Apical Boundary Research Articles

Mechanism of interdigitation formation at apical boundary of MDCK cell

It has been reported that the MDCK cell tight junction shows stochastic fluctuation and forms the interdigitation structure, but the mechanism of the pattern formation remains to be elucidated. In the present study, we first quantified the shape of the cell-cell boundary at the initial phase of pattern formation. We found that the Fourier transform of the boundary shape shows linearity in the log-log plot, indicating the existence of scaling. Next, we tested several working hypotheses and found that the Edwards-Wilkinson equation, which consists of stochastic movement and boundary shortening, can reproduce the scaling property. Next, we examined the molecular nature of stochastic movement and found that myosin light chain puncta may be responsible. Quantification of boundary shortening indicates that mechanical property change may also play some role. Physiological meaning and scaling properties of the cell-cell boundary are discussed.

CLINICAL DIFFICULTIES IN ENDODONTIC TREATMENT OF PREMOLARS WITH ATYPICAL ANATOMY

To achieve high levels of success in endodontic treatment requires in-depth knowledge of the anatomy and morphology of the root canal system. Premolars are a group of teeth which shows a wide spectrum of different variations, reaching the apical opening. The frequency of one root canal at the apical tip for the maxillary first premolars is 8.66%, while 89.64% of the examined teeth show two root canals at the apical tip. The frequency of two or more root canals in the mandibular first premolars varies between 2.7% to 62.5%, while the frequency of two or more root canals in the mandibular second premolars varies between 0% and 34.3%. Some of the clinical aspects in the treatment of such aberrated premolars include implementing detailed preoperative evaluation- X-Rays and CBCT, sufficient opening of the endodontic access and careful three-dimentional cleaning of the root canal system with combination of different endodontic instruments and hybrid techniques of work and following hermetic sealing of all portals of exits. A 3D printing after a CBCT scan is a method of choice when a clinician wants to master the anatomy of a premolar. Knowledge of the complex root canal system is essential for understanding the principles and problems of shaping and, cleaning, determining the apical boundaries and dimensions of the root canals.

Unveiling the polarity of actin filaments by cryo-electron tomography

SummaryThe actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in intact cells would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies, and therefore the polarity of actin filaments cannot be directly measured. Here, we developed a method that enables us to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.

WE‐AB‐202‐09: Feasibility and Quantitative Analysis of 4DCT‐Based High Precision Lung Elastography

Purpose:The purpose of this project is to derive high precision elastography measurements from 4DCT lung scans to facilitate the implementation of elastography in a radiotherapy context.Methods:4DCT scans of the lungs were acquired, and breathing stages were subsequently registered to each other using an optical flow DIR algorithm. The displacement of each voxel gleaned from the registration was taken to be the ground‐truth deformation. These vectors, along with the 4DCT source datasets, were used to generate a GPU‐based biomechanical simulation that acted as a forward model to solve the inverse elasticity problem. The lung surface displacements were applied as boundary constraints for the model‐guided lung tissue elastography, while the inner voxels were allowed to deform according to the linear elastic forces within the model. A biomechanically‐based anisotropic convergence magnification technique was applied to the inner voxels in order to amplify the subtleties of the interior deformation. Solving the inverse elasticity problem was accomplished by modifying the tissue elasticity and iteratively deforming the biomechanical model. Convergence occurred when each voxel was within 0.5 mm of the ground‐truth deformation and 1 kPa of the ground‐truth elasticity distribution. To analyze the feasibility of the model‐guided approach, we present the results for regions of low ventilation, specifically, the apex.Results:The maximum apical boundary expansion was observed to be between 2 and 6 mm. Simulating this expansion within an apical lung model, it was observed that 100% of voxels converged within 0.5 mm of ground‐truth deformation, while 91.8% converged within 1 kPa of the ground‐truth elasticity distribution. A mean elasticity error of 0.6 kPa illustrates the high precision of our technique.Conclusion:By utilizing 4DCT lung data coupled with a biomechanical model, high precision lung elastography can be accurately performed, even in low ventilation regions of the lungs.This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1144087.

