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Journal of Visualized Experiments : JoVE May 2021Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form...
Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.
Topics: Actin Cytoskeleton; Cell Membrane; Cell Surface Extensions; Microscopy, Electron, Scanning; Pinocytosis
PubMed: 34125102
DOI: 10.3791/62658 -
Anatomical Record (Hoboken, N.J. : 2007) Aug 2021The blood circulates through the hepatic sinusoids delivering nutrients and oxygen to the liver parenchyma and drains into the hepatic central vein, yet the structures... (Review)
Review
The blood circulates through the hepatic sinusoids delivering nutrients and oxygen to the liver parenchyma and drains into the hepatic central vein, yet the structures and phenotypes of these vessels are distinctively different. Sinusoidal endothelial cells are uniquely fenestrated, lack basal lamina and possess organelles involved in endocytosis, pinocytosis, degradation, synthesis and secretion. Hepatic central veins are nonfenestrated but are also active in synthesis and secretion. Endothelial cells of sinusoids and central veins secrete angiocrines that play respective roles in hepatic regeneration and metabolic homeostasis. The list of markers for identifying sinusoidal endothelial cells is long and their terminologies are complex. Further, their uses vary in different investigations and, in some instances, could be confusing. Central vein markers are fewer but more distinctive. Here we analyze and categorize the molecular pathways/modules associated with the sinusoid-mediated liver regeneration in response to partial hepatectomy and chemical-induced acute or chronic injury. Similarly, we highlight the findings that central vein-derived angiocrines interact with Wnt/β-catenin in perivenous hepatocytes to direct gene expression and maintain pericentral metabolic zonation. The proposal that perivenous hepatocytes behave as stem/progenitor cells to provoke hepatic homeostatic cell renewal is reevaluated and newer concepts of broad zonal distribution of hepatocyte proliferation in liver homeostasis and regeneration are updated. Thus, this review integrates the structures, biology and physiology of liver sinusoids and central veins in mediating hepatic regeneration and metabolic homeostasis.
Topics: Biomarkers; Capillaries; Endothelial Cells; Hepatocytes; Homeostasis; Humans; Liver; Liver Regeneration
PubMed: 33135318
DOI: 10.1002/ar.24560 -
Physiological Reports Feb 2021Piglets must acquire passive immunity through colostrum within hours after birth to survive. How colostral macromolecules traverse the small intestinal epithelium may...
Piglets must acquire passive immunity through colostrum within hours after birth to survive. How colostral macromolecules traverse the small intestinal epithelium may include nonselective pinocytosis and paracellular transport through tight junction proteins located between epithelial cells. Claudin proteins-3 and -4 contribute to the epithelial tight junctions (TJs) on the apical aspect of lateral surfaces of intestinal epithelial cells (IECs) where they help regulate ion and macromolecule movement across the intestinal epithelium. Throughout the small intestine of newborn piglets, Claudin-3 was localized to the lateral and basolateral surface of intestinal epithelial cells as well as the membrane of large vacuoles. In the duodenum and jejunum, Claudin-4 was localized to the apical surface independent of tight junction regions. In the ileum, Claudin-4 was localized to the lateral and basolateral surfaces indicating region-specific differences and noncanonical patterns of Claudin-4 localization independent of tight junction regions. Understanding the timing of changes in surface localization of Claudin-3 and Claudin-4 and how they may coincide with changes in small intestinal permeability may help develop new protective strategies against infectious diseases within newborn piglets.
Topics: Animals; Animals, Newborn; Biological Transport; Claudin-3; Claudin-4; Colostrum; Immunity; Intestinal Mucosa; Intestine, Small; Permeability; Sus scrofa; Tight Junctions
PubMed: 33523589
DOI: 10.14814/phy2.14717 -
Frontiers in Immunology 2019There are several lines of evidence indicating that the physiopathological bases of bullous pemphigoid (BP), the most common subepidermal autoimmune bullous disease, are... (Review)
Review
There are several lines of evidence indicating that the physiopathological bases of bullous pemphigoid (BP), the most common subepidermal autoimmune bullous disease, are hallmarked by the production of autoantibodies directed against the hemidesmosomal anchoring proteins BP180 and BP230. In contrast to the robustness of the latter assumption, the multifaceted complexity of upstream and downstream mechanisms implied in the pathogenesis of BP remains an area of intense speculation. So far, an imbalance between T regulatory cells and autoreactive T helper (Th) cells has been regarded as the main pathogenic factor triggering the autoimmune response in BP patients. However, the contributory role of signaling pathways fostering the B cell stimulation, such as Toll-like receptor activation, as well as that of ancillary inflammatory mechanisms responsible for blister formation, such as Th17 axis stimulation and the activation of the coagulation cascade, are still a matter of debate. In the same way, the pathomechanisms implied in the loss of dermal-epidermal adhesion secondary to autoantibodies binding are not fully understood. Herein, we review in detail the current concepts and controversies on the complex pathogenesis of BP, shedding light on the most recent theories emerging from the literature.
