Microvillus Membrane Research Articles

Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model.

Many years ago, asbestos fibers were banned and replaced by synthetic vitreous fibers because of their carcinogenicity. However, the toxicity of the latter fibers is still under debate, especially when it concerns the early fiber interactions with biological cell membranes. Here, we aimed to investigate the effects of a synthetic vitreous fiber named FAV173 on the Xenopus laevis oocyte membrane, the cell model we have already used to characterize the effect of crocidolite asbestos fiber exposure. Using an electrophysiological approach, we found that, similarly to crocidolite asbestos, FAV173 was able to stimulate a chloride outward current evoked by step membrane depolarizations, that was blocked by the potent and specific TMEM16A channel antagonist Ani9. Exposure to FAV173 fibers also altered the oocyte cell membrane microvilli morphology similarly to crocidolite fibers, most likely as a consequence of the TMEM16A protein interaction with actin. However, FAV173 only partially mimicked the crocidolite fibers effects, even at higher fiber suspension concentrations. As expected, the crocidolite fibers' effect was more similar to that induced by the co-treatment with (Fe3+ + H2O2), since the iron content of asbestos fibers is known to trigger reactive oxygen species (ROS) production. Taken together, our findings suggest that FAV173 may be less harmful that crocidolite but not ineffective in altering cell membrane properties.

Mechanical force activates the light-dependent channels TRP and TRPL in excised patches from the rhabdomere of Drosophila photoreceptors

Drosophila phototransduction in light-sensitive microvilli involves a metabotropic signaling cascade. Photoisomerized rhodopsin couples to G-protein, activating phospholipase C, which cleaves phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate, diacylglycerol (DAG) and a proton. DAG is converted into phosphatidic acid by DAG-kinase and metabolized to L-linoleoyl glycerol (2-LG) by DAG-lipase. This complex enzyme cascade ultimately opens the light-dependent transient receptor potential channels, TRP and TRPL. PIP2, DAG, H+ and 2-LG are possible channel activators, either individually or combined, but their direct participation in channel-gating remains unresolved. Molecular interaction with the channels, modification of the channels’ lipid moiety and mechanical force on the channels by changes in the membrane structure derived from light-dependent changes in lipid composition are possible gating agents. In this regard, mechanical activation was suggested, based on a rapid light-dependent contraction of the photoreceptors mediated by the phototransduction cascade. Here, we further examined this possibility by applying force to inside-out patches from the microvilli membrane by changing the pressure in the pipette or pulling the membrane with a magnet through superparamagnetic nanospheres. The channels were opened by mechanical force, while mutant lacking both channels was insensitive to mechanical stimulation. Atomic Force Microscopy showed that the stiffness of an artificial phospholipid bilayer was increased by arachidonic acid and diacylglycerol whereas elaidic acid was ineffective, mirroring their relative effects in channel activity previously observed electrophysiologically. Together, the results are consistent with the notion that light-induced changes in lipid composition alter the membrane structure, generating mechanical force on the channels leading to channel opening.

Composition and abundance of midgut plasma membrane proteins in two major hemipteran vectors of plant viruses, Bemisia tabaci and Myzus persicae.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut.

Juno and CD9 protein network organization in oolemma of mouse oocyte.

Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson's correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Hyperstabilization of T cell microvilli contacts by chimeric antigen receptors.

T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

Discrete localization of phospholipase Cβ3 and diacylglycerol kinase ι along the renal proximal tubules of normal rat kidney and gentamicin-induced changes in their expression.

