase their antitumour activity and equip them to overcome resistance. Some of these methods have been translated into clinical-grade platforms and support clinical trials of NK cell infusions in patients with haematological malignancies or solid tumours, whichhave yielded encouraging results so far. The next generation of NK cell products will be engineered to enhance activating signals and proliferation, suppress inhibitory signals and promote their homing to tumours. These modifications promise to significantly increase their clinical activity. Finally, there is emerging evidence of increased NK cell-mediated tumour cell killing in the context of molecularly targeted therapies. These observations, in addition to the capacity of NK cells to magnify immune responses, suggest that NK cells are poised to become key components of multipronged therapeutic strategies for cancer.

Evaluation of lipid coverage and high spatial resolution MALDI-imaging capabilities of oversampling combined with laser post-ionisation

Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualising the spatial locations of lipids in biological tissues. However, a major challenge in interpreting the biological significance of local lipid compositions and distributions detected using MALDI-MSI is the difficulty in associating spectra with cellular lipid metabolism within the tissue. By-and-large this is due to the typically limited spatial resolution of MALDI-MSI (30–100 μm) meaning individual spectra represent the average spectrum acquired from multiple adjacent cells, each potentially possessing a unique lipid composition and biological function. The use of oversampling is one promising approach to decrease the sampling area and improve the spatial resolution in MALDI-MSI, but it can suffer from a dramatically decreased sensitivity. In this work we overcome these challenges through the coupling of oversampling MALDI-MSI with laser post-ionisation (MALDI-2). We demonstrate the ability to acquire rich lipid spectra from pixels as small as 6 μm, equivalent to or smaller than the size of typical mammalian cells. Coupled with an approach for automated lipid identification, it is shown that MALDI-2 combined with oversampling at 6 μm pixel size can detect up to three times more lipids and many more lipid classes than even conventional MALDI at 20 μm resolution in the positive-ion mode. Applying this to mouse kidney and human brain tissue containing active multiple sclerosis lesions, where 74 and 147 unique lipids are identified, respectively, the localisation of lipid signals to individual tubuli within the kidney and lipid droplets with lesion-specific macrophages is demonstrated.Graphical abstract

A Pseudo-Spectrum Approach for Weak Target Detection and Tracking

Conventional velocity-filtering-based track-before-detect (VF-TBD) methods integrate the energy of a cell in a frame with that of the cell closest to the predicted target position in the last frame of the processing batch, assuming a certain target velocity. However, the target may not exactly be on the quantized cell and its echo envelope may occupy multiple adjacent cells. This often leads to significant energy loss and echo envelope degradation. In this paper, a novel VF-TBD method based on pseudo-spectrum (PS-VF-TBD) is presented to address this problem. For every cell, a pseudo-spectrum is constructed around the predicted position according to the assumed velocity using a truncated point spread function. Samples of the pseudo-spectrum on the cells that are located in the truncated spread area are added onto the last frame of the processing batch to integrate the target energy within multiple cells. Due to the use of the point spread model and the accurate sampling of the predicted spectrum, energy loss can be mitigated and the echo envelope is well maintained. This approach simultaneously maximizes the signal-to-noise ratio (SNR) gain and enables improved parameter estimation utilizing the envelope characteristics. The procedure for pseudo-spectrum construction and multiframe accumulation is derived in detail and the output SNR is analyzed theoretically. It is found that the proposed PS-VF-TBD can achieve an SNR gain greater than that by the conventional VF-TBD method. To deal with a target with unknown velocity, a bank of pseudo-spectrum-based velocity filters is proposed. The signal gain loss resulting from velocity mismatch is investigated and the $\mu$ -width of the envelope in the velocity domain is analyzed. Finally, a method for improved position and velocity estimation is presented. Simulation results demonstrate the superiority of the proposed method in terms of SNR gain, detection probability, and estimation accuracy at the expense of increased computational complexity.

TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells

Persistent microbial infection promotes the fusion of several kinds of somatic cells, such as macrophages and endothelial cells, leading to the formation of multinucleated giant cells (MGCs). However, the molecular mechanisms of MGCs formation are still poorly understood. By laser confocal microscope, we discovered that TRIM34 increased the efficiency of cell fusion in Human Embryonic Kidney cells (HEK293T). By means of DiD cell membrane probes, LysoTracker Deep Red or MitoTracker Deep Red staining, we also demonstrated that TRIM34 stimulated cell fusion in paraformaldehyde fixed or living HEK293T cells. Moreover, we discovered that the nuclei shapes of MGCs induced by TRIM34 were diversiform, such as horseshoe shape, ring like shape etc. Through 3D reconstruction of confocal z-stacks images, we found that TRIM34-EGFP proteins could form macromolecule aggregates in the central area of MGCs, while the nuclei were arranged in ring like shape and distributed around the plasma membrane. Cell fusion assay showed that cocultured TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells could fuse to form MGCs. We speculate that the formation of MGCs can be divided into two phase: primary multinucleated cells (PMCs) and secondary multinucleated cells (SMCs). Firstly, TRIM34 induced fusion of multiple adjacent cells resulting in PMCs formation, and then PMCs were endowed with the capacity of phagocytosis and turned into SMCs. Collectively, these results suggest that TRIM34 proteins contribute to the formation of MGCs by promoting cell fusion and phagocytosis in epithelial cells.

The Complex Cell Wall Composition of Syncytia Induced by Plant Parasitic Cyst Nematodes Reflects Both Function and Host Plant.

Plant–parasitic cyst nematodes induce the formation of specialized feeding structures, syncytia, within their host roots. These unique plant organs serve as the sole nutrient resource for development and reproduction throughout the biotrophic interaction. The multinucleate syncytium, which arises through local dissolution of cell walls and protoplast fusion of multiple adjacent cells, has dense cytoplasm containing numerous organelles, surrounded by thickened outer cell walls that must withstand high turgor pressure. However, little is known about how the constituents of the syncytial cell wall and their conformation support its role during nematode parasitism. We used a set of monoclonal antibodies, targeted to a range of plant cell wall components, to reveal the microstructures of syncytial cell walls induced by four of the most economically important cyst nematode species, Globodera pallida, Heterodera glycines, Heterodera avenae and Heterodera filipjevi, in their respective potato, soybean, and spring wheat host roots. In situ fluorescence analysis revealed highly similar cell wall composition of syncytia induced by G. pallida and H. glycines. Both consisted of abundant xyloglucan, methyl-esterified homogalacturonan and pectic arabinan. In contrast, the walls of syncytia induced in wheat roots by H. avenae and H. filipjevi contain little xyloglucan but are rich in feruloylated xylan and arabinan residues, with variable levels of mixed-linkage glucan. The overall chemical composition of syncytial cell walls reflected the general features of root cell walls of the different host plants. We relate specific components of syncytial cell walls, such as abundant arabinan, methyl-esterification status of pectic homogalacturonan and feruloylation of xylan, to their potential roles in forming a network to support both the strength and flexibility required for syncytium function.

Human Three-Dimensional Endometrial Epithelial Cell Model To Study Host Interactions with Vaginal Bacteria and Neisseria gonorrhoeae.

