Lateral Plasma Membrane Research Articles

Expression of cell adhesion molecule 1 in human and murine endometrial glandular cells and its increase during the proliferative phase by estrogen and cell density

AimsCell adhesion molecule 1 (CADM1) mediates interepithelial adhesion and is upregulated in crowded epithelial monolayers. This study aimed to examine CADM1 expression in the human endometrium of proliferative and secretory phases, and its transcriptional regulation in terms of estrogen stimuli and higher cellularity. Main methodsCADM1 immunohistochemistry was conducted on endometrial tissues from women in their 40s and adult mice subcutaneously injected with estradiol following ovariectomy. Dual-luciferase reporter assays were conducted using human endometrial HEC-50B and HEC-1B cells and reporter plasmids harboring the human CADM1 3.4-kb promoter and its deleted and mutated forms. Cells were transfected with estrogen receptor α cDNA and reporter plasmids, and treated with estradiol before luciferase activity measurement. Key findingsImmunohistochemistry revealed that CADM1 was clearly expressed on the lateral membranes of the simple columnar glandular cells in the proliferative phase, but not in the secretory phase, from both women and the mouse model. The glandular cell density increased two-fold in the proliferative phase. Reporter assays identified three Sp1-binding sites as estradiol-responsive elements in the proximal region (from −223 to −84) of the transcription start site (+1) in HEC-50B cells. When the cell culture was started at eight-fold higher cell density, the CADM1 3.4-kb promoter was transactivated at a two-fold higher level in HEC-50B cells. This cell density effect was not detected for the CADM1 2.3-kb or 1.6-kb promoter. SignificanceTwo (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase.

Evaluation of E-Cadherin and β-Catenin Immunoreactivity for Determining Undifferentiated Cells in Anaplastic Thyroid Carcinoma

Introduction: An immunohistochemical study has occasionally been performed to diagnose anaplastic thyroid carcinoma (ATC). However, antibodies to confirm the undifferentiated nature of ATC have not yet been evaluated. The aim of this study was to evaluate E-cadherin and β-catenin expressions in immunoreactivity to determine undifferentiated carcinoma cells in the diagnosis of ATC. Methods: We immunohistochemically examined 29 ATCs, 30 poorly differentiated thyroid carcinomas (PDTCs), 22 well-differentiated thyroid carcinomas (WDTCs), and 3 squamous cell carcinomas. Antibodies for thyroid transcription factor-1 (TTF-1), paired-box gene 8 (PAX8), β-catenin, and E-cadherin were used. Results: All WDTCs tested positive for TTF-1, PAX8, and E-cadherin. The positive rates of TTF-1, PAX8, and E-cadherin were 93.3, 93.3, and 100%, respectively, in PDTCs and 17.2, 51.7, and 10.3%, respectively, in ATCs. WDTC expressed the lateral cell membrane staining for β-catenin and E-cadherin, whereas PDTC showed circumferential cell membranous expression (fishnet pattern). β-catenin cell membrane expression in ATCs is lost or discontinuous. Carcinoma cells with β-catenin nuclear expression without cell membranous expression were scattered in 72.4% of ATCs but were not observed in the other carcinomas. Conclusion: We propose 3 immunohistochemical findings to determine undifferentiated carcinoma cells in the diagnosis of ATC: (1) β-catenin nuclear expression with no or reduced cell membranous expression, (2) the loss or discontinuous pattern of E-cadherin expression, and (3) the loss of PAX8 nuclear expression.

Functional Parameters of Prestin Are Not Correlated With the Best Hearing Frequency.

Among the vertebrate lineages with different hearing frequency ranges, humans lie between the low-frequency hearing (1 kHz) of fish and amphibians and the high-frequency hearing (100 kHz) of bats and dolphins. Little is known about the mechanism underlying such a striking difference in the limits of hearing frequency. Prestin, responsible for cochlear amplification and frequency selectivity in mammals, seems to be the only candidate to date. Mammalian prestin is densely expressed in the lateral plasma membrane of the outer hair cells (OHCs) and functions as a voltage-dependent motor protein. To explore the molecular basis for the contribution of prestin in hearing frequency detection, we collected audiogram data from humans, dogs, gerbils, bats, and dolphins because their average hearing frequency rises in steps. We generated stable cell lines transfected with human, dog, gerbil, bat, and dolphin prestins (hPres, dPres, gPres, bPres, and nPres, respectively). The non-linear capacitance (NLC) of different prestins was measured using a whole-cell patch clamp. We found that the Qmax/Clin of bPres and nPres was significantly higher than that of humans. The V1/2 of hPres was more hyperpolarized than that of nPres. The z values of hPres and bPres were higher than that of nPres. We further analyzed the relationship between the high-frequency hearing limit (Fmax) and the functional parameters (V1/2, z, and Qmax/Clin) of NLC among five prestins. Interestingly, no significant correlation was found between the functional parameters and Fmax. Additionally, a comparative study showed that the amino acid sequences and tertiary structures of five prestins were quite similar. There might be a common fundamental mechanism driving the function of prestins. These findings call for a reconsideration of the leading role of prestin in hearing frequency perception. Other intriguing kinetics underlying the hearing frequency response of auditory organs might exist.

