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Microvillus inclusion disease (MVID) is a congenital diarrheal disorder resulting in life-threatening secretory diarrhea in newborns. Inactivating and nonsense mutations in myosin Vb (MYO5B) have been identified in MVID patients. Work using patient tissues, cell lines, mice, and pigs has led to critical insights into the pathology of MVID and a better understanding of both apical trafficking in intestinal enterocytes and intestinal stem cell differentiation. These studies have demonstrated that loss of MYO5B or inactivating mutations lead to loss of apical sodium and water transporters, without loss of apical CFTR, accounting for the major pathology of the disease. In addition, loss of MYO5B expression induces the formation of microvillus inclusions through apical bulk endocytosis that utilizes dynamin and PACSIN2 and recruits tight junction proteins to the sites of bulk endosome formation. Importantly, formation of microvillus inclusions is not required for the induction of diarrhea. Recent investigations have demonstrated that administration of lysophosphatidic acid (LPA) can partially reestablish apical ion transporters in enterocytes of MYO5B KO mice. In addition, further studies have shown that MYO5B loss induces an imbalance in Wnt/Notch signaling pathways that can lead to alterations in enterocyte maturation and tuft cell lineage differentiation. Inhibition of Notch signaling leads to improvements in those cell differentiation deficits. These studies demonstrate that directed strategies through LPA receptor activation and Notch inhibition can bypass the inhibitory effects of MYO5B loss. Thus, effective strategies may be successful in MVID patients and other congenital diarrhea syndromes to reestablish proper apical membrane absorption of sodium and water in enterocytes and ameliorate life-threatening congenital diarrhea.

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.

Aberrant expression of tight junction proteins has recently been focused on in the cancer research field. We previously showed that claudin-1 is aberrantly expressed from an early stage of uterine cervical adenocarcinoma and contributes to malignant potentials. To elucidate the molecular mechanisms underlying tumor-promoting roles of claudin-1, we established and analyzed claudin-1 knockout cells. Knockout of claudin-1 suppressed conventional tight junctional functions, barrier and fence functions, and expression of cell adhesion-associated proteins including E-cadherin. Comparative proteome analysis revealed that expression of claudin-1 affected expression of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling. Interactome analysis of the identified proteins revealed that E-cadherin and focal adhesion kinase play central roles in the claudin-1-dependently affected protein network. Moreover, knockout of claudin-1 significantly suppressed microvilli formation and activity of Ezrin/Radixin/Moesin. Taken together, the results indicate that expression of claudin-1 affects not only conventional tight junction function but also expression and activity of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling, to contribute to malignant potentials and microvilli formation in cervical adenocarcinoma cells.

Filopodia, microvilli and stereocilia represent an important group of plasma membrane protrusions. These specialized projections are supported by parallel bundles of actin filaments and have critical roles in sensing the external environment, increasing cell surface area, and acting as mechanosensors. While actin-associated proteins are essential for actin-filament elongation and bundling in these protrusions, myosin motors have a surprising role in the formation and extension of filopodia and stereocilia and in the organization of microvilli. Actin regulators and specific myosins collaborate in controlling the length of these structures. Myosins can transport cargoes along the length of these protrusions, and, in the case of stereocilia and microvilli, interactions with adaptors and cargoes can also serve to anchor adhesion receptors to the actin-rich core via functionally conserved motor-adaptor complexes. This review highlights recent progress in understanding the diverse roles myosins play in filopodia, microvilli and stereocilia.

Herein, we investigated the functional association of the serotonin transporter (SERT) with syntaxin-3 (STX3). We first overexpressed SERT and STX3 in various cells and examined their interaction, localization, and functional association. Immunoprecipitation studies revealed that STX3 interacted with SERT when expressed in COS-7 cells. Immunocytochemical studies revealed that SERT and STX3 were colocalized in the endoplasmic reticulum (ER) and Golgi apparatus. STX3 overexpression significantly reduced the uptake activity of SERT by attenuating its plasma membrane expression, suggesting that overexpressed STX3 anchors SERT in the ER and Golgi apparatus. STX3 knockdown did not affect the uptake activity of SERT but altered its glycosylation state. To elucidate the association of STX3 with SERT under physiological conditions, rather than overexpressing cells, we investigated this interaction in polarized Caco-2 cells, which endogenously express both proteins. Immunocytochemical studies revealed that SERT and STX3 were localized in microvilli-like structures at the apical plasma membrane. STX3 knockdown marginally but significantly decreased the serotonin uptake activity of Caco-2 cells, suggesting that STX3 positively regulates SERT function in Caco-2 cells, as opposed to SERT regulation by STX3 in overexpressing cells. Collectively, STX3 may colocalize with SERT during SERT membrane trafficking and regulate SERT function in an STX3-expressing lesion-dependent manner.

BACKGROUND: Chronic alcohol use often leads to malnutrition. However, how the intestinal absorption of nutrients such as glucose may be affected during moderate ethanol use has not been investigated. Glucose is absorbed via sodium (Na)-dependent glucose co-transport (SGLT1; SLC5A1) along the brush border membrane (BBM) of intestinal absorptive villus cells. OBJECTIVE: The aim of this study was to investigate how moderate alcohol consumption affects the absorption of glucose via SGLT1. METHODS: Intestinal epithelial cells (IEC-18; rat) were exposed to 8.64 mM ethanol over 1, 3, 6, and 12 h. Rats (16-wk-old, male, Sprague-Dawley) were administered 2 g/kg ethanol over 1, 3, and 6 h. Na-dependent (3)H-O-methyl-d-glucose uptake was measured to assess SGLT1 activity. Na-K-ATPase activity was measured as a function of inorganic phosphate release. Protein expression was analyzed by Western blot analysis and immunohistochemical staining. RESULTS: Ethanol significantly decreased Na-dependent glucose absorption in enterocytes in vitro (ethanol treatment: 48.4% of controls at 1 h; P