Many signaling enzymes have multiple isozymes that are localized discretely at varying molecular levels in different compartments of cells where they play specific roles. In this study, among the various isozymes of phospholipase C (PLC) and diacylglycerol kinase (DGK), which work sequentially in the phosphoinositide cycle, both PLCβ3 and DGKι were found in renal brush-border microvilli, but found to replace each other along the proximal tubules: PLCβ3 in the proximal straight tubules (PST) of the outer stripe of the outer medulla (OSOM) and the medullary ray (MR), and DGKι in the proximal convoluted tubules (PCT) in the cortex and partially in the PST of the MR. Following daily injection of gentamicin for 1 week, the expression of PLCβ3 and DGKι was transiently enhanced, as demonstrated by western blot, and the increases were found to most likely occur in their original sites, that is, in the brush borders of the PST for PLCβ3 and in the PCT for DGKι. These findings showing differences in expression along the tubules suggest that the exertion of reabsorption and secretion through various ion channels and transporters in the microvillus membranes and the maintenance of microvillus turnover are regulated by a PLC-mediated signal with the balance shifted toward relative augmentation of the DAG function in the PST, and by a DGK-mediated signal with the balance shifted to relative augmentation of the phosphatidic acid function in the PCT. Our results also suggest the possibility that these isozymes are potential diagnostic signs for the early detection of acute kidney injury caused by gentamicin.

Cell polarity signaling in the regulation of syncytiotrophoblast homeostasis and inflammatory response

Maintenance of cell polarity and the structure of the apical surface of epithelial cells is a tightly regulated process necessary for tissue homeostasis. The syncytiotrophoblast of the human placenta is an entirely unique epithelial layer. It is a single giant multinucleate syncytial layer that comprises the maternal-facing surface of the human placenta. Like other epithelia, the syncytiotrophoblast is highly polarized with the apical surface dominated by microvillar membrane protrusions. Syncytiotrophoblast dysfunction is a key feature of pregnancy complications like preeclampsia. Preeclampsia is commonly associated with a heightened maternal immune response and pro-inflammatory environment. Importantly, reports have observed disruption of syncytiotrophoblast apical microvilli in placentas from preeclamptic pregnancies, indicating a loss of apical polarity, but little is known about how the syncytiotrophoblast regulates polarity.Here, we review the evolutionarily conserved mechanisms that regulate apical-basal polarization in epithelial cells, and the emerging evidence that PAR polarity complex components are critical regulators of syncytiotrophoblast homeostasis and apical membrane structure. Pro-inflammatory cytokines have been shown to disrupt the expression of polarity regulating proteins. We also discuss initial data showing that syncytiotrophoblast apical polarity can be disrupted by the addition of the pro-inflammatory cytokine tumor necrosis factor-α, revealing that physiologically relevant signals can modulate syncytiotrophoblast polarization. Since disrupted polarity is a feature of preeclampsia, further elucidation of the syncytiotrophoblast-specific polarity signaling network and testing whether the disruption of polarity-factor signaling networks may contribute to the development of preeclampsia is warranted.

Transmembrane proteins tetraspanin 4 and CD9 sense membrane curvature

Multiple membrane-shaping and remodeling processes are associated with tetraspanin proteins by yet unknown mechanisms. Tetraspanins constitute a family of proteins with four transmembrane domains present in every cell type. Prominent examples are tetraspanin4 and CD9, which are required for the fundamental cellular processes of migrasome formation and fertilization, respectively. These proteins are enriched in curved membrane structures, such as cellular retraction fibers and oocyte microvilli. The factors driving this enrichment are, however, unknown. Here, we revealed that tetraspanin4 and CD9 are curvature sensors with a preference for positive membrane curvature. To this end, we used a biomimetic system emulating membranes of cell retraction fibers and oocyte microvilli by membrane tubes pulled out of giant plasma membrane vesicles with controllable membrane tension and curvature. We developed a simple thermodynamic model for the partitioning of curvature sensors between flat and tubular membranes, which allowed us to estimate the individual intrinsic curvatures of the two proteins. Overall, our findings illuminate the process of migrasome formation and oocyte microvilli shaping and provide insight into the role of tetraspanin proteins in membrane remodeling processes.

The Sensilla-Specific Expression and Subcellular Localization of SNMP1 and SNMP2 Reveal Novel Insights into Their Roles in the Antenna of the Desert Locust Schistocerca gregaria.