Colonization of the endometrium by pathogenic bacteria ascending from the lower female reproductive tract (FRT) is associated with many gynecologic and obstetric health complications. To study these host-microbe interactions in vitro, we developed a human three-dimensional (3-D) endometrial epithelial cell (EEC) model using the HEC-1A cell line and the rotating wall vessel (RWV) bioreactor technology. Our model, composed of 3-D EEC aggregates, recapitulates several functional/structural characteristics of human endometrial epithelial tissue, including cell differentiation, the presence of junctional complexes/desmosomes and microvilli, and the production of membrane-associated mucins and Toll-like receptors (TLRs). TLR function was evaluated by exposing the EEC aggregates to viral and bacterial products. Treatment with poly(I·C) and flagellin but not with synthetic lipoprotein (fibroblast-stimulating lipoprotein 1 [FSL-1]) or lipopolysaccharide (LPS) significantly induced proinflammatory mediators in a dose-dependent manner. To simulate ascending infection, we infected EEC aggregates with commensal and pathogenic bacteria: Lactobacillus crispatus, Gardnerella vaginalis, and Neisseria gonorrhoeae All vaginal microbiota and N. gonorrhoeae efficiently colonized the 3-D surface, localizing to crevices of the EEC model and interacting with multiple adjacent cells simultaneously. However, only infection with pathogenic N. gonorrhoeae and not infection with the other bacteria tested significantly induced proinflammatory mediators and significant ultrastructural changes to the host cells. The latter observation is consistent with clinical findings and illustrated the functional specificity of our system. Additionally, we highlighted the utility of the 3-D EEC model for the study of the pathogenesis of N. gonorrhoeae using a well-characterized ΔpilT mutant. Overall, this study demonstrates that the human 3-D EEC model is a robust tool for studying host-microbe interactions and bacterial pathogenesis in the upper FRT.

Intraductal Fenestration of Biliary Metal Stents Using Argon Plasma to Facilitate Bilateral Hilar Stenting

Background: When placed across the biliary confluence, uncovered self-expanding metal stents (SEMS) may impede access to the contralateral intrahepatic ducts. In particular it is difficult to place additional plastic or metal stents across multiple adjacent cells of a hilar SEMS. Argon plasma has been used to trim biliary SEMS. However, the use of argon plasma for intraductal fenestration of biliary metal stents has not been described. Case report: A 38-year-old man with hilar cholanogiocarcinoma presented with cholangitis and right lobe liver abscesses. He had been previously treated with placement of multiple closed-cell, uncovered SEMS extending from duodenum to left hepatic duct. A percutaneous transhepatic catheter was placed into an abscess cavity, which communicated with right hepatic ducts. Subsequently a percutaneous transhepatic guidewire was advanced through the hilar SEMS interstices and into the duodenum. During ERCP a temporary plastic stent was placed into the left main duct for venting of argon gas. The percutaneous guidewire was then grasped with a snare and brought out via the mouth. Choledochoscopy was then performed using a pediatric gastroscope which was advanced over the guidewire into the existing SEMS up to the level of the hilum. Metal struts of the embedded SEMS were seen crisscrossing over the right duct orifice. The visible SEMS wires were fenestrated using a pediatric argon plasma coagulation probe at power of 80 W and flow 0.3 L/min, and further unwoven with a pediatric biopsy forceps. Argon gas vented from the percutaneous biliary sheath as well as the left hepatic duct stent. Subsequently plastic stents were placed via a duodenoscope into 4 separate right intrahepatic ducts, each of which was accessed across adjacent interstices of the SEMS. The patient had rapid clinical improvement and a follow-up computed tomography at 4 weeks showed resolution of liver abscesses. Conclusion: Fenestration of hilar biliary SEMS using argon plasma under direct endoscopic visualization can facilitate bilateral hilar stent placement in patients with complex malignant obstruction of the central intrahepatic ducts. Argon plasma has been used to trim biliary and rectal SEMS. The use of argon plasma for endoscopic fenestration of duodenal stents to facilitate ERCP has also been reported. Our case extends the application of this technique by describing intraductal fenestration of an indwelling biliary SEMS to accomplish bilateral hilar stenting.

SEU Tolerant Memory Using Error Correction Code

With decreasing circuit lithography dimensions and increasing memory densities, an SEU may affect multiple adjacent memory cells. This paper presents an SEU hardened memory using error correction code that can correct single errors, double-adjacent errors, triple-adjacent errors and double-almost-adjacent errors. The proposed memory introduces small area, power and delay overheads.