BACKGROUND & AIM: Myosin VB (MYO5B) is an essential trafficking protein for membrane recycling in gastrointestinal epithelial cells. The inactivating mutations of MYO5B cause the congenital diarrheal disease, microvillus inclusion disease (MVID). MYO5B deficiency in mice causes mislocalization of SGLT1 and NHE3, but retained apical function of CFTR, resulting in malabsorption and secretory diarrhea. Activation of lysophosphatidic acid (LPA) receptors can improve diarrhea, but the effect of LPA on MVID symptoms is unclear. We investigated whether LPA administration can reduce the epithelial deficits in MYO5B-knockout mice. METHODS: Studies were conducted with tamoxifen-induced, intestine-specific knockout of MYO5B (VilCre(ERT2);Myo5b(flox/flox)) and littermate controls. Mice were given LPA, an LPAR2 agonist (GRI977143), or vehicle for 4 days after a single injection of tamoxifen. Apical SGLT1 and CFTR activities were measured in Üssing chambers. Intestinal tissues were collected, and localization of membrane transporters was evaluated by immunofluorescence analysis in tissue sections and enteroids. RNA sequencing and enrichment analysis were performed with isolated jejunal epithelial cells. RESULTS: Daily administration of LPA reduced villus blunting, frequency of multivesicular bodies and levels of cathepsins in intestinal tissues of MYO5B-knockout mice compared to vehicle administration. LPA partially restored the brush border height and the localization of SGLT1 and NHE3 in small intestine of MYO5B-knockout mice and enteroids. The SGLT1-dependent short-circuit current was increased and abnormal CFTR activities were decreased in jejunum from MYO5B-knockout mice given LPA compared with vehicle. CONCLUSIONS: LPA may regulate a MYO5B-independent trafficking mechanism and brush border maturation, and therefore be developed for treatment of MVID.

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.

Background/Objectives Malignant pancreatobiliary strictures are in many cases clinically indistinguishable and present a major problem to endoscopy specialists. Intraductal sampling procedures such as brush cytology are commonly used for diagnosis with a sensitivity that is low for a diagnostic test used in daily clinical practice. MicroRNA (miR) alterations detected in many cancers are disease-specific, which can be utilized in clinical applications. The aim of the present study was to analyze whether determination of miR expression levels in intraductal brush cytology specimens is a feasible approach to improve the diagnosis of pancreatobiliary cancer. Methods Brush cytology specimens have been collected during endoscopic retrograde cholangio-pancreatography (ERCP) and analyzed by routine cytology and ancillary miR assays. Total RNA was extracted using the miRNeasy Mini Kit and the expression of miRs frequently dysregulated in pancreatobiliary cancer (miR-16, miR-21, miR-196a, miR-221) were analyzed by quantitative real-time PCR using RNU6B as internal control. Results Routine cytology resulted in no false positive diagnoses, however, the combined sensitivity remained at 53.8%. Expression (ΔCt values) of miR-16 (p = 0.0039), miR-196a (p = 0.0003) and miR-221 (p = 0.0049) showed a clear statistical significance between malignant and benign pancreatobiliary specimens (n = 35). Malignancy could be detected combining routine cytology and the miR-196a single marker expression levels with a sensitivity of 84.6% (92.9% in biliary strictures) with no false positives. Conclusions The results offer the first direct demonstration that microRNAs are readily detectable in brush cytology specimens obtained during ERCP, and have the potential to help the cytological diagnosis of pancreatobiliary malignancy.

Liver dysfunction reduces systemic clearance of drugs that are primarily eliminated by the liver. However, liver dysfunction can cause a reduction in the plasma concentration profiles of certain drugs, including several tyrosine kinase inhibitors, after oral administration. The aim of the present study was to clarify the reduction in oral absorption of a tyrosine kinase inhibitor, imatinib, and the mechanisms of action involved under conditions of hepatic dysfunction, focusing on intestinal transporters. The maximum plasma concentration of imatinib after oral administration in mice subjected to bile duct ligation (BDL) was lower than that in sham-operated mice, whereas the plasma concentration profile after intravenous administration was essentially unaffected by BDL. The change in maximum plasma concentration was compatible with a reduction in small intestinal permeability of imatinib observed in the in situ closed loop. Gene expression of abcg2 was increased in BDL mice compared with that in sham-operated mice. Expression of breast cancer resistance protein and P-glycoprotein in the small intestinal brush border membrane fraction from BDL mice was also increased compared with that in sham-operated mice. In summary, the intestinal absorption and permeability of imatinib was decreased in BDL mice, and this may be attributed to the up-regulation of the efflux transporter(s).

Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) is mainly controlled with synthetic insecticides such as chlorantraniliprole. However, these compounds may affect non-target organs of insect metabolism. The objective of this study was to evaluate the toxic effect in the midgut goblet cells of A. gemmatalis caterpillars exposed to chlorantraniliprole. The midgut of these caterpillars, which ingested the insecticide in medium-lethal dose (LD50), was dissected and evaluated by transmission electron microscopy. The goblet cells microvilli, after exposure to the insecticide, were disorganized and degenerated. This can compromise ionic homeostasis and nutrient absorption, impair physiological mechanisms of detoxification, and reduce the movement of food boluses throughout the insect midgut.

Microvilli are membrane protrusions enabling the increase of the cell surface area by over hundreds fold, thereby enhancing nutrition absorption. However, the correlation between the morphology of the microvilli and absorption capability of cells remains elusive. Herein, by combining atomic force microscopy with fluorescence microscopy, we explored the effects of starvation on the morphology of microvilli in HeLa cells at the single cell level. We found that there is an increasing signal of dextran absorption after starvation, and importantly, we observed a significant increase in the number of single microvillus and the lamella-shaped microvilli on the surface of HeLa cells. In addition, we also found reversible changes in the morphology of microvilli under starvation stress and after relief from starvation. These phenomena indicate that the morphology of the microvilli plays a crucial role in absorption capacity of HeLa cells. Our finding provides direct evidence for comprehensive understanding the function of microvilli.

Human RPE cell lines, especially the ARPE-19 cell line, are widely-used in eye research, as well as general epithelial cell studies. In comparison with primary RPE cells, they offer relative convenience and consistency, but cultures derived from these lines are typically not well differentiated. We describe a simple, rapid method to establish cultures from ARPE-19 cells, with significantly improved epithelial cell morphology and cytoskeletal organization, and RPE-related functions. We identify the presence of nicotinamide, a member of the vitamin B family, as an essential factor in promoting the observed differentiation, indicating the importance of metabolism in RPE cell differentiation.

Myosins are a class of motors that participate in a wide variety of cellular functions including organelle transport, cell adhesion, endocytosis and exocytosis, movement of RNA, and cell motility. Among the emerging roles for myosins is regulation of the assembly, morphology, and function of actin protrusions such as microvilli. The intestine harbors an elaborate apical membrane composed of highly organized microvilli. Microvilli assembly and function are intricately tied to several myosins including Myosin 1a, non-muscle Myosin 2c, Myosin 5b, Myosin 6, and Myosin 7b. Here, we review the research progress made in our understanding of myosin mediated apical assembly.