Distinct Apical and Basolateral Mechanisms Drive Planar Cell Polarity-Dependent Convergent Extension of the Mouse Neural Plate

The mechanisms of tissue convergence and extension (CE) driving axial elongation in mammalian embryos, and in particular, the cellular behaviors underlying CE in the epithelial neural tissue, have not been identified. Here we show that mouse neural cells undergo mediolaterally biased cell intercalation and exhibit both apical boundary rearrangement and polarized basolateral protrusive activity. Planar polarization and coordination of these two cell behaviors are essential for neural CE, as shown by failure of mediolateral intercalation in embryos mutant for two proteins associated with planar cell polarity signaling: Vangl2 and Ptk7. Embryos with mutations in Ptk7 fail to polarize cell behaviors within the plane of the tissue, whereas Vangl2 mutant embryos maintain tissue polarity and basal protrusive activity but are deficient in apical neighbor exchange. Neuroepithelial cells in both mutants fail to apically constrict, leading to craniorachischisis. These results reveal a cooperative mechanism for cell rearrangement during epithelial morphogenesis.

Anisotropy of Crumbs and aPKC Drives Myosin Cable Assembly during Tube Formation

SummaryThe formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, I find that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized.

Serrano (Sano) Functions with the Planar Cell Polarity Genes to Control Tracheal Tube Length

Epithelial tubes are the functional units of many organs, and proper tube geometry is crucial for organ function. Here, we characterize serrano (sano), a novel cytoplasmic protein that is apically enriched in several tube-forming epithelia in Drosophila, including the tracheal system. Loss of sano results in elongated tracheae, whereas Sano overexpression causes shortened tracheae with reduced apical boundaries. Sano overexpression during larval and pupal stages causes planar cell polarity (PCP) defects in several adult tissues. In Sano-overexpressing pupal wing cells, core PCP proteins are mislocalized and prehairs are misoriented; sano loss or overexpression in the eye disrupts ommatidial polarity and rotation. Importantly, Sano binds the PCP regulator Dishevelled (Dsh), and loss or ectopic expression of many known PCP proteins in the trachea gives rise to similar defects observed with loss or gain of sano, revealing a previously unrecognized role for PCP pathway components in tube size control.

Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crabCallinectes sapidus

Two isoforms of the enzyme carbonic anhydrase (CA) in the blue crab gill, CasCAg and CasCAc, were identified, sequenced, and found to match the membrane-associated and cytoplasmic isoforms, respectively. The membrane-associated isoform is present in much higher levels of mRNA expression in both anterior and posterior gills in crabs acclimated to high salinity (35 p.p.t.), but expression of the cytoplasmic isoform in the posterior gill undergoes a significantly greater degree of up-regulation after exposure to low salinity (15 p.p.t.). CasCAc has the largest scope of induction (100-fold) reported for any transport-related protein in the gill, and this may be necessary to overcome diffusion limitations between gill cytoplasm and the apical boundary layer. Furthermore, the timing of the changes in expression of CasCAc corresponds to the timing of the induction of protein-specific CA activity and CA protein concentration. No changes in CA mRNA expression or activity occur in the anterior gills. The pattern of up-regulation of expression of mRNA of the alpha-subunit of the Na+/K+-ATPase is similar to that for CasCAc, and both precede the establishment of the new acclimated physiological state of the crab in low salinity. A putative ;housekeeping' gene, arginine kinase, also showed about a threefold increase in expression in response to low salinity, but only in the posterior gills. These results suggest that for studies of expression in crustacean gill tissue, a control tissue, such as the anterior gill, be used until an adequate control gene is identified.

Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis

The primary mechanism by which the kidneys mediate net acid excretion is through ammonia metabolism. In the current study, we examined whether chronic metabolic acidosis, which increases ammonia metabolism, alters the cell-specific and/or the subcellular expression of the ammonia transporter family member, Rhcg, in the outer medullary collecting duct in the inner stripe (OMCDi). Chronic metabolic acidosis was induced in normal SD rats by HCl ingestion for 7 days; controls were pair-fed. The subcellular distribution of Rhcg was determined using immunogold electron microscopy and morphometric analyses. In intercalated cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane. Intracellular Rhcg decreased significantly, and basolateral Rhcg was unchanged. Because apical plasma membrane length increased in parallel with apical Rhcg immunolabel, apical plasma membrane Rhcg density was unchanged. In principal cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane while decreasing the intracellular proportion. In contrast to the intercalated cell, chronic metabolic acidosis did not significantly alter apical boundary length; accordingly, apical plasma membrane Rhcg density increased. In addition, basolateral Rhcg immunolabel increased in response to chronic metabolic acidosis. These results indicate that in the rat OMCDi 1) chronic metabolic acidosis increases apical plasma membrane Rhcg in both the intercalated cell and principal cell where it may contribute to enhanced apical ammonia secretion; 2) increased apical plasma membrane Rhcg results from both increased total protein and changes in the subcellular distribution of Rhcg; 3) the mechanism of Rhcg subcellular redistribution differs in intercalated and principal cells; and 4) Rhcg may contribute to regulated basolateral ammonia transport in the principal cell.

Temporary DPOAE level shifts, ABR threshold shifts and histopathological damage following below-critical-level noise exposures

DPOAE temporary level shift (TLS) at 2 f 1 − f 2 and f 2 − f 1, ABR temporary threshold shift (TTS), and detailed histopathological findings were compared in three groups of chinchillas that were exposed for 24 h to an octave band of noise (OBN) centered at 4 kHz with a sound pressure level (SPL) of 80, 86 or 92 dB ( n = 3, 4, 6). DPOAE levels at 39 frequencies from f 1 = 0.3 to 16 kHz ( f 2/ f 1 = 1.23; L 2 and L 1 = 55, 65 and 75 dB, equal and differing by 10 dB) and ABR thresholds at 13 frequencies from 0.5 to 20 kHz were collected pre- and immediately post-exposure. The functional data were converted to pre- minus post-exposure shift and overlaid upon the cytocochleogram of cochlear damage using the frequency-place map for the chinchilla. The magnitude and frequency place of components in the 2 f 1− f 2 TLS patterns were determined and group averages for each OBN SPL and L 1, L 2 combination were calculated. The f 2 − f 1 TLS was also examined in ears with focal lesions equal to or greater than 0.4 mm. The 2 f 1 − f 2 TLS (plotted at f 1) and TTS aligned with the extent and location of damaged supporting cells. The TLS patterns over frequency had two features which were unexpected: (1) a peak at about a half octave above the center of the OBN with a valley just above and below it and (2) a peak (often showing enhancement) at the apical boundary of the supporting-cell damage. The magnitudes of the TLS and TTS generally increased with increasing SPL of the exposure. The peaks of the TLS and TTS, as well as the peaks and valleys of the TLS pattern moved apically as the SPL of the OBN was increased. However, there was little consistency in the pattern relations with differing L 1, L 2 combinations. In addition, neither the 2 f 1 − f 2 nor f 2 − f 1 TLS for any L 1, L 2 combination reliably detected focal lesions (100% OHC loss) from 0.4 to 1.2 mm in size. Often, the TLS went in the opposite direction from what would be expected at focal lesions. Recovery from TLS and TTS was also examined in seven animals. Both TLS and TTS recovered partially or completely, the magnitude depending upon exposure SPL.

Delta-catenin, an adhesive junction-associated protein which promotes cell scattering.

The classical adherens junction that holds epithelial cells together consists of a protein complex in which members of the cadherin family linked to various catenins are the principal components. delta-catenin is a mammalian brain protein in the Armadillo repeat superfamily with sequence similarity to the adherens junction protein p120(ctn). We found that delta-catenin can be immunoprecipitated as a complex with other components of the adherens junction, including cadherin and beta-catenin, from transfected cells and brain. The interaction with cadherin involves direct contact within the highly conserved juxtamembrane region of the COOH terminus, where p120(ctn) also binds. In developing mouse brain, staining with delta-catenin antibodies is prominent towards the apical boundary of the neuroepithelial cells in the ventricular zone. When transfected into Madin-Darby canine kidney (MDCK) epithelial cells delta-catenin colocalized with cadherin, p120(ctn), and beta-catenin. The Arm domain alone was sufficient for achieving localization and coimmunoprecipitation with cadherin. The ectopic expression of delta-catenin in MDCK cells altered their morphology, induced the elaboration of lamellipodia, interfered with monolayer formation, and increased scattering in response to hepatocyte growth factor treatment. We propose that delta-catenin can regulate adhesion molecules to implement the organization of large cellular arrays necessary for tissue morphogenesis.