Ultrastructure of Midgut of Scrabeus sacer (Coleoptera:Scrabaeidae)

The midgut of the adult Scrabeus sacer was divided into three parts, anterior, middle and posterior midgut. In the anterior part of the midgut, the apical part showed microvilli long and slender. These microvilli appear open in other parts. Numerous organelles appeared, well-developed nucleus mitochondria, rough endoplasmic reticulum. A thin basement membrane was observed and well-developed basal labyrinth. Regenerative cells were observed and well-developed circular muscle fibers. Tracheae and mitochondria were also observed. In the median portion of the midgut, well-developed long and slender microvilli were observed. Polymorphic mitochondria, well-developed nucleus, rough endoplasmic reticulum and other organelles were coming out of the cell to the lumen through the apical part of the middle midgut. A basement membrane, well-developed basal labyrinth, circular muscles, tracheae and vacuoles were observed in the basal part of the median midgut. The rear part of the midgut showed a well-developed brush border of microvilli. Polymorphic mitochondria, rods of bacteria, nucleus and other organelles are projecting in the lumen of the cell. Lateral cell membranes and numerous crescent shape mitochondria were observed in the posterior midgut. Gap junctions were observed in the posterior midgut. A well-developed basal labyrinth was observed enclosing mitochondria and tracheae. Regenerative cells are obvious in the basal part of the posterior region. The rough endoplasmic reticulum is not restricted to a defined part of the cell. No peritrophic membrane was observed throughout the midgut of the adult beetle Scrabeus sacer. Multivesicular bodies were observed in different parts of the midgut.

Feline lymphoplasmacytic rhinitis (FLPCR): Severity of inflammation correlates with reduced mucosal IgA expression

Feline lymphoplasmacytic rhinitis (FLPCR) is a rare disease with an unclear pathogenesis characterized by lymphoplasmacytic (LPC) inflammation and progressive tissue destruction. Aims were to evaluate specific FLPCR clinical and pathological features to gain insights into disease pathogenesis. Signalment, clinical signs, serology and 47 pin. h biopsies were retrospectively collected from 33 FLPCR and 3 normal cats. Microscopical lesions and immunohistochemistry results utilizing anti-CD3, anti-CD20, anti-FOXP3, anti-feline-IgA, IgG, IgE and anti-FeLV (p27 and gp70), FIV, FCV and, FHV were scored and most were analyzed statistically. The majority of cats were domestic short haired (26/31) with median age of 11 years and a 0.35 F/M ratio. Serology evidenced 3/22 FIV and 1/22 FeLV positive cats. Immunohistochemistry evidenced 1/33 FeLV-p27 positive cats. Common clinical signs were sneezing (19/24 [79 %]), mucous discharge (13/24 [54 %]) and stertor (10/24 [42 %]). In normal tissues, IgAs were expressed in mucin, apical and lateral cell membrane of columnar cells and in periglandular plasma cells. IgGs were expressed in 20–30 % of columnar cells. Number of clinical signs was statistically significantly higher in female cats (p < 0.0001) and was significantly correlated with chronicity (p = 0.004), and IgG scores (p = 0.01). LPC severity scores correlated positively with infiltration of neutrophils (p = 0.015), gland destruction (p = 0.019) and angiogenesis (p = 0.016) and negatively with fibrosis (p < 0.0001). LPC severity scores were also significantly associated to female sex (p = 0.01) and to IgA (p = 0.03), with higher IgA scores associated to lower LPC scores.FLPCR associated to disruption of mucosal defense mechanisms generating cycles of tissue inflammation, tissue damage and repair with progressive loss of function independent from viral infections.