Orotate, a nutritional compound typically utilized as an intermediate in pyrimidine synthesis, has been suggested to undergo renal reabsorption. However, the detailed mechanisms involved in the process remain unclear, with only urate transporter 1 (URAT1/SLC22A12) being indicated as a transporter involved in its tubular uptake. As an attempt to identify transporters involved in that to help clarify the mechanisms, we examined a possibility that organic anion transporter 10 (OAT10/SLC22A13), which is present at the brush border membrane in renal tubular epithelial cells, could transport orotate. The operation of human OAT10 for orotate transport was demonstrated indeed and analyzed in detail in Madin-Darby canine kidney II cells introduced with this transporter by stable transfection. Orotate transport by OAT10 was found to be kinetically saturable with a biphasic characteristic and dependent on Cl

Microvilli are conserved actin-based surface protrusions that have been repurposed throughout evolution to fulfill diverse cell functions. In the case of transporting epithelia, microvilli are supported by a core of actin filaments bundled in parallel by villin, fimbrin, and espin. Remarkably, microvilli biogenesis persists in mice lacking all three of these factors, suggesting the existence of unknown bundlers. We identified Mitotic Spindle Positioning (MISP) as an actin binding factor that localizes specifically to the rootlet end of the microvillus. MISP promotes rootlet elongation in cells, and purified MISP exhibits potent filament bundling activity in vitro. MISP-bundled filaments also recruit fimbrin, which further elongates and stabilizes bundles. MISP confinement to the rootlet is enforced by ezrin, which prevents decoration of the membrane-wrapped distal end of the core bundle. These discoveries reveal how epithelial cells optimize apical membrane surface area and offer insight on the remarkable robustness of microvilli biogenesis.

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.

Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. We investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Here, through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, we demonstrate that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, we show that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. Our results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, our findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). the main mechanism for cellular folate uptake at physiologic pH in mammalian cells, We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight

“Cell penetrating peptides” (CPPs) are natural or synthetic peptides with the ability to interact with cell membranes in order to enter cells and/or deliver cargo. They attract considerable interest as permeation enhancers for oral delivery of therapeutic drugs with poor bioavailability, such as proteins or DNA. A main barrier is the intestinal epithelium where passage needs to proceed through a paracellular -and/or a transcellular pathway. Using an organ cultured mucosal explant model system and a selection of fluorescent polar -and lipophilic tracers, the aim of the present study was to investigate the interaction of two CPPs, melittin and Hiv-1 Tat, with the enterocyte brush border. Melittin belongs to the amphipathic class of CPPs, and within 0.5–1 h it bound to, and penetrated, the enterocyte brush border, causing leakage into the cytosol and increased paracellular passage into the lamina propria. Surprisingly, melittin also abolished endocytosis of tracers from the brush border into early endosomes in the terminal web region (TWEEs), excluding any permeation enhancing effect via such an uptake mechanism. Electron microscopy revealed that melittin caused an elongation of the brush border microvilli and a reduction in their diameter. HIV-1 Tat is a cationic CPP that is internalized by cells due to a sequence, mainly of arginines, from residue 49 to 57, and a peptide containing this sequence permeabilized enterocytes to a polar tracer by a leakage into the cytosol. In conclusion, the CPPs studied acted by causing leakage of tracers into the enterocyte cytosol, not by inducing endocytosis.

An increasing number of studies are suggesting that plant viruses, including southern rice black-streaked dwarf virus (SRBSDV), can adversely affect biological characteristics of insect vectors by unknown mechanisms. To study the adverse effect of SRBSDV at cellular level on the insect vector, we promoted viral infection by the disruption of the small interfering RNA (siRNA) pathway. The transmission electron microscopy was utilized to describe the ultrastructural changes that occurred in insects when the core component of the siRNA pathway, Dicer-2, was knocked down. The increasing accumulation of SRBSDV in virus-infected vector, the white-backed planthoppers, caused severe cytopathology in the alimentary canal. Similar cytopathology changes in the midgut ultrastructure were characterized in the virus-infected incompetent vector, the small brown planthopper. These results not only add support to the existing evidence suggesting that the siRNA pathway has an antiviral effect, but also reveal the universal and potential ability of SRBSDV to cause damage to the insect tissues of both the vector and non-vector.

Although the general structure of the barrier between the gut and the blood is well known, many details are still missing. Here, we analyse the literature and our own data related to lipid transcytosis through adult mammalian enterocytes, and their absorption into lymph at the tissue level of the intestine. After starvation, the Golgi complex (GC) of enterocytes is in a resting state. The addition of lipids in the form of chyme leads to the initial appearance of pre-chylomicrons (ChMs) in the tubules of the smooth endoplasmic reticulum, which are attached at the basolateral plasma membrane, immediately below the 'belt' of the adhesive junctions. Then pre-ChMs move into the cisternae of the rough endoplasmic reticulum and then into the expansion of the perforated Golgi cisternae. Next, they pass through the GC, and are concentrated in the distensions of the perforated cisternae on the trans-side of the GC. The arrival of pre-ChMs at the GC leads to the transition of the GC to a state of active transport, with formation of intercisternal connections, attachment of cis-most and trans-most perforated cisternae to the medial Golgi cisternae, and disappearance of COPI vesicles. Post-Golgi carriers then deliver ChMs to the basolateral plasma membrane, fuse with it, and secret ChMs into the intercellular space between enterocytes at the level of their interdigitating contacts. Finally, ChMs are squeezed out into the interstitium through pores in the basal membrane, most likely due to the function of the actin-myosin 'cuff' around the interdigitating contacts. These pores appear to be formed by protrusions of the dendritic cells and the enterocytes per se. ChMs are absorbed from the interstitium into the lymphatic capillaries through the special oblique contacts between endothelial cells, which function as valves through the contraction-relaxation of bundles of smooth muscle cells in the interstitium. Lipid overloading of enterocytes results in accumulation of cytoplasmic lipid droplets, an increase in diameter of ChMs, inhibition of intra-Golgi transport, and fusion of ChMs in the interstitium. Here, we summarise and analyse recent findings, and discuss their functional implications.

Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda . In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The LC–MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda .

Myosin-5B is a ubiquitous molecular motor that transports cargo vesicles of the endomembrane system in intracellular recycling pathways. Myosin-5B malfunction causes the congenital enteropathy microvillus inclusion disease, underlining its importance in cellular homeostasis. Here we describe the interaction of myosin-5B with F-actin, nucleotides, and the pyrazolopyrimidine compound myoVin-1. We show that single-headed myosin-5B is an intermediate duty ratio motor with a kinetic ATPase cycle that is rate-limited by the release of phosphate. The presence of a second head generates strain and gating in the myosin-5B dimer that alters the kinetic signature by reducing the actin-activated ADP release rate to become rate-limiting. This kinetic transition into a high-duty ratio motor is a prerequisite for the proposed transport function of myosin-5B in cellular recycling pathways. Moreover, we show that the small molecule compound myoVin-1 inhibits the enzymatic and functional activity of myosin-5B in vitro. Partial inhibition of the actin-activated steady-state ATPase activity and sliding velocity suggests that caution should be used when probing the effect of myoVin-1 on myosin-5–dependent transport processes in cells.

The stomach cancer-associated protein tyrosine phosphatase 1 (SAP-1) is a receptor-type protein tyrosine phosphatase that is specifically expressed on the apical membrane of the intestinal epithelium. SAP-1 is known to maintain the balance of phosphorylation of proteins together with protein kinases; however, its biological function and impact on pharmacokinetics in the intestine remain unclear. The present study, therefore, aimed at clarifying the relationship between SAP-1 and the intestinal absorption behaviors of typical transporter substrates and macromolecules. The endogenous levels of glucose and total cholesterol in the blood were similar between wild-type and SAP-1-deficient mice (Sap1-/-), suggesting no contribution of SAP-1 to biogenic influx. Moreover, in vitro transport study with everted ileal sacs demonstrated that there was no difference in the absorption of breast cancer resistance protein, P-glycoprotein, and peptide transporter substrates between both mice. However, absorptive clearance of macromolecular model dextrans (FD-4 and FD-10) in Sap1-/- mice was significantly higher than that in wild-type mice, and this was confirmed by the trend of increased FD-4 absorption from colonic loops of Sap1-/- mice. Therefore, the results of this study suggest the partial contribution of SAP-1 to the regulated transport of hydrophilic macromolecules through paracellular tight junctions.

High levels of resistance to Bt toxin Cry2Ab have been identified to be genetically linked with loss of function mutations of an ABC transporter gene (ABCA2) in two lepidopteran insects, Helicoverpa armigera and Helicoverpa punctigera. To further confirm the causal relationship between the ABCA2 gene (HaABCA2) and Cry2Ab resistance in H. armigera, two HaABCA2 knockout strains were created from the susceptible SCD strain with the CRISPR/Cas9 genome editing system. One strain (SCD-A2KO1) is homozygous for a 2-bp deletion in exon 2 of HaABCA2 created by non-homologous end joining (NHEJ). The other strain (SCD-A2KO2) is homozygous for a 5-bp deletion in exon 18 of HaABCA2 made by homology-directed repair (HDR), which was produced to mimic the r2 resistance allele of a field-derived Cry2Ab-resistant strain from Australia. Both knockout strains obtained high levels of resistance to both Cry2Aa (>120-fold) and Cry2Ab (>100-fold) compared with the original SCD strain, but no or very limited resistance to Cry1Ac (

Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1–50 μg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5–9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins.

Little is known about the role of oxytocin (OT) in colostrum during early gut colonization. We previously showed that transient OT receptor (OTR) expression on newborn rat enterocytes coincides with the milk-suckling period, and that OT activates endoplasmic reticulum stress sensors in cultured enterocytes. Here, we explored whether colostrum-OT attenuates stress in newborn villi primed and unprimed by colostrum by measuring levels of stress markers including BiP (an ER chaperone), eIF2a (translation initiation factor), and pPKR (eIF2a kinase). We also measured two inflammation-signaling proteins NF-κB and its inhibitor IκB. To test the impact of colostrum on autophagy, we measured a marker of autophagy initiation, LC3A. Colostrum increased inactive p-eIF2a, p-PKR and IκB and reduced p-IκB, BiP and LC3A. LPS increased and OT decreased p-IkB. BiP (GRP78) was higher in unprimed than primed villi. Together, these data suggest that colostrum OT attenuates the impact of inflammation on postnatal gut villi and that OT enhances autophagy to protect against amino acid insufficiency-induced stress during the interval between birth and the first feeding.

Chitosan is a polycationic polysaccharide consisting of β-(1-4)-linked glucosamine units and due to its mucoadhesive properties, chemical derivatives of chitosan are potential candidates as enhancers for transmucosal drug delivery. Recently, glycol chitosan (GC), a soluble derivative of chitosan, was shown to bind specifically to lipid raft domains in model bilayers. The small intestinal brush border membrane has a unique lipid raft composition with high amounts of glycolipids cross-linked by lectins, and the aim of the present work therefore was to study the interaction of FITC-conjugated GC (FITC-GC) with the small intestinal epithelium. Using organ culture of pig jejunal mucosal explants as a model system, we observed widespread binding of luminal FITC-GC to the brush border. Only little uptake via constitutive endocytosis into apical early endosomes occurred, unless endocytosis was induced by the simultaneous presence of cholera toxin B subunit (CTB). Biochemically, GC bound to microvillus membrane vesicles and caused a change in the density profile of detergent resistant membranes (DRMs). Collectively, the results showed that FITC-GC binds passively to lipid raft domains in the brush border, i.e. without inducing endocytosis like CTB. Instead, and unlike CTB, FITC-GC seems to exert a stabilizing, detergent-protective effect on the lipid raft organization of the brush border.

Microvilli are actin-bundle-supported surface protrusions that play essential roles in diverse epithelial functions. To develop our understanding of microvilli biogenesis, we used live imaging to directly visualize protrusion growth at early stages of epithelial differentiation. Time-lapse data revealed that specific factors, including epidermal growth factor pathway substrate 8 (EPS8) and insulin-receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1), appear in diffraction-limited puncta at the cell surface and mark future sites of microvillus growth. New core actin bundles elongate from these puncta in parallel with the arrival of ezrin and subsequent plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Moreover, we found that nascent microvilli can also collapse, characterized first by loss of membrane wrapping and ezrin enrichment, followed by a sharp decrease in distal tip EPS8 and IRTKS levels, and ultimately disassembly of the core actin bundle itself. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight factors that participate in this process; they also provide important insights on the growth of apical specializations that will likely apply to diverse epithelial contexts.