Characterization of ATPases of apical membrane fractions from Locusta migratoria Malpighian tubules

Apical and basal membrane fractions from Locusta Malpighian tubules were prepared and were characterized by marker enzyme analysis. The apical membranes contained an azide- and orthovanadate-insensitive ATPase activity that was inhibited by bafilomycin A 1 (IC 50=0.44 nM) and NEM (IC 50=2.15 μM), and thus was characterized as a putative V-type ATPase. The enzyme was stimulated by a variety of monovalent cations (Tris>K=Na>choline>Li=Rb) maximal stimulation occurring at 30-40 mM. It was also stimulated by a variety of monovalent anions (maximal activation 30-40 mM), but was strongly inhibited by nitrate and thiocyanate. SDS-PAGE separation of proteins present in the various membrane fractions was carried out. The apical membrane fraction alone contained a 28 kDa protein band that bound a monoclonal antibody specific for a 28 kDa peptide which was a component of the V-type ATPase from midgut of Manduca sexta and, in native gels, possessed ATPase activity which was also sensitive to both bafilomycin and NEM but not to azide or orthovanadate. Binding of the fluorescent monoclonal antibody was located at the apical boundary of the tubule cells. It was concluded that a V-type ATPase is present at the apical surface of Locusta Malpighian tubule cells and that it is involved in their secretory functioning.

Drosophila C virus cycle during the development of two Dorosphila melanogaster strains (Charolles and Champetie`res) after larval contamination by food

Dorosphila C virus (DCV) cycle during Drosophila melanogaster development was studied after feeding contamination at the first, most sensitive, instar (L1). Two Drosophila strains were examined and compared. Presence of DCV C in apparently healthy animals (L3 larvae bred on a contaminated rearing medium and adults coming from larvae which were grown on medium containing DCV C) was demonstrated by biological tests. Using the immunofluorescence technique, DCV was exhibited in the diseased Charolles larvae, in the lumen of the digestive tract and in the basal part of gut cells which is in contact with the haemolymph. On the contrary, in Charolles larvae which seemed ‘healthy’, DCV was exhibited only in the lumen of the digestive tract at the apical boundary of the gut cells. But DCV typical protein capsid was not shown in the tissue of Drosophila L3 and adults. However, C virus remained in Drosophila tissues even after host metamorphosis and would seem to interact with Drosophila cells. Hypotheses are proposed concerning the intracellular state of Drosophila C virus in this case.

Single-tone and two-tone responses of cochlear-nerve fibers: changes after kanamycin and noise treatments

Responses to single tones and two-tone inhibition (TTI) above CF were determined in untreated Mongolian gerbils and after kanamycin or impulse-noise treatment. Kanamycin produced maximum hair-cell damage in the basal cochlear turn, the noise, in the mid-cochlear region and sometimes in the base. Fibers associated with 100% loss of outer hair cells (OHC) in the basal turn showed elevated CF thresholds, reduced slopes of tuning curves, no TTI, steep rate-intensity curves above CF, and excitation during basilar-membrane displacement towards scala vestibuli. In the presence of 50–95% OHC loss restricted to 2–4 mm of the midcochlear region, corresponding fibers exhibited less homogeneous characteristics. CF thresholds corresponding to the basal boundary of the lesion were nearly normal and increased toward and beyond the apical boundary. The tuning curves tended to be somewhat broadened and TTI, though reduced, was still present in fibers with elevated CF thresholds. Fiber excitation usually occurred during basilar-membrane displacement towards scala vestibuli. Some fibers from both treated groups showed increased sensitivity below CF despite elevated CF thresholds. Tuning curves associated with no TTI had high-frequency slopes between 70–200 dB/oct.