Simple SummaryThe desert locust, Schistocerca gregaria, can form gigantic swarms of millions of individuals that devastate the vegetation of invaded landscapes. Locust food search, reproduction, and aggregation behaviors are triggered and controlled by complex olfactory signals. Insects detect odorants through different types of olfactory sensilla on the antenna that house olfactory sensory neurons and associated support cells, both of which express the proteins required for olfactory signaling. Among these proteins, two members of the CD36 lipid transporter/receptor family, named sensory neuron membrane proteins 1 and 2 (SNMP1 and SNMP2), are indicated to be of vital importance. Towards a better understanding of the role of the two SNMPs in the olfactory system of S. gregaria, we have analysed their antennal topography and subcellular localization using specific antibodies. The results indicate sensilla type- and cell type-specific distribution patterns of the SNMP proteins. SNMP1 was located in the receptive dendrites of subpopulations of olfactory sensory neurons as well as in the microvilli of associated support cells, suggesting a dual function of this protein, both in olfactory signal detection and in sensillum lymph maintenance, respectively. In contrast, SNMP2 was found solely in support cells and their microvilli membranes, suggesting a role limited to sensillum lymph recovery processes. Insect olfactory sensilla house olfactory sensory neurons (OSNs) and supports cells (SCs). The olfactory sensory processes require, besides the odorant receptors (ORs), insect-specific members of the CD36 family, named sensory neuron membrane proteins (SNMPs). While SNMP1 is considered to act as a coreceptor in the OR-mediated detection of pheromones, SNMP2 was found to be expressed in SCs; however, its function is unknown. For the desert locust, Schistocerca gregaria, we previously visualized mRNA for SNMP1 in OSNs and SNMP2 mRNA in cells associated with OSN clusters. Towards an understanding of their functional implication, it is imperative to explore the cellular and the subcellular localization the SNMP proteins. Therefore, we have generated polyclonal antibodies against SNMP1 and SNMP2 and used fluorescence immunohistochemistry (FIHC) to visualize the SNMP proteins. We found SNMP1 in the somata and respective dendrites of all OSNs in trichoid sensilla and in subsets of OSNs in basiconic sensilla. Notably, SNMP1 was also detected in SCs of these sensilla types. In contrast, SNMP2 protein was only visualized in SCs of basiconic and coeloconic sensilla, but not of trichoid sensilla. Exploring the subcellular localization by electron microscopy using anti-SNMP1-ab and anti-SNMP2-ab revealed an immunogold labelling of SC microvilli bordering the sensillum lymph. Together our findings suggest a dual role of SNMP1 in the antenna of S. gregaria, in some OSN subpopulations in odor detection as well as in functions of some SCs, whereas the role of SNMP2 is limited to the functions of support cells.

In Vitro and In Vivo Biocompatibility Studies on Engineered Fabric with Graphene Nanoplatelets.

To produce clothes made with engineered fabrics to monitor the physiological parameters of workers, strain sensors were produced by depositing two different types of water-based inks (P1 and P2) suitably mixed with graphene nanoplatelets (GNPs) on a fabric. We evaluated the biocompatibility of fabrics with GNPs (GNP fabric) through in vitro and in vivo assays. We investigated the effects induced on human keratinocytes by the eluates extracted from GNP fabrics by the contact of GNP fabrics with cells and by seeding keratinocytes directly onto the GNP fabrics using a cell viability test and morphological analysis. Moreover, we evaluated in vivo possible adverse effects of the GNPs using the model system Caenorhabditis elegans. Cell viability assay, morphological analysis and Caenorhabditis elegans tests performed on smart fabric treated with P2 (P2GNP fabric) did not show significant differences when compared with their respective control samples. Instead, a reduction in cell viability and changes in the membrane microvilli structure were found in cells incubated with smart fabric treated with P1. The results were helpful in determining the non-toxic properties of the P2GNP fabric. In the future, therefore, graphene-based ink integrated into elastic fabric will be developed for piezoresistive sensors.

A proteomic approach to identify digestive enzymes, their exocytic and microapocrine secretory routes and their compartmentalization in the midgut of Spodoptera frugiperda.