Made available in DSpace on 2021-06-25T11:04:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) The anthranilic diamide, chlorantraniliprole is a systemic insecticide affecting ryanodine receptors. This insecticide is used to control caterpillars in soybean crops because it has low toxicity to non-target organisms. The objective was to identify side-effects of chlorantraniliprole on midgut histopathology, respiration and behavior of the velvetbean caterpillar Anticarsia gemmatalis in laboratoty. Chlorantraniliprole has LC50 = 0.61 (0.58–0.64) mg mL−1 for A. gemmatalis fourth instar caterpillars after 96 h. The insecticide causes severe histopathological effects in the midgut with epithelial disorganization, microvilli degeneration, cytoplasm vacuolization, cell fragmentation, and peritrophic matrix disorganization. The respiratory rate and the walking speed decrease, whereas the resting period increase for caterpillars exposed to this insecticide. Chlorantraniliprole is toxic to A. gemmatalis at median lethal concentrations causing severe histological and ultrastructural changes with degeneration of the midgut epithelium, reduction of respiratory rates and inducing an arresting behavioral response of this insect. Departamento de Entomologia/BIOAGRO Universidade Federal de Viçosa, Viçosa Departamento de Biologia Geral Universidade Federal de Viçosa, Viçosa Programa de Pós-Graduação Em Produção Vegetal Universidade Federal Dos Vales Jequitinhonha e Mucuri Departamento de Proteção Vegetal Universidade Estadual Paulista (UNESP), São Paulo Instituto de Ciências Agrárias Universidade Federal de Uberlândia, Monte Carmelo Departamento de Proteção Vegetal Universidade Estadual Paulista (UNESP), São Paulo

In order to know the catalytic activities of the disaccharidases expressed in the mammalian small intestinal brush-border membrane vesicles (BBMV) high concentrated solutions of sucrose, maltose, isomaltulose, trehalose and the mixture sucrose:lactose were incubated with pig small intestine disaccharidases. The hydrolysis and transglycosylation reactions generated new di- and trisaccharides, characterized and quantified by GC–MS and NMR, except for trehalose where only hydrolysis was detected. In general, α-glucosyl-glucoses and α-glucosyl-fructoses were the most abundant structures, whereas no fructosyl-fructoses or fructosyl-glucoses were found. The in-depth structural characterization of the obtained carbohydrates represents a new alternative to understand the potential catalytic activities of pig small intestinal disaccharidases. The hypothesis that the oligosaccharides synthesized by glycoside hydrolases could be also hydrolysed by the same enzymes was confirmed. This information could be extremely useful in the design of new non-digestible or partially digestible oligosaccharides with potential prebiotic properties., This work has been financed by the Spanish Ministry of Economy, Industry and Competitiveness (Project AGL2017-84614-C2-1-R). O.H-H. thanks to the Spanish Ministry of Science, Innovation and Universities for the JIN project (Project RTI2018-101273-J-I00). C.J-G. thanks the governorship of Bolívar-Colombia and CeiBA Foundation for the scholarship granted in the project “Bolívar Gana con Ciencia”.

Microvillus inclusion disease (MVID) is a life-threatening enteropathy characterised by malabsorption and incapacitating fluid loss due to chronic diarrhoea. Histological analysis has revealed that enterocytes in MVID patients exhibit reduction of microvilli, presence of microvillus inclusion bodies and intestinal villus atrophy, whereas genetic linkage analysis has identified mutations in myosin Vb gene as the main cause of MVID. In order to understand the cellular basis of MVID and the associated formation of inclusion bodies, an animal model that develops ex utero and is tractable genetically as well as by microscopy would be highly useful. Here we report that the intestine of the zebrafish goosepimples (gsp)/myosin Vb (myoVb) mutant shows severe reduction in intestinal folds - structures similar to mammalian villi. The loss of folds is further correlated with changes in the shape of enterocytes. In striking similarity with MVID patients, zebrafish gsp/myoVb mutant larvae exhibit microvillus atrophy, microvillus inclusions and accumulation of secretory material in enterocytes. We propose that the zebrafish gsp/myoVb mutant is a valuable model to study the pathophysiology of MVID. Furthermore, owing to the advantages of zebrafish in screening libraries of small molecules, the gsp mutant will be an ideal tool to identify compounds having therapeutic value against MVID., Highlights • myosin Vb is expressed in the zebrafish intestine. • goosepimples/myosin Vb function is essential for epithelial morphogenesis in the zebrafish intestine. • The goosepimples mutant recapitulates pathognomonic features of microvillus inclusion disease. • The function of myosin Vb in the intestine is conserved between fish and mammals.

Intestinal M (microfold) cells are specialized epithelial cells overlying lymphoid tissues in the small intestine. Unlike common enterocytes, M cells lack an organized apical brush border, and are able to transcytose microparticles across the mucosal barrier to underlying antigen-presenting cells. We found that in both the dextran sodium sulfate and Citrobacter rodentium models of colitis, significantly increased numbers of Peyer's patch (PP) phenotype M cells were induced at the peak of inflammation in colonic epithelium, often accompanied by loosely organized lamina propria infiltrates. PP type M cells are thought to be dependent on cytokines, including tumor necrosis factor (TNF)-α and receptor activator of nuclear factor kappa-B ligand; these cytokines were also found to be induced in the inflamed tissues. The induction of M cells was abrogated by anti-TNF-α blockade, suggesting that anti-TNF-α therapies may have similar effects in clinical settings, although the functional consequences are not clear. Our results suggest that inflammatory cytokine-induced PP type M cells may be a useful correlate of chronic intestinal inflammation.

Diabetic kidney disease (DKD) is a dominant factor standing for kidney impairments during diabetes. In this study, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to disclose the diabetes-induced structural changes in the kidney and evaluate the effects of selenium on diabetes. The increase in the area of the olefinic band indicated increased amount of lipid peroxidation end products in diabetic kidney brush border cell membrane. Moreover, saturated lipid content of this cell membrane considerably diminished. DKD was found to disrupt lipid order and cause a decrease in membrane dynamics. However, the administration of selenium at low and medium doses was shown to improve these conditions by changing the lipid contents toward control values, restoring the ordered structure of the lipids and membrane dynamics. Curve-fitting and artificial neural network (ANN) analyses of secondary structures of proteins demonstrated a relative increase in α-helix and reduction in the β-sheet during diabetes in comparison to the control group, which were ameliorated following selenium treatment at low and medium doses. These findings were further confirmed by applying hierarchical cluster analysis (HCA) and principal component analysis (PCA). A clear separation of the experimental groups was obtained with high heterogeneity in the lipid and protein regions. These chemometric analyses showed that the low and medium doses of selenium-treated diabetic groups are successfully segregated from the diabetic group and clustered closer to the control. The study suggests that medium and, more predominantly, low-dose selenium treatment can be efficient in eliminating diabetes-induced structural alterations.