Rab13 and Desmosome Redistribution in Uterine Epithelial Cells During Early Pregnancy.

The luminal uterine epithelial cells are the first point of contact with the implanting blastocyst. Dramatic changes occur in the structure and function of these cells at the time of receptivity including changes in the lateral junctional complex. While these morphological changes are important for uterine receptivity, currently there is no known mechanism of regulation of the lateral junctional complexes. Rab13, a member of the Rab (Ras-related in the brain) family of GTPases has a critical role in endosomal trafficking to the lateral plasma membrane and is involved in modulation of the tight junction in several cell types. The aim of this study is to investigate the role of Rab13 in changes to the lateral junctional complex at the time of receptivity. Immunofluorescence microscopy demonstrated no association between Rab13 and ZO-1 (a tight junction protein) or Rab13 and E-cadherin (an integral component of adherens junctions). Co-localisation was demonstrated between Rab 13 and desmoglein-2 at the time of fertilization and also at receptivity suggesting involvement of Rab13 in relocalisation of desmoglein-2 and formation of giant desmosomes in the apical part of the lateral plasma membrane at the time of uterine receptivity. We suggest that despite the loss of the adherens junction at the time of receptivity, the presently reported redistribution of desmosomes regulated by Rab13 allows the uterine epithelium to maintain structural integrity.

Proper E-cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer.

We reported that Disabled-2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E-cadherin. Dab2 depletion in Caco-2 cells led to E-cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium-induced colitis increased Dab2/E-cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E-cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E-cadherin delocalization. Epithelial Dab2 decrement preceded that of E-cadherin. We suggest that Dab2, by inhibiting E-cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.

An In Vitro Study on Prestin Analog Gene in the Bullfrog Hearing Organs.

The prestin-based active process in the mammalian outer hair cells (OHCs) is believed to play a crucial role in auditory signal amplification in the cochlea. Prestin belongs to an anion transporter family (SLC26A). It is densely expressed in the OHC lateral plasma membrane and functions as a voltage-dependent motor protein. Analog genes can be found in the genome of nonmammalian species, but their functions in hearing are poorly understood. In the present study, we used the gerbil prestin sequence as a template and identified an analog gene in the bullfrog genome. We expressed the gene in a stable cell line (HEK293T) and performed patch-clamp recording. We found that these cells exhibited prominent nonlinear capacitance (NLC), a widely accepted assay for prestin functioning as a motor protein. Upon close examination, the key parameters of this NLC are comparable to that conferred by the gerbil prestin, and nontransfected cells failed to display NLC. Lastly, we performed patch-clamp recording in HCs of all three hearing organs in bullfrog. HCs in both the sacculus and the amphibian papilla exhibited a capacitance profile that is similar to NLC while HCs in the basilar papilla showed no sign of NLC. Whether or not this NLC-like capacitance change is involved in auditory signal amplification certainly requires further examination; our results represent the first and necessary step in revealing possible roles of prestin in the active hearing processes found in many nonmammalian species.

Expression and Immunolocalization of Xenobiotic Transporters at the Human Blood‐Testis Barrier

The blood‐testis barrier (BTB) is formed by basal tight junctions between adjacent Sertoli cells of the seminiferous tubules and functions as a physical barrier to protect developing germ cells in the adluminal compartment from reproductive toxicants. Multiple xenobiotics are known to cross into the male genital tract (MGT) including male contraceptives, cancer chemotherapeutics, and environmental toxicants, although the mechanism(s) for their movement into the MGT is(are) largely unknown. Previous studies showed that several ATP‐binding cassette (ABC) and solute carrier (SLC) superfamily transporters are expressed in the testes and could potentially facilitate the disposition of xenobiotics into the MGT through concurrent uptake and efflux mechanisms. This study aimed to determine the expression and localization of xenobiotic transporters that may facilitate transepithelial transport across the human BTB. We used immunofluorescence staining to determine the cellular localization of these transporters in the several cell types found in human testes. OAT1, OAT2, OCTN1, and MRP3 were primarily localized to the basal membrane of Sertoli cells, whereas OCTN2, MRP6, and MRP7 were localized to both the basal membrane of Sertoli cells and the peritubular myoid cells that surround the basal membrane of Sertoli cells. Interestingly, CNT2 was localized to the lateral membranes of Sertoli cells and the peritubular myoid cells. OCT1, OCT2, and OCT3 were localized to the peritubular myoid cells and the Leydig cells, although there was moderate staining of spermatogenic cell membranes. However, definitive staining of CNT1, OAT3, MATE1, MATE2, OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2A1, OATP2B1, OATP3A1, OATP4A1, and OATP6A1 was not observed due to non‐specific antibody staining or an absence of staining. The localization of CNT2, OAT1, OAT2, OCT1, OCT2, OCT3, OCTN1, OCTN2, MRP3, MRP6, and MRP7 suggests these transporters may contribute to the mechanism(s) by which xenobiotics can cross the BTB. Taken together, the localization of these pharmacologically relevant transporters provides insight into the selectivity of drug disposition across the BTB and may be useful in developing tools to understand and overcome the pharmacokinetic and pharmacodynamic difficulties presented by the BTB.Support or Funding Information5R01GM123643‐045R01GM129777‐02P30CA023074