A proteomic approach was used to identify the digestive enzymes secreted by exocytosis and by microapocrine vesicles and enzyme midgut compartmentalization in Spodoptera frugiperda larvae. For this, proteomic analyses were performed in isolated midgut enterocyte microvillar membrane, in a fraction enriched in microapocrine vesicles (separated in soluble and membrane fractions), in the washings of the peritrophic membrane to isolate its loosely- and tightly-bound proteins, and in the peritrophic membrane contents. PM washings correspond to proteins extracted from the mucus layer surrounding PM. Serine endopeptidases (trypsins, chymotrypsins and serine endopeptidase homologs that have substitutions in the catalytic residues) and lipases are mainly secreted by exocytosis. Aminopeptidases are mainly microvillar enzymes and some are secreted membrane-bound to microapocrine vesicles, whereas carboxypeptidase isoforms follow different secretory routes. The results also showed that most polymer hydrolases (such as amylase and endopeptidases) are not retained in the ectoperitrophic fluid (found in PM washings but absent from PM contents). On the contrary, most enzymes involved in intermediate digestion (exemplified by carboxypeptidase and aminopeptidase) do not pass through the peritrophic membrane. Finally, the data revealed that the protein composition of PM includes peritrophins classified as peritrophic membrane proteins, PMP, and chitin deacetylase.

Probing sulfatide-tissue lectin recognition with functionalized glycodendrimersomes.

SummaryThe small 3-O-sulfated galactose head group of sulfatides, an abundant glycosphingolipid class, poses the (sphinx-like) riddle on involvement of glycan bridging by tissue lectins (sugar code). First, synthesis of head group derivatives for functionalization of amphiphilic dendrimers is performed. Aggregation of resulting (biomimetic) vesicles, alone or in combination with lactose, demonstrates bridging by a tissue lectin (galectin-4). Physiologically, this can stabilize glycolipid-rich microdomains (rafts) and associate sulfatide-rich regions with specific glycoproteins. Further testing documents importance of heterobivalency and linker length. Structurally, sulfatide recognition by galectin-8 is shown to involve sphingosine's OH group as substitute for the 3′-hydroxyl of glucose of lactose. These discoveries underscore functionality of this small determinant on biomembranes intracellularly and on the cell surface. Moreover, they provide a role model to examine counterreceptor capacity of more complex glycans of glycosphingolipids and to start their bottom-up glycotope surface programming.

Intracellular calcium phosphate deposits contribute to transcellular calcium transport within the hepatopancreas of Porcellio scaber

Like in most Crustacea, the cuticle of terrestrial isopods is hardened by a calcareous mineral phase. This rigid cuticle is frequently shed during a process called moulting. To reduce calcium loss, Porcellio scaber eats the shed cuticle, the exuviae, and absorb the calcium from it through large tubular diverticula of the intestine, called the mid gut glands or hepatopancreas. After moulting the absorbed calcium should be transported immediately into the hemolymph from which it is used to rapidly mineralize the new cuticle. This suggests that the hepatopancreas epithelium transports calcium from the lumen to the hemolymph. We used TEM, energy-filtered TEM and electron-probe X-ray microanalysis to analyse the distribution of elevated calcium within the hepatopancreas cells of P. scaber. We used animals in the postmoult stage that have eaten their exuviae and, as a control, those that have not ingested the exuviae. To minimize calcium loss within the samples, we used high pressure frozen and freeze substituted samples and propane-1–3-diol as floatation medium for thin-sectioning. The results reveal intracellular dense deposits containing calcium, phosphorus and oxygen at the apical microvillus membrane, within the cytoplasm, attached to vesicles and to the basolateral membrane, as well as extracellular between cells and the basal lamina. Control animals were devoid of these deposits. The results indicate that calcium from the exuviae is absorbed and transported across the epithelium into the hemolymph. We propose that during transport, intracellular calcium is bound to phosphate avoiding toxic effects of high concentrations of ionized calcium.