Topics: Autoantibodies; Autoantigens; Autoimmunity; Blood Coagulation; Comorbidity; Complement Activation; Dystonin; Humans; Leukocytes; Neurodegenerative Diseases; Non-Fibrillar Collagens; Pemphigoid, Bullous; Pinocytosis; Thrombosis; Vitamin D Deficiency; Collagen Type XVII
PubMed: 31312206
DOI: 10.3389/fimmu.2019.01506 -
Journal of Nanobiotechnology Aug 2021Based on the concept of "multimodal analgesia", a novel dual drug delivery system was designed to achieve synergistic analgesia between najanajaatra venom protein (αCT)...
BACKGROUND
Based on the concept of "multimodal analgesia", a novel dual drug delivery system was designed to achieve synergistic analgesia between najanajaatra venom protein (αCT) and resveratrol (Res). In order to meet the joint loading of two drugs with different physicochemical properties without affecting each other, an oral Janus nanoparticle (JNP) with a unique cavity structure and synergistic drug delivery was constructed using an improved double emulsion solvent evaporation method, and combined with low-molecular-weight chitosan/sodium alginate and PLGA to achieve its pH-responsive.
RESULTS
The synthesized αCT/Res-JNPs are homogeneous in shape, with a two-compartment structure, approximately 230 nm in size, and zeta potential of 23.6 mV. Drug release assayed in vitro show that JNP was stable in simulated gastric juice (pH = 1.2) but was released in phosphate buffer saline (pH = 7.4). After intragastric administration in rats, PK evaluation showed that αCT/Res-JNPs could significantly improve the oral bioavailability, and the simultaneous encapsulation of the two drugs had no significant interaction on PK parameters. An obvious synergistic analgesic effects of αCT/Res-JNPs was confirmed in a spinal cord injury and acute pain model. Confocal laser scanning microscopy and single-pass intestinal perfusion model provided strong evidence that αCT/Res-JNPs could pass through intestinal epithelial cells, and the endocytosis pathway was mainly involved in the mediation and pinocytosis of reticulin. The concentrations of αCT and Res from αCT/Res-JNP in lymphatic transport were only about 8.72% and 6.08% of their blood concentrations at 1 h, respectively, which indicated that lymphatic transport in the form of JNP has limited advantages in improving the oral bioavailability of Res and αCT. Cellular uptake efficiency at 4 h was about 10-15% in Caco-2 cell lines for αCT/Res-JNP, but was reduced to 7% in Caco-2/HT29-MTX co-culture models due to the hindrance by the mucus layers. Approximately 12-17% of αCT/Res-JNP were transported across Caco-2/HT29-MTX/Raji monolayers. The cumulative absorption of JNP in three cell models was higher than that of free drug.
CONCLUSIONS
This study investigated the contribution of Janus nanoparticles in oral absorption, and provide a new perspective for oral administration and analgesic treatment of dual drug delivery system containing peptide drugs.
Topics: Administration, Oral; Alginates; Analgesia; Animals; Biological Availability; Caco-2 Cells; Chitosan; Drug Carriers; Drug Delivery Systems; Drug Liberation; Female; Humans; Hydrogen-Ion Concentration; Male; Multifunctional Nanoparticles; Nanoparticles; Rats; Rats, Sprague-Dawley; Resveratrol
PubMed: 34362394
DOI: 10.1186/s12951-021-00974-6 -
The Korean Journal of Physiology &... Jul 2022Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add...
Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity the glycine (Gly)/glycine receptor (GlyR) α2/TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca] increase in neutrophils that had taken up E. coli . Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion the GlyRα2/ROS/calcium/p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.
PubMed: 35766001
DOI: 10.4196/kjpp.2022.26.4.229 -
Journal of Virology Apr 2023Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its...
Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.
Topics: Virus Internalization; Alphacoronavirus; rab GTP-Binding Proteins; Endosomes; Coronavirus Infections; Hydrogen-Ion Concentration; Dynamins; Caveolae; Cholesterol; Clathrin; Pinocytosis; Vero Cells; Chlorocebus aethiops; Animals
PubMed: 36975780
DOI: 10.1128/jvi.00210-23 -
Journal of Extracellular Vesicles Apr 2024Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical...
Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.
Topics: Proton Pump Inhibitors; Extracellular Vesicles; Endocytosis; Pinocytosis; Adenosine Triphosphatases
PubMed: 38532609
DOI: 10.1002/jev2.12426 -
Autophagy May 2021Poorly vascularized tumors embedded within a thick desmoplastic stroma, like pancreatic ductal adenocarcinoma (PDAC), are nutritionally stressed. Such tumors are also...
Poorly vascularized tumors embedded within a thick desmoplastic stroma, like pancreatic ductal adenocarcinoma (PDAC), are nutritionally stressed. Such tumors are also hypoxic and rely on a number of adaptive responses, including macroautophagy/autophagy and macropinocytosis (MP), to support their bioenergetic needs. Whereas autophagy enables starved cells to recycle intracellular macromolecules via lysosomal degradation and use the liberated amino acids (AA) to fuel their metabolism, MP allows cells to take up extracellular proteins via fluid-phase endocytosis and use them as an energy source. However, how any MP-enabled organism, including the prototypical cancer cell, coordinately regulates and balances autophagy and MP is not fully understood. We recently found that inhibition of autophagy results in upregulation of MP, which enables cancer cells to overcome autophagy deficiency and continue to support their bioenergetic demands. The NFE2L2/NRF2-driven induction of MP-related genes (MRGs) is responsible for the upregulation of MP in autophagy inhibited, hypoxic, and oxidatively stressed-exposed cancer cells. Concurrent autophagy and MP blockade effectively cuts off the cancer cell's nutrient and supplies, leading to rapid tumor regression. These findings suggest MP to be an important target in cancer treatment and that shutting off the energy spigot is a promising therapeutic strategy. AA: amino acids; ADCs: autophagy deficient-cells; AI: autophagy inhibition; ALB: albumin; CHUK/IKKα: component of inhibitor of nuclear factor kappa B kinase complex; CQ: chloroquine; ECM: extracellular matrix; HCQ: hydroxychloroquine; MI: MP inhibition; MP: macropinocytosis; MRGs: MP-related genes; MRPs: MP-related proteins; PDAC: pancreatic ductal adenocarcinoma.
Topics: Autophagy; Carcinoma, Pancreatic Ductal; Humans; NF-E2-Related Factor 2; Pancreatic Neoplasms; Pinocytosis
PubMed: 33879021
DOI: 10.1080/15548627.2021.1919969 -
Microbiome Feb 2021Sponges are increasingly recognised as key ecosystem engineers in many aquatic habitats. They play an important role in nutrient cycling due to their unrivalled capacity...
BACKGROUND
Sponges are increasingly recognised as key ecosystem engineers in many aquatic habitats. They play an important role in nutrient cycling due to their unrivalled capacity for processing both dissolved and particulate organic matter (DOM and POM) and the exceptional metabolic repertoire of their diverse and abundant microbial communities. Functional studies determining the role of host and microbiome in organic nutrient uptake and exchange, however, are limited. Therefore, we coupled pulse-chase isotopic tracer techniques with nanoscale secondary ion mass spectrometry (NanoSIMS) to visualise the uptake and translocation of C- and N-labelled dissolved and particulate organic food at subcellular level in the high microbial abundance sponge Plakortis angulospiculatus and the low microbial abundance sponge Halisarca caerulea.
RESULTS
The two sponge species showed significant enrichment of DOM- and POM-derived C and N into their tissue over time. Microbial symbionts were actively involved in the assimilation of DOM, but host filtering cells (choanocytes) appeared to be the primary site of DOM and POM uptake in both sponge species overall, via pinocytosis and phagocytosis, respectively. Translocation of carbon and nitrogen from choanocytes to microbial symbionts occurred over time, irrespective of microbial abundance, reflecting recycling of host waste products by the microbiome.
CONCLUSIONS
Here, we provide empirical evidence indicating that the prokaryotic communities of a high and a low microbial abundance sponge obtain nutritional benefits from their host-associated lifestyle. The metabolic interaction between the highly efficient filter-feeding host and its microbial symbionts likely provides a competitive advantage to the sponge holobiont in the oligotrophic environments in which they thrive, by retaining and recycling limiting nutrients. Sponges present a unique model to link nutritional symbiotic interactions to holobiont function, and, via cascading effects, ecosystem functioning, in one of the earliest metazoan-microbe symbioses. Video abstract.
Topics: Animals; Carbon; Microbiota; Nitrogen; Nutrients; Porifera; Symbiosis
PubMed: 33583434
DOI: 10.1186/s40168-020-00984-w