The cages, dynamics, and structuring of incipient methane clathrate hydrates

Interest in describing clathrate hydrate formation mechanisms spans multiple fields of science and technical applications. Here, we report findings from multiple molecular dynamics simulations of spontaneous methane clathrate hydrate nucleation and growth from fully demixed and disordered two-phase fluid systems of methane and water. Across a range of thermodynamic conditions and simulation geometries and sizes, a set of seven cage types comprises approximately 95% of all cages formed in the nucleated solids. This set includes the ubiquitous 5(12) cage, the 5(12)6(n) subset (where n ranges from 2-4), and the 4(1)5(10)6(n) subset (where n also ranges from 2-4). Transformations among these cages occur via water pair insertions/removals and rotations, and may elucidate the mechanisms of solid-solid structural rearrangements observed experimentally. Some consistency is observed in the relative abundance of cages among all nucleation trajectories. 5(12) cages are always among the two most abundant cage types in the nucleated solids and are usually the most abundant cage type. In all simulations, the 5(12)6(n) cages outnumber their 4(1)5(10)6(n) counterparts with the same number of water molecules. Within these consistent features, some stochasticity is observed in certain cage ratios and in the long-range ordering of the nucleated solids. Even when comparing simulations performed at the same conditions, some trajectories yield swaths of multiple adjacent sI unit cells and long-range order over 5 nm, while others yield only isolated sI unit cells and little long-range order. The nucleated solids containing long-range order have higher 5(12)6(2)/5(12) and 5(12)6(3)/4(1)5(10)6(2) cage ratios when compared to systems that nucleate with little long-range order. The formation of multiple adjacent unit cells of sI hydrate at high driving forces suggests an alternative or addition to the prevailing hydrate nucleation hypotheses which involve formation through amorphous intermediates.

Coordinated and distributed MIMO [Guest Editorial

The impetus for this feature topic was spurred by the technical trend in wireless communications: the need for high bit rates (~1 Gb/s) in future wireless systems. However, due to the limited transmit power, the transmission distance cannot be long if the desired transmission rate is very high. In order to have a reasonable coverage area in each cell, antennas centrally attached to the base station in conventional mobile systems should be distributed through the entire cell via wires (or fibers) so that the wireless transmission distance between mobile units and base stations can be shortened. Moreover, reception of one signal at different locations provides spatial diversity against fading. Such network architecture is called a distributed antenna system (DAS). In order to reduce the transmission distance and provide diversity gain, the distributed multiple-input multiple-output (MIMO) concept is introduced to achieve broadband wireless transmission. In addition, coordination from multiple adjacent cells is needed to efficiently use the distributed MIMO. Therefore, coordinated and distributed MIMO becomes crucial. The key to implement coordinated and distributed MIMO is to have the mobiles communicate simultaneously with several remote antennas with perfect coordination between these antennas.

Evaluation of Downlink Performance of a Multiple-Cell, Rake Receiver Assisted CDMA Mobile System

In wireless Code Division Multiple Access (CDMA) system, the use of power control is indispensable to combat near-far and fading problems. Signals transmitted over a multipath propagation channel which exhibits inter-path interference and fading. The receiver has to employ measures to mitigate these effects or it will incur severe performance degradation. A classic approach in CDMA communications is the rake receiver. In this paper, the downlink performance is estimated for a CDMA mobile system at the vertex of multiple adjacent cells. At the base station the received signal is coherently dispread and demodulated using a rake receiver. The effects of power control, error correction and rake receiver were also investigated on the assumption that the received signals undergo Rayleigh fading, lognormal shadowing, and frequency selective fading. The evaluation of performance measures of base to mobile link (downlink) of a multiple-cell CDMA mobile system is presented. This study demonstrates that significant performance improvements are achievable with combined use of power control, rake receiver and error correction scheme.

Region-based Shadow Registration Scheme for Efficient Mobility Management in the SIP

In the session-initiated protocol (SIP) for wireless networks, delays and disruptions that occur during the handoff of nodes are related to authentication, authorization, and accounting (AAA). Various schemes have been proposed to reduce these handoff delays, and one such proposal, shadow registration in adjacent cells, has been adopted. Shadow registration reduces handoff delays by preregistering a cell with all of its neighboring cells. However, preregistering with multiple adjacent cells significantly increases both the amount of network traffic and the corresponding waste of network resources. This paper introduces region-based shadow registration (RSR) for efficient mobility management in mobile SIP environments. The proposed scheme performs shadow registration with neighboring cells selectively, only when it is necessary at the FAAA level. In an experiment implementing the system, we show that it reduces handoff latency while decreasing excessive signaling traffic during shadow registration.