In addition to its principle function as a calcium regulator, vitamin D can affect cell and tissue morphology. The intestine is an important target tissue of vitamin D, as it must ensure the efficient transport of nutrients across the epithelium while excluding the passage of harmful molecules and bacteria into the organism. These functions require a highly organized morphology, which may be modified by vitamin D deficiency. To elucidate the role of vitamin D in gut morphology and barrier function, we compared the enterocyte microstructures, gut permeability, and cytoskeletal and cell junction protein expression in vitamin D receptor (VDR) knockout (KO) and wild-type (WT) mice. We found that the duodenal epithelial cells in the VDR-KO mice had longer microvilli (+19%) than those of the WT mice (P < .05). Interestingly, microvilli elongation in the VDR-KO mice was associated with higher messenger RNA and protein expression of ezrin, which is involved in the regulation of microvillus morphogenesis. Intestinal tight junction width and permeability were assessed by measuring the fluorescein isothiocyanate dextran concentrations in plasma; the concentrations were comparable between the 2 groups of mice. We further observed a decrease in the messenger RNA and protein expression of the calcium-transporting tight junction protein claudin-2 in the VDR-KO mice compared with the WT mice (P < .05). In conclusion, the mice lacking VDR had longer enterocyte microvilli, likely as a result of increased ezrin expression. However, the morphology of the tight junctions and the intestinal permeability for large molecules were not affected.

Pinocytosis at the small intestinal brush border was studied in postweaned porcine cultured mucosal explants, using the fluorescent polar probes Alexa hydrazide (AH, MW 570), Texas red dextran (TRD, MW ~ 3000), and Cascade blue dextran (CBD, MW ~ 10,000). Within 1 h, AH appeared in a string of subapical punctae in enterocytes, indicative of an ongoing constitutive pinocytosis. By comparison, TRD was taken up less efficiently into the same compartment, and no intracellular labeling of CBD was detectable, indicating that only small molecules are pinocytosed from the postweaned gut lumen. AH remained in the terminal web region in EEA-1-positive endosomes (“TWEEs”) for at least 2 h, implying that the pinocytic uptake does not proceed towards a transcytic pathway. Like AH, cholera toxin B subunit (CTB) was readily internalized, but the two probes appeared in completely non-overlapping subapical compartments, indicating the existence of two different uptake mechanisms operating simultaneously at the brush border. CTB is internalized by clathrin-dependent receptor mediated endocytosis, but surprisingly the toxin also caused a rapid disappearance from the apical cell surface of two major brush border enzymes, alkaline phosphatase and aminopeptidase N, demonstrating the disruptive effect of this pathway. By immunofluorescence, caveolin-1 was hardly detectable in enterocytes, arguing against a caveolae-mediated uptake of AH, whereas the pinocytosis/phagocytosis inhibitors dimethyl amiloride and cytochalasin D both arrested AH uptake. We propose that the constitutive pinocytic mechanism visualized by AH contributes to maintenance of membrane homeostasis and to enrich the contents of lipid raft constituents at the brush border.

SummaryTransporting and sensory epithelial cells shape apical specializations using protocadherin-based adhesion. In the enterocyte brush border, protocadherin function requires a complex of cytoplasmic binding partners, although the composition of this complex and logic governing its assembly remain poorly understood. We found that ankyrin repeat and sterile α motif domain containing 4B (ANKS4B) localizes to the tips of adherent brush border microvilli and is essential for intermicrovillar adhesion. ANKS4B interacts with USH1C and MYO7B, which link protocadherins to the actin cytoskeleton. ANKS4B and USH1C also bind to the MYO7B cargo-binding tail at distinct sites. However, a tripartite complex only forms if ANKS4B and MYO7B are first activated by USH1C. This study uncovers an essential role for ANKS4B in brush border assembly, reveals a hierarchy in the molecular interactions that drive intermicrovillar adhesion, and informs our understanding of diseases caused by mutations in USH1C and ankyrin repeat proteins, such as Usher syndrome.

Background & Aim Myosin VB (MYO5B) is an essential trafficking protein for membrane recycling in gastrointestinal epithelial cells. The inactivating mutations of MYO5B cause the congenital diarrheal disease, microvillus inclusion disease (MVID). MYO5B deficiency in mice causes mislocalization of SGLT1 and NHE3, but retained apical function of CFTR, resulting in malabsorption and secretory diarrhea. Activation of lysophosphatidic acid (LPA) receptors can improve diarrhea, but the effect of LPA on MVID symptoms is unclear. We investigated whether LPA administration can reduce the epithelial deficits in MYO5B-knockout mice. Methods Studies were conducted with tamoxifen-induced, intestine-specific knockout of MYO5B (VilCreERT2;Myo5bflox/flox) and littermate controls. Mice were given LPA, an LPAR2 agonist (GRI977143), or vehicle for 4 days after a single injection of tamoxifen. Apical SGLT1 and CFTR activities were measured in Ussing chambers. Intestinal tissues were collected, and localization of membrane transporters was evaluated by immunofluorescence analysis in tissue sections and enteroids. RNA sequencing and enrichment analysis were performed with isolated jejunal epithelial cells. Results Daily administration of LPA reduced villus blunting, frequency of multivesicular bodies, and levels of cathepsins in intestinal tissues of MYO5B-knockout mice compared with vehicle administration. LPA partially restored the brush border height and the localization of SGLT1 and NHE3 in small intestine of MYO5B-knockout mice and enteroids. The SGLT1-dependent short-circuit current was increased and abnormal CFTR activities were decreased in jejunum from MYO5B-knockout mice given LPA compared with vehicle. Conclusions LPA may regulate a MYO5B-independent trafficking mechanism and brush border maturation, and therefore be developed for treatment of MVID.

Epithelial mesenchymal transition (EMT) in development, tissue repair and carcinogenesis involves cellular plasticity with varying degrees of epithelial and mesenchymal properties. Several recent studies have focused on EMT phenotypic dynamism; however, information on cellular interaction in the context of EMT is inadequate. In our previous study, we investigated EMT phenotypic plasticity and anticipated it as a population driven interactive process. Present study has characterized cellular connectivity as a representative of interactivity during EMT in epithelial normal and cancer cell. It has also explored dynamism of connectivity and phenotype employing Markov model. Further, plasticity was substantiated with cell surface microvilli and molecular marker. The study unveiled interplay between phenotype and connectivity too. Findings have revealed that intercellular connectivity fueled EMT plasticity and its dynamism was more prominent in cancer population. However, normal cells are more vibrant in transition and phenotypic plasticity. We have proposed connectivity plasticity as a hallmark of EMT and needs to be studied in depth. Present study also paves the way in translating in vitro EMT findings in histopathological practices.