N-glycosylation in Spodoptera frugiperda (Lepidoptera: Noctuidae) midgut membrane-bound glycoproteins.

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 H218O 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 25N-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 Hex3HexNAc4dHex4 and the paucimannose structure that contains only the doubly-fucosylated trimannosylchitobiose core Hex3HexNAc2dHex2, each appearing in 22 occupied sites (13.8%). These findings enlighten aspects of the glycobiology of lepidopteran midgut microvilli.

Inter-individual variations and modulators of MDR1 transport activity in human placenta

IntroductionATP binding cassette (ABC) membrane transporter multidrug resistance 1 (MDR1) is one of the most important efflux transporters in the human placenta protecting the fetus from exposure to xenobiotic toxicity. Recent studies have focused on placental MDR1 expression, but few studies have analyzed placental MDR1 transport activity. The purpose of this study was to investigate placental MDR1 transport activity using a relatively large sample size of human placentas. Furthermore, the effect of ABCB1 gene polymorphisms were investigated along with physiological factors including maternal age, times of pregnancy, BMI, delivery mode or pregnancy complications on placental MDR1 transport activity. MethodsA total of 252 human placentas were obtained after delivery. MDR1 transport activity was detected by N-methyl quinidine uptake in placental microvillus membrane vesicles (MVMVs). Nine common single nucleotide polymorphisms (SNPs) in ABCB1 genes were determined by Snapshot. The association between ABCB1 gene polymorphisms, maternal age, times of pregnancy, BMI, delivery mode or pregnancy complications, and transporter activity was investigated. ResultsInter-individual variations of MDR1 transport activity were observed among 252 subjects. The per unit protein activity was ranged from 0.05 to 0.15/mg. Nine SNPs in ABCB1 gene didn't exhibit significant association with transporter activity of MDR1. Likewise, neither age, times of pregnancy, delivery mode nor pregnancy complications showed any significant effect of placental MDR1 transport activity. But placental MDR1 transport activity in obese pregnant women was lower than those in non-obese pregnant women. ConclusionInter-individual variations of MDR1 transport activity existed in human placentas. This may contribute to variations in drug exposure to the fetus affecting clinical outcomes. Maternal age, times of pregnancy, delivery mode nor pregnancy complications included in this study maybe not significantly impact placental MDR1 transport activity, but maternal obese could inhibit placental MDR1 activity.

Tetraspanin 33 (TSPAN33) regulates endocytosis and migration of human B lymphocytes by affecting the tension of the plasma membrane.

B lymphocytes are a leukocyte subset capable of developing several functions apart from differentiating into antibody-secreting cells. These processes are triggered by external activation signals that induce changes in the plasma membrane properties, regulated by the formation of different lipid-bilayer subdomains that are associated with the underlying cytoskeleton through different linker molecules, thus allowing the functional specialization of regions within the membrane. Among these, there are tetraspanin-enriched domains. Tetraspanins constitute a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity and localization. In this study, we identified TSPAN33 as an active player during B-lymphocyte cytoskeleton and plasma membrane-related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility. By using an overexpression model of TSPAN33 in human Raji cells, we detected a specific distribution of this protein that includes membrane microvilli, the Golgi apparatus, and extracellular vesicles. Additionally, we identified diminished phagocytic ability and altered cell adhesion properties due to the aberrant expression of integrins. Accordingly, these cells presented an enhanced migratory phenotype, as shown by its augmented chemotaxis and invasion rates. When we evaluated the mechanic response of cells during fibronectin-induced spreading, we found that TSPAN33 expression inhibited changes in roughness and membrane tension. Contrariwise, TSPAN33 knockdown cells displayed opposite phenotypes to those observed in the overexpression model. Altogether, our data indicate that TSPAN33 represents a regulatory element of the adhesion and migration of B lymphocytes, suggesting a novel implication of this tetraspanin in the control of the mechanical properties of their plasma membrane.

Midgut fluxes and digestive enzyme recycling in Musca domestica: A molecular approach

Insects are reported to have water midgut countercurrents fluxes powering enzyme recovery before excretion, usually known as enzyme recycling. Up to now there is a single, and very incomplete, attempt to relate transporters and channels with countercurrent fluxes. In this work, M. domestica midgut water fluxes were inferred from the concentration of ingested and non absorbable dye along the midgut, which anterior midgut was divided in two sections (A1, A2), the middle in one (M) and the posterior midgut in four (P1, P2, P3, and P4), which led to the finding of additional sites of secretion and absorption. Water is secreted in A1 and A2 and absorbed at the middle midgut (M), whereas in posterior midgut, water is absorbed at P2 and secreted in the other sections, mainly at P4. Thus, a countercurrent flux is formed from P4 to P2. To disclose the involvement of the known water transporters Na+:K+:2Cl− (NKCC) and K+:Cl− (KCC), as well as the water channels aquaporins in water fluxes, their expression was evaluated by RNA-seq analyses from triplicate samples of seven sections along the midgut. MdNKCC1 was expressed in A1, MdNKCC2 was expressed in M1 and P2 and MdKCC in middle and in the most posterior region, thus apparently involved in secretion, absorption and both, respectively. MdNKCC2, MdKCC and aquaporins MdDRIP1 and 2 were confirmed as being apical by proteomics of purified microvillar membranes. The role of NKCC and KCC on midgut water fluxes was tested observing the effect of the inhibitor furosemide. The change of trypsin distribution along the posterior midgut and the increase of trypsin excretion in the presence of furosemide lend support to the proposal that countercurrent fluxes power enzyme recycling and that the fluxes are caused by NKCC and KCC transporters helped by aquaporins.

Transport of Docosahexaenoic Acid via the Human Placenta: A Theoretical Study.

The high demand of the fetus for Docosahexaenoic acid, DHA, is satisfied by a concert of several mechanisms that take place in the placental terminal villi. Scarcity of laboratory data makes the detailed description of these mechanisms elusive. Here, the DHA transport across the placenta is modeled as a boundary value problem that accounts for diffusion, reactions with fatty acids binding proteins, FABPs, and metabolic consumption within the Syncytiotrophoblast, ST. For any given DHA fluxes at the bordering membranes, analytical and numerical solutions yield the DHA concentration profile within the ST. We find that in order to comply with adequate DHA delivery to the fetus and with physiological DHA concentrations in the maternal and fetal circulations, it is essential to have a significant rise of DHA concentration at the microvillus membrane, MVM and a rapid dissociation of the DHA from the FABP. The model establishes the relations between the concentrations of the FABPs in the ST, their equilibrium dissociation constant from the DHA, and the placental DHA metabolic degradation rate, hitherto unknown. We conclude that the bound (to the protein) DHA molecule is degraded at a rate of 0.3-0.45s-1.

Molecular mechanisms associated with acidification and alkalization along the larval midgut of Musca domestica

To disclose the molecular mechanisms involved in luminal midgut buffering of M. domestica, we used RNA-seq analyses from triplicate samples of seven sections along the midgut to evaluate the expression levels of genes coding for selected manually curated protein sequences. Channels, pumps and transporters were confirmed as being apical by proteomics of purified microvillar membranes. Midgut pH determinations with a microsensor and a pH indicator were carried out in larvae in different diets with or without added compounds to evaluate the role of proteins in buffering. The data suggested that acidification occurs at middle midgut by the action of H+ V-ATPase with protons produced by carbonic anhydrase, followed by chloride ions transported by a K+Cl− symporter. K+ ions are recovered by an apical K+ channel and K+ homeostasis maintained by a basolateral Na+/K+-ATPase. Acidification is also affected by a Na+/H+ exchanger and a multidrug resistance protein. Posterior midgut alkalization results from the action of a NH3 transporter and H+-coupled peptide transporter, mainly in a diet rich in free peptides. A working model was proposed for the midgut luminal acidification and alkalization, as well as for mucosal protection against acid by a neutralized mucus layer.