Golgi α1,2-mannosidase I induces clustering and compartmentalization of CD147 during epithelial cell migration

ABSTRACT CD147 is a widely expressed matrix metalloproteinase inducer involved in the regulation of cell migration. The high glycosylation and ability to undergo oligomerization have been linked to CD147 function, yet there is limited understanding on the molecular mechanisms behind these processes. The current study demonstrates that the expression of Golgi α1,2-mannosidase I is key to maintaining the cell surface organization of CD147 during cell migration. Using an in vitro model of stratified human corneal epithelial wound healing, we show that CD147 is clustered within lateral plasma membranes at the leading edge of adjacent migrating cells. This localization correlates with a surge in matrix metalloproteinase activity and an increase in the expression of α1,2-mannosidase subtype IC (MAN1C1). Global inhibition of α1,2-mannosidase I activity with deoxymannojirimycin markedly attenuates the glycosylation of CD147 and disrupts its surface distribution at the leading edge, concomitantly reducing the expression of matrix metalloproteinase-9. Likewise, treatment with deoxymannojirimycin or siRNA-mediated knockdown of MAN1C1 impairs the ability of the carbohydrate-binding protein galectin-3 to stimulate CD147 clustering in unwounded cells. We conclude that the mannose-trimming activity of α1,2-mannosidase I coordinates the clustering and compartmentalization of CD147 that follows an epithelial injury.

Ultrastructure of the genital papillae in the fresh-water mite Limnochares aquatica (L., 1758) (Acariformes, Limnocharidae)

The genital papillae of the fresh-water mite, Limnochares aquatica (L., 1758) were studied using TEM and SEM methods. The papillae are multiple organs spreading in large number over the ventral body surface. Each papilla is composed of the basal internal portion immersed into the body cavity and the external bowl-shaped portion placed among cuticular protuberances. A semi-spherical cap bearing minute pores covers the papillae bowl. An electron-lucent chamber is present between the cell apexes and the cuticle of the papillae cap. The latter is formed of the endo- and the exocuticle pierced by pores. The papillae are built of columnar cells with nuclear zone occupying the basal portion and the apical zone extending within the bowl and forming plications of the apical plasma membrane. Numerous electron-clear vesicles detach from both the basal and the lateral plasma membrane of papillae cells. The mitochondrial pool is quite moderate. The cells also contain numerous axial microtubules, glycogen particles, single residual bodies as well as minute apical vesicles supposedly releasing secretion into the lucent chamber. This secretion is accumulated underneath the cuticle of the cap and moving out through the pores to the outside. Following TEM organization, the papillae function in vesicular transportation of exceeding water and solutions across the organ from the organism to the external medium. The reverse active path of ions through the apical plasma membrane is also possible. In general, the papillae organization corresponds to chloride cells of aquatic insects taking part in ion–water balance but seems to be more primitive than analogous organs of other mites studied.

CagA Effector Protein in Helicobacter pylori-Infected Human Gastric Epithelium in Vivo: From Bacterial Core and Adhesion/Injection Clusters to Host Cell Proteasome-Rich Cytosol.