Spodoptera frugiperda is a widely distributed agricultural pest. It has previously been established that glycoproteins in the midgut microvillar membrane of insects are targets for toxins produced by different organisms as well as plant lectins. However, there is still little information about the N-glycome of membrane-bound midgut glycoproteins in Lepidoptera and other insect groups. The present study used mass spectrometry-based approaches to characterize the N-glycoproteins present in the midgut cell microvilli of Spodoptera frugiperda. We subjected midgut cell microvilli proteins to proteolytic digestion and enriched the resulting glycopeptides prior to analysis. We also performed endoglycosidase release of N-glycans in the presence of H(2)(18)O determining the compositions of released N-glycans by MALDI-TOF MS analysis and established the occupancy of the potential N-glycosylation sites. We report here a total of 160 glycopeptides, representing 25 N-glycan compositions associated with 70 sites on 35 glycoproteins. Glycan compositions consistent with oligomannose, paucimannose and complex/hybrid N-glycans represent 35, 30 and 35% of the observed glycans, respectively. The two most common N-glycan compositions were the complex/hybrid Hex(3)HexNAc(4)dHex(4) and the paucimannose structure that contains only the doubly-fucosylated trimannosylchitobiose core Hex(3)HexNAc(2)dHex(2), each appearing in 22 occupied sites (13.8%). These findings enlighten aspects of the glycobiology of lepidopteran midgut microvilli.

Summary During development, intestinal epithelia undergo dramatic morphogenesis mediated by mesenchymal signaling to form villi, which are required for efficient nutrient absorption and host defense. Although both smooth-muscle-induced physical forces and mesenchymal cell clustering beneath emerging villi are implicated in epithelial folding, the underlying cellular mechanisms are unclear. Hedgehog (Hh) signaling can mediate both processes. We therefore analyzed its direct targetome and revealed GLI2 transcriptional activation of atypical cadherin and planar cell polarity (PCP) genes. By examining Fat4 and Dchs1 knockout mice, we demonstrate their critical roles in villus formation. Analyses of PCP-mutant mice and genetic interaction studies show that the Fat4-Dchs1 axis acts in parallel to the core-Vangl2 PCP axis to control mesenchymal cell clustering. Moreover, live light-sheet fluorescence microscopy and cultured PDGFRα+ cells reveal a requirement for PCP in their oriented cell migration guided by WNT5A. Therefore, mesenchymal PCP induced by Hh signaling drives cell clustering and subsequent epithelial remodeling.

Enzymatic transgalactosylation, in different concentrated carbohydrate solutions, was investigated using brush border membrane vesicles (BBMV) from the pig small intestine. When lactulose was incubated with BBMV, the hydrolytic activity of the enzyme towards the disaccharide was observed to be very low compared to that towards the lactose, but the linkage specificity β-(1 → 3), previously observed in lactose solutions, was not significantly affected. As in the case of lactose, lactulose transgalactosylation by BBMV synthesizes the corresponding 3′-galactosyl derivative (β-Gal-(1 → 3)-β-Gal-(1 → 4)-β-Fru). Fructose released during lactulose hydrolysis was found to be good acceptor for the transgalactosylation reaction, giving rise to the synthesis of the disaccharide β-Gal-(1 → 5)-Fru. When incubating an 80/20 mixture of lactulose/galactose, the presence of galactose did not affect the qualitative composition of the transglycosylated substrate but enhanced the synthesis of β-Gal-(1 → 5)-Fru and decreased the synthesis of β-(1 → 3) glycosidic bonds. The marked tendency for synthesizing this linkage indicates that under hydrolytic conditions, β-Gal-(1 → 3)-Gal- and β-Gal-(1 → 5)-Fru glycosidic bonds would be preferentially digested., This work has been financed by the Spanish Ministry of Economy, Industry and Competitiveness (Project AGL2017-84614-C2-1-R) and the Spanish Ministry of Science, Innovation and Universities (Project RTI2018-101273-J-I00). Julio-González thanks the governorship of Bolivar-Colombia and CeiBA Foundation for the scholarship granted in the project “Bolívar Gana con Ciencia”.

In vitro studies showed that 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5) increases P-glycoprotein (P-gp) expression and activity in Caco-2 cells, preventing xenobiotic toxicity. The present study aimed at investigating TX5 effects on P-gp expression/activity using Wistar Han rats: a) in vivo, evaluating intestinal P-gp activity; b) ex vivo, evaluating P-gp expression in ileum brush border membranes (BBM) and P-gp activity in everted intestinal sacs; c) ex vivo, evaluating P-gp activity in everted intestinal sacs of the distal and proximal ileum. TX5 (30 mg/kg, b.w.), gavage, activated P-gp in vivo, given the significant decrease in the AUC of digoxin (0.25 mg/kg, b.w.). The efflux of rhodamine 123 (300 μM), a P-gp fluorescent substrate, significantly increased in TX5-treated everted sacs from the distal portion of the rat ileum, when P-gp activity was evaluated in the presence of TX5 (20 μM), an effect abolished by the P-gp inhibitor verapamil (100 μM). No increases on P-gp expression or activity were found in TX5-treated BBM of the distal ileum and everted distal sacs, respectively, 24 h after TX5 (10 mg/kg, b.w.) administration. In vivo, no differences were found on digoxin portal concentration between control (digoxin 0.025 mg/kg, b.w., intraduodenal) and TX5-treated (digoxin+TX5 20 μM, intraduodenal) rats. The observed discrepancies in digoxin results can be related to differences in TX5 dose administered and used methodologies. Thus, the results show that TX5 activates P-gp at the distal portion of the rat ileum, and, at the higher dose tested (30 mg/kg, b.w.), seems to modulate in vivo the AUC of P-gp substrates. Fil: Rocha-Pereira, Carolina. Universidad de Porto; Portugal Fil: Ghanem, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentina Fil: Silva, Renata. Universidad de Porto; Portugal Fil: Casanova, Alfredo G.. Universidad de Salamanca; España Fil: Duarte Araújo, Margarida. Universidad de Porto; Portugal Fil: Gonçalves Monteiro, Salomé. Universidad de Porto; Portugal Fil: Sousa, Emília. Universidad de Porto; Portugal Fil: Bastos, Maria de Lourdes. Universidad de Porto; Portugal Fil: Remião, Fernando. Universidad de Porto; Portugal

Transporting epithelial cells generate arrays of microvilli, known as a brush border, to enhance functional capacity. To understand brush border formation, we used live cell imaging to visualize apical remodeling early in this process. Strikingly, we found individual microvilli exhibit persistent active motility, translocating across the cell surface at ~0.2 μm/min. Perturbation with inhibitors and photokinetic experiments revealed microvillar motility is driven by actin assembly at the barbed-ends of core bundles, which in turn is linked to robust treadmilling of these structures. Actin regulatory factors IRTKS and EPS8 localize to the barbed-ends of motile microvilli where they control the kinetics and nature of movement. As the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately clustering and consolidation into higher order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.