An Epithelial Niche in the Drosophila Ovary Undergoes Long-Range Stem Cell Replacement

Adult epithelial stem cells are thought to reside in specific niches, where they are maintained by adhesion to stromal cells and by intercellular signals. In niches that harbor multiple adjacent stem cells, such as those maintaining Drosophila germ cells, lost stem cells are replaced by division of neighboring stem cells or reversion of transit cells. We have characterized the Drosophila follicle stem cell (FSC) niche as a model of the epithelial niche to learn whether nonneighboring cells can also generate stem cell replacements. Exactly two stroma-free FSC niches holding single FSCs are located in fixed locations on opposite edges of the Drosophila ovariole. FSC daughters regularly migrate across the width of the ovariole to the other niche before proliferating and contributing to the follicle cell monolayer. Crossmigrating FSC daughters compete with the resident FSC for niche occupancy and are the source of replacement FSCs. The ability of stem cell daughters to target a distant niche and displace its resident stem cell suggests that precancerous mutations might spread from niche to niche within stem cell-based tissues.

How do malaria ookinetes cross the mosquito midgut wall?

The route taken by malaria ookinetes to cross the midgut wall of the mosquito vector has been (and continues to be) controversial. In this article, we attempt to reinterpret and integrate the apparently conflicting observations of the past using the recently proposed Time Bomb model of ookinete invasion as a unifying framework. We argue that parasite entry into the midgut epithelium is always intracellular, occurs at sites where multiple adjacent host cells converge, and that parasite exit from the midgut epithelium can occur by either an intracellular or intercellular route, depending on the manner in which invaded host cells are extruded from the midgut wall. We also propose a novel hypothesis, the Cellular Treadmill model, to explain the movement of ookinetes through multiple adjacent midgut cells.

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The route taken by malaria ookinetes to cross the midgut wall of the mosquito vector has been (and continues to be) controversial. In this article, we attempt to reinterpret and integrate the apparently conflicting observations of the past using the recently proposed Time Bomb model of ookinete invasion as a unifying framework. We argue that parasite entry into the midgut epithelium is always intracellular, occurs at sites where multiple adjacent host cells converge, and that parasite exit from the midgut epithelium can occur by either an intracellular or intercellular route, depending on the manner in which invaded host cells are extruded from the midgut wall. We also propose a novel hypothesis, the Cellular Treadmill model, to explain the movement of ookinetes through multiple adjacent midgut cells.

Development of intercellular communication during the epithelial reorganization of a renal cell line (LLC-PK1).

Junctional permeability determinations after microinjection of the fluorescent tracer, Lucifer Yellow CH, show that the cells in confluent monolayers of the renal epithelial cell lines LLC-PK1 and A6 are interconnected by intercellular junctions. This cell-to-cell communication network permits the fluorescent dye to diffuse from the microinjected cell into multiple adjacent neighboring cells. Cell-to-cell diffusion of the fluorescent dye was not observed at pH 6.0. Full recovery occurred, however, when the pH of the extracellular medium was adjusted to 7.4. To provide a sensitive index of the averaged efficacy of junctional communication, we measured the number of cells that survived ouabain treatment in a 50% mixture of wild and ouabain-resistant mutant LLC-PK1 cells. Electron probe microanalysis in uncoupled cells showed that ouabain treatment produced two populations of cells, with totally different intracellular Na+ and K+ content. Under this condition, only 50% of the population survived after 48 h of treatment. When ouabain treatment was initiated 24 h after plating, however, 100% survival was observed, and the cells contained uniform intracellular Na+ and K+ concentration. This finding is consistent with the theory that this protective effect is mediated through the presence of the functional communicating intercellular junctions. When ouabain was applied at different times after plating, full protection is reached by 2 h. The early development of cell-to-cell communication, which precedes the development of the occluding junctions and several transport systems by several hours, is consistent with the involvement of the intercellular junctions in the synchronization of the polarization process.