A key role in the carcinogenic action of Helicobacter pylori is played by the effector protein CagA, the first identified oncoprotein of the bacterial world. However, the present knowledge in regard to the bacterial injection of CagA into epithelial cells (through a type IV secretion system) and its intracellular fate is based primarily on experimental studies in vitro. Our study was aimed to investigate, in H. pylori-infected human gastric epithelium, CagA delivery and intracellular distribution in order to identify any in vivo counterpart of the cell injection mechanism described in vitro and any intracellular cytoplasmic site of preferential CagA distribution, thus shedding light on the natural history of CagA in vivo. By transmission electron microscopy and ultrastructural immunocytochemistry (which combine precise molecule localization with detailed analysis of bacterial-host cell interaction and epithelial cell ultrastructure), we investigated endoscopic biopsies of gastric antrum from H. pylori-infected dyspeptic patients. Our findings provide support for CagA direct injection into gastric epithelial cells at bacterial adhesion sites located on the lateral plasma membrane and for its cytosolic intracellular distribution with selective concentration inside peculiar proteasome-rich areas, which might be site not only of CagA degradation but also of CagA-promoted crucial events in gastric carcinogenesis.

Syncytiotrophoblast of Placentae from Women with Zika Virus Infection Has Altered Tight Junction Protein Expression and Increased Paracellular Permeability.

The cytotrophoblast of human placenta transitions into an outer multinucleated syncytiotrophoblast (STB) layer that covers chorionic villi which are in contact with maternal blood in the intervillous space. During pregnancy, the Zika virus (ZIKV) poses a serious prenatal threat. STB cells are resistant to ZIKV infections, yet placental cells within the mesenchyme of chorionic villi are targets of ZIKV infection. We seek to determine whether ZIKV can open the paracellular pathway of STB cells. This route is regulated by tight junctions (TJs) which are present in the uppermost portion of the lateral membranes of STB cells. We analyzed the paracellular permeability and expression of E-cadherin, occludin, JAMs –B and –C, claudins -1, -3, -4, -5 and -7, and ZO-1, and ZO-2 in the STB of placentae from ZIKV-infected and non-infected women. In ZIKV-infected placentae, the pattern of expression of TJ proteins was preserved, but the amount of claudin-4 diminished. Placentae from ZIKV-infected women were permeable to ruthenium red, and had chorionic villi with a higher mean diameter and Hofbauer hyperplasia. Finally, ZIKV added to the basolateral surface of a trophoblast cell line reduced the transepithelial electrical resistance. These results suggest that ZIKV can open the paracellular pathway of STB cells.

Fungal plasma membrane domains.

The plasma membrane (PM) performs a plethora of physiological processes, the coordination of which requires spatial and temporal organization into specialized domains of different sizes, stability, protein/lipid composition and overall architecture. Compartmentalization of the PM has been particularly well studied in the yeast Saccharomyces cerevisiae, where five non-overlapping domains have been described: The Membrane Compartments containing the arginine permease Can1 (MCC), the H+-ATPase Pma1 (MCP), the TORC2 kinase (MCT), the sterol transporters Ltc3/4 (MCL), and the cell wall stress mechanosensor Wsc1 (MCW). Additional cortical foci at the fungal PM are the sites where clathrin-dependent endocytosis occurs, the sites where the external pH sensing complex PAL/Rim localizes, and sterol-rich domains found in apically grown regions of fungal membranes. In this review, we summarize knowledge from several fungal species regarding the organization of the lateral PM segregation. We discuss the mechanisms of formation of these domains, and the mechanisms of partitioning of proteins there. Finally, we discuss the physiological roles of the best-known membrane compartments, including the regulation of membrane and cell wall homeostasis, apical growth of fungal cells and the newly emerging role of MCCs as starvation-protective membrane domains.

Modification of genital kidney nephrons for sperm transport in a plethodontid salamander, Eurycea longicauda.

Salamanders possess kidneys with two distinct regions: a caudal pelvic portion and cranial genital portion. Nephrons of the pelvic region are responsible for urine formation and transport. Nephrons of the genital region transport sperm from testes to Wolffian ducts; however, nephrons of the genital region possess all the same functional regions found in pelvic kidney nephrons that are involved with urine formation and transport (renal corpuscles, proximal tubules, distal tubules, and collecting ducts). Morphological similarities between pelvic and genital regions stimulated past researchers to hypothesize that nephrons of genital kidneys possess dual function; that is, sperm transport and urine formation/transport. Considering size of glomeruli is directly related to the total amount of blood plasma filtered into the Bowman's space, we tested the hypothesis that nephrons of genital kidneys have reduced urine formation function by comparing glomerular size between nephrons of pelvic and genital kidney regions in Eurycea longicauda with general histological techniques. Light microscopy analysis revealed that glomeruli of pelvic kidneys were significantly larger than those measured from genital kidneys. Transmission electron microscopy analysis also revealed modifications in genital kidney nephrons when compared to pelvic kidney nephrons that suggested a decrease in urine formation function in genital kidneys. Such modifications included a decrease in basal and lateral plasma membrane folding in genital kidney proximal and distal tubules compared to that of pelvic kidney proximal and distal tubules. Genital kidney proximal tubules were also ciliated, which was not observed in pelvic kidney proximal tubules. In conclusion, although structurally similar at the histological level, it appears that nephrons of genital kidneys have decreased urine formation function based on glomerular size comparison and nephron ultrastructure.