In this study, we report that two types of embryos, normal and pseudogerm, are generated from a single egg of the polyembryonic larval endoparasitoid Macrocentrus cingulum (Braconidae). M. cingulum larvae develop in the host hemocoel, emerging from the host to pupate. After egg cleavage and embryo proliferation dozens of normal embryos and thousands of pseudogerms are generated in the host larva. The difference between normal embryos and pseudogerms is that the former develop into larvae while the latter do not. The primordium that develops in normal embryos is surrounded by an extraembryonic membrane that originates from the syncytium. Pseudogerms in contrast consist only of a syncytium containing many large nuclei and are continuously generated during embryonic development. Both pseudogerms and early embryos possess dense microvilli that function to absorb nutrients from the host. After eclosion wasp larvae produced from normal embryos feed on pseudogerms. Therefore, two types of embryos originating from the same egg serve different functions. These results contribute to our understanding of the development of polyembryonic parasitoids.

Article, Previous studies have shown that overexpression of enzymatically active GFP-HAS induces the growth of long, slender protrusions that share many features of both filopodia and microvilli. These protrusions are dependent on continuing hyaluronan synthesis, and disrupt upon digestion of hyaluronan by hyaluronidase. However, complete understanding of their nature is still missing. This work shows that the protrusions on rat peritoneal surface are ultrastructurally indistinguishable from those induced by GFP-HAS3 in MCF-7 cells. Analysis of the actin-associated proteins villin, ezrin, espin, fascin, and Myo10 indicated that the HAS3-induced protrusions share most cytoskeletal features with filopodia, but they do not require adherence to the substratum like traditional filopodia. GFP-HAS3 overexpression was found to markedly enhance filamentous actin in the protrusions and their cortical basis. Analysis of the protrusion dynamics after enzymatic digestion of hyaluronan revealed that while GFP-HAS3 escape from the protrusions and the protrusion collapse takes place immediately, the complete retraction of the protrusions occurs more slowly. This finding also suggests that hyaluronan chain maintains HAS3 in the plasma membrane. The results of this work suggest that protrusions similar to those of HAS3 overexpressing cells in vitro exist also in cells with active hyaluronan synthesis in vivo. These protrusions are similar to common filopodia but are independent of substratum attachment due to the extracellular scaffolding by the hyaluronan coat that accounts for the growth and maintenance of these structures, previously associated to invasion, adhesion and multidrug resistance., final draft, http://purl.org/eprint/status/PeerReviewed

Located at the anterior end of the turkey hen's vagina are numerous discrete tubular invaginations of the surface epithelium, collectively referred to as the sperm storage tubules (SSTs). After mating or artificial insemination, sperm ascend the vagina, enter the SSTs, and over the ensuing days and weeks, gradually exit the SSTs and are transported to the anterior end of the oviduct to fertilize a daily succession of ova. Little is known regarding the cellular and molecular mechanisms responsible for sperm subsistence in the lumen of the SST. In this study, the origin of microvillus blebs (MvBs) on the apical tips of SST epithelial cells was examined, and their possible role in sperm survival was discussed. Regardless, if sperm are present or not, transmission electron microscopy revealed two types of microvilli differentiated by the presence or absence of pleomorphic unilaminar MvBs localized to their apical tips. Although some MvBs appeared to be discharging their contents into the SST lumen, others appeared to have pinched off the microvillus stem. When SSTs contained clusters of densely packed sperm, the sperm heads of those sperm adjacent to the SST epithelial cell surface were surrounded by the microvilli. Associated with the plasmalemma of sperm throughout the SST lumina were membrane fragments and small vesicles (30-130 nm in diameter), some of which appeared to have fused with sperm. It is concluded that the MvBs are a form of shedding vesicle released from the SST epithelial cell microvilli by apocrine secretion. On the basis of observations described herein and those of other authors, it is suggested that the MvBs contribute to sustained sperm storage in the SSTs by (1) supplying metabolic substrates used by resident sperm, (2) serving as fusogenic vehicles providing exogenous macromolecules that reversibly suppress sperm functions associated with fertilization (decapacitation?) and stabilize the sperm plasmalemma, and (3) acting as transport vesicles actively transporting fluid from the SST epithelial cells to the SST lumen.

Dietary phosphorus (P) is known as a main modulator of phosphate (Pi) transporter expression. The effect of supplemented mineral P with or without phytase on protein expression of two sodium-dependent Pi (NaPi) transporters and a calcium channel was studied in the small intestine of broilers. Thirty-six broilers were randomly assigned to six different diets at 15 days of age. Two levels of total P (tP, adjusted by monocalcium phosphate (MCP) supplementation), 0.39% (BD-) and 0.47% (BD+) were fed until day 25; and at each tP level, three levels of phytase were used with 0, 500, and 12,500 FTU/kg of an E. coli phytase. Mucosa samples from jejunum and ileum were taken and apical membranes were isolated by MgCl2 precipitation. Protein expression of NaPi IIb, NaPi type III (PiT1) and the calcium channel TRPV6 were semiquantitatively measured by Western blotting and jejunal mucosal phytase activity by measurement of Pi release. The jejunal NaPi IIb transporter was expressed with two distinct bands, which were modulated differently by diet. NaPi IIb Band1 increased (P < 0.05) and Band2 decreased (P < 0.05) with phytase supplementation but was not affected by MCP supplementation. This inverse modulation of Band1 and Band2 was significantly related to the amount of net absorbed P with higher expression of Band1 at higher amounts of net absorbed P. In addition, a second Pi transporter, PiT1, was detected in which ileal expression decreased (P < 0.05) in response to higher phytase supplementation. The expression of the calcium channel TRPV6 was increased in BD+ groups. A trend for an interaction between MCP and phytase supplementation on mucosal phytase activity was observed (P = 0.079) with a decrease in activity when BD+ with 12,500 FTU/kg phytase was fed. Chicken intestinal epithelial cells responded to dietary supplemented phytase and MCP by changing the Pi transporter expression in apical membranes. In conclusion, availability of Pi is most likely the key modulator of transporter protein expression. However, a contribution of lower inositol phosphates generated by phytases and other phosphatases may also be relevant.

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.