Ultrastructure of the rectum of the soil-spraying larva in Bittacus cirratus (Mecoptera: Bittacidae).

The larvae of Bittacidae have an interesting behavior of spraying soil particles on their body surface through the anus. However, the hindgut specialization associated with this behavior has rarelybeen studied hitherto. Here, we investigated the fine structure of the larval rectum in the hangingfly Bittacus cirratus Tjeder using light and transmission electron microscopy. The results show that the larvae of B. cirratus have a tubular rectum without rectal pads or papillae. The rectum consists of well-developed visceral muscle layers, a non-cellular basal lamina, a single-layer epithelium with a cuticular intima, and a central lumen. The folded rectal epithelium consists of two types of flattened epithelial cells: electron-dense type I cells and electron-lucent type II cells. The apical and basal plasma membranes are infolded and are associated with mitochondria in the epithelial cells. The epithelial cells are held by septate and scalariform junctions. The lateral cell membranes are combined with mitochondria among type I cells and generate mitochondria-scalariform junction complexes. These features suggest that the epithelial cells are active in water and ion reabsorption. We conclude that the absence of rectal pads or papillae and the presence of developed circular muscles are likely morphological adaptations of these larvae to the soil-spraying behavior.

During Ca2+‐dependent gastric epithelial repair, Ca2+ is sourced from both Ca2+ uptake and intracellular Ca2+ release

BackgroundCalcium (Ca2+) is a known accelerator for gastric repair. However the mechanism by which Ca2+ mobilizes to promote repair remains unclear, and cannot be readily evaluated in vivo. Using gastric organoids derived from transgenic mice expressing a fluorescent Ca2+ reporter (yellow cameleon‐nano15; YC‐Nano), we previously observed intracellular Ca2+ increases in cells directly adjacent to a damaged cell. Using this Ca2+ sensor, we investigate the potential sources of intracellular Ca2+ essential for repair.MethodsGastroids generated from YC‐Nano mouse stomach corpus were cultured 4–5 days prior to experiments. Photodamage and resultant cell death was induced to 1–2 gastroid epithelial cells by ~3 sec high intensity 840 nm light. YC‐Nano reports Förster resonance energy transfer (FRET) from CFP to YFP in response to increased intracellular Ca2+. Change in intracellular Ca2+ was measured as FRET/CFP ratio, in cells adjacent to damaged cells. Inhibitors were used to test roles of Ca2+ channels (10 μM verapamil, 20 μM YM58483), intracellular Ca2+ (50 μM BAPTA/AM), Phospholipase C (10 μM U73122), and IP3R (50 μM 2‐APB).ResultsUnperturbed gastroid cells maintain a constant FRET ratio, indicating stable Ca2+ levels. In response to photodamage of the gastroid epithelium, increased levels of Ca2+ were observed specifically within the lateral membranes of cells neighboring the damaged cell. Chelation of intracellular Ca2+ by BAPTA/AM resulted in significant dampening of Ca2+ response, as well as blocking prompt repair. Inhibition of L‐type channels (verapamil) or store operated Ca2+ entry (YM58483) resulted in delaying repair and dampening intracellular Ca2+ response. Furthermore, inhibition of PLC (U73122) or IP3R (2‐APB) resulted in delayed repair and dampened Ca2+ response.ConclusionThese results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ that stimulates repair. The findings implicate an intracellular Ca2+ raise mediated via Ca2+ uptake via plasma membrane Ca2+ channels and intracellular Ca2+ release from the ER. Collectively this work indicates the usefulness of YC‐Nano to further assess intracellular Ca2+ dynamics and further investigate the signaling cascade behind Ca2+‐mediated repair.Support or Funding InformationThis work was supported by the National Institutes of Health (NIH) R01DK102551 (Montrose; Aihara) and F31DK115126 (Engevik). This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Taurine Protects Against the Neurotoxic Effects of Manganese on the Dopaminergic Control of Lateral Cell Membrane Potential and Ciliary Response in Gill of Crassostrea virginica

Manganese is a neurotoxin causing manganism, a Parkinson's‐like disease involving disruption of dopaminergic neurotransmission. The mechanism by which manganese produces this dysfunction is not fully resolved. Unlike Parkinson's disease, reports postulate that manganese neurotoxicity is more likely related to downstream neuronal pathways rather than deficits in nigrostriatal function. Lack of effective treatment for manganism has been a major obstacle in its clinical management. The cilia of lateral cell in gill of Crassostrea virginica are controlled by serotonergic‐dopaminergic innervations from their ganglia. Dopamine (DA) hyperpolarizes the cell and causes cilio‐inhibition, while serotonin depolarizes and causes cilio‐excitation. Previous work of our lab showed acute and short‐term manganese treatments blocked the cilio‐inhibitory effect of DA. This neurotoxic action of manganese could be prevented by the drug p‐aminosalicylic acid or by taurine, an amino acid with efficacy in other neurodegenerative disorders. In this study we hypothesize that taurine would effectively prevent the toxic actions of manganese on both dopaminergic membrane hyperpolarization and lateral cilia inhibition. To test this we conducted acute experiments on DA dose responses (10−6 – 10−3 M) of manganese treated excised gill in the presence or absence of taurine. Ciliary activity of gill lateral cells was measured by stroboscopic microscopy and expressed as beats/min ± sem. Cell membrane potentials were simultaneously measured using the fluorescent dye DiBAC4(3). In control gill, the lateral cells responded normally to the DA dose response showing the appropriate decrease in cilia beating rates and membrane hyperpolarization. Gills treated with manganese (2 × 10−4M) had a disrupted DA response, cilio‐inhibition was impaired and the membrane did not hyperpolarize. Taurine treatment (2 × 10−4M) effectively prevented these neurotoxic effects in manganese treated gill. This physiological study in C. virginica shows that manganese disrupts the dopaminergic post‐synaptic response of lateral cells preventing membrane hyperpolarization as well disrupting cilio‐inhibition, and that taurine effectively blocked these neurotoxic actions. These findings are helpful in furthering the understanding of the mechanism of manganese neurotoxicity and provides evidence suggesting taurine be further investigated as a potential therapeutic agent for manganism.Support or Funding InformationThis work was supported in part by grant 2R25GM06003 of the Bridge Program of NIGMS, NIH grant K12GM093854‐07A1 IRACDA Program of Rutgers University and 604060048 of PSC‐CUNY.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ultrastructural study of uterine epithelium in the domestic cat during prenatal development

The study describes the sequence of ultrastructural changes of the endometrial lining epithelium, first in the primordial paramesonephric ducts, then in the differentiating uterine horns, and finally in the mature uterus. The research material comprised female fetuses of the domestic cat, aged 33–63 days post conceptionem (p.c.), and mature females. The ultrastructural observations of the developing epithelium were conducted using transmission electron microscopy (TEM) and light microscopy (LM). The results revealed two basic stages in the development of the uterine mucosal epithelium in the domestic cat: the development of the epithelium of the uterine segment of the paramesonephric ducts, lasting from day 33 to day 42 p.c., and the differentiation of this epithelium into the mucosal epithelium of the uterus, starting after day 42 p.c. and lasting until the end of the prenatal period. The epithelium of the uterine segments of the paramesonephric ducts is pseudostratified with a flat surface, all cells being characterized by the same distribution of desmosomes, ribosomes, rough endoplasmic reticulum (rER) cisternae, and mitochondria in the cytoplasm. The differentiating epithelium of the uterus after day 51 p.c. is pseudostratified with a varied height. In the epithelial cells there are increases of (i) the number and complexity of the junctional complexes, (ii) the number of invaginations of the lateral cell membranes, and (iii) the number of elongated mitochondria, and also there appear distended cisternae of the rER, lipid droplets and clusters of glycogen, which suggest increasing metabolism and secretory activity of the cells during the transformation of the epithelium.

Campylobacter jejuni enters gut epithelial cells and impairs intestinal barrier function through cleavage of occludin by serine protease HtrA

Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81–176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells.