Fluid-phase Marker Research Articles

Mannose receptor (MRC1) mediates uptake of dextran in macrophages via receptor-mediated endocytosis.

Macrophages constantly survey and clear tissues by specifically and non-specifically internalizing debris and solutes. However, the molecular mechanisms and modes of regulation of these endocytic and macropinocytic processes are not well understood. Here, CRISPR/Cas9 whole genome screens were used to identify genes regulating uptake of dextran, a sugar polymer that is frequently used as a marker macropinocytosis, and compared with Lucifer yellow, a fluorescent dye with no known receptors. The authors identified the mannose receptor as well as other proteins regulating expression of the mannose receptor as top hits in the screen. Targeted disruption of Mrc1 , the gene that encodes mannose receptor, greatly diminished dextran uptake but had no effect on cellular uptake of Lucifer yellow. Furthermore, exposure to the cytokine IL4 upregulated mannose receptor expression on the cell surface and increased uptake of dextran with little effect on Lucifer yellow uptake. Studies seeking to understand regulation of macropinocytosis in macrophages will be confounded by the use of dextran as a fluid-phase marker. MRC1 is a marker of alternatively activated/anti-inflammatory macrophages and is a potential target for delivery of therapeutics to macrophages. This work provides the basis for mechanistic underpinning of how MRC1 contributes to the receptor-mediated uptake of carbohydrates and glycoproteins from the tissue milieu and distinguishes genes regulating receptor-mediated endocytosis from those regulating the bona fide fluid-phase uptake of fluids and solutes by macropinocytosis.

High Resolution Reconstruction of the Proximal Tubule Apical Endocytic Pathway

Kidney proximal tubule (PT) cells are specialized for effcient apical uptake of albumin and other proteins that escape the glomerular filtration barrier. The apical endocytic pathway is uniquely organized to maintain highly dynamic retrieval of these proteins via binding to the multiligand receptors megalin and cubilin. Ultrastructural studies following the fate of internalized markers have provided a temporal model for the delivery of receptor-bound ligands and fluid phase markers to lysosomes. In this model, internalization from the apical membrane occurs via budding of irregular clathrin-coated invaginations that form at the base of microvilli. After uncoating, budded vesicles fuse with apical early endosomes (AEEs) where acidification triggers dissociation of ligands from their receptors. AEEs further mature and expand into apical vacuoles (AVs) that ultimately deliver ligands and fluid to lysosomes. Receptor recycling to the apical membrane is thought to occur via dense apical tubules (DATs) that have a uniform diameter and membrane thickness and which appear to bud from both AEEs and AVs. We used Zeiss Arivis Pro technology to annotate and reconstruct apical membrane invaginations and endocytic compartments in a 3D volume of mouse kidney with isotropic voxels of 4 nm, acquired using focused ion-beam scanning electron microscopy (FIB-SEM). Strikingly, 3D reconstruction of DATs revealed a large and highly interconnected network of tubules in the subapical region of PT cells. Connections were observed between the DAT network and endosomal compartments, as well as between DATs and apical membrane invaginations. Our data suggest the possibility that DATs exist as a stable compartment that interacts transiently with the apical membrane to enable receptor recycling. Current studies are underway to better understand how PT cells modulate endocytic entry and recycling via the same portals. National Institute of Health: 5T32GM133353-03, R01 DK118726, R01 DK125049, U54 DK137329. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Validation of dot blot immunoassay for measurement of complement opsonization of nanoparticles

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field.

SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages via PKC-NADPH Oxidase Signaling.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While recent studies have demonstrated that SARS-CoV-2 may enter kidney and colon epithelial cells by inducing receptor-independent macropinocytosis, it remains unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated lung pneumonia, such as alveolar epithelial cells and macrophages. The goal of our study was to investigate the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in human alveolar epithelial cells and primary human and murine macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor-binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages in an angiotensin-converting enzyme 2 (ACE2)-independent manner. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike-protein-induced fluid-phase marker internalization in macrophages both in vitro and in vivo. High-resolution scanning electron microscopy (SEM) imaging confirmed that spike protein subunits promote the formation of membrane ruffles on the dorsal surface of macrophages. Mechanistic studies demonstrated that SARS-CoV-2 spike protein stimulated macropinocytosis via NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) generation. In addition, inhibition of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) in macrophages blocked SARS-CoV-2 spike-protein-induced macropinocytosis. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and COVID-19 pathogenesis.

HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice.

The HIV-1 core consists of a cone-shaped capsid shell made of capsid protein (CA) hexamers and pentamers encapsulating the viral genome. HIV-1 capsid disassembly, referred to as uncoating, is important for productive infection; however, the location, timing, and regulation of uncoating remain controversial. Here, we employ amber codon suppression to directly label CA. In addition, a fluid phase fluorescent probe is incorporated into the viral core to detect small defects in the capsid lattice. This double-labeling strategy enables the visualization of uncoating of single cores in vitro and in living cells, which we found to always proceed through at least two distinct steps─the formation of a defect in the capsid lattice that initiates gradual loss of CA below a detectable level. Importantly, intact cores containing the fluid phase and CA fluorescent markers enter and uncoat in the nucleus, as evidenced by a sequential loss of both markers, prior to establishing productive infection. This two-step uncoating process is observed in different cells, including a macrophage line. Notably, the lag between the release of fluid phase marker and terminal loss of CA appears to be independent of the cell type or reverse transcription and is much longer (>5-fold) for nuclear capsids compared to cell-free cores or cores in the cytosol, suggesting that the capsid lattice is stabilized by capsid-binding nuclear factors. Our results imply that intact HIV-1 cores enter the cell nucleus and that uncoating is initiated through a localized defect in the capsid lattice prior to a global loss of CA.

Abstract 15524: SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages

Coronavirus Disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of this virus has led to millions of deaths and the long-term cardiovascular and respiratory consequences of COVID-19 continue to pose a massive threat to public health. While recent studies have demonstrated that SARS-CoV-2 may enter kidney epithelial cells via angiotensin converting enzyme 2 (ACE2)-independent mechanisms by inducing receptor-independent macropinocytosis, it is currently unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated cardiovascular disease or pneumonia. Here, we investigated the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in alveolar epithelial cells and macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages, but not in human lung epithelial cells. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike protein-induced macropinocytosis in macrophages both in vitro and in vivo . High resolution scanning electron microscopy (SEM) imaging confirmed spike protein-induced plasma membrane activities characteristic of macropinocytosis. Mechanistic studies demonstrated that inhibition of protein kinase C and phosphoinositide 3-kinase in macrophages blocks SARS-CoV-2 spike protein-induced macropinocytosis. Further, pharmacological blockade of ACE2 did not inhibit macropinocytosis in SARS-CoV-2 spike protein-treated cells. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and its cardiovascular and respiratory complications.

Human Immunodeficiency Virus 1 Preferentially Fuses with pH-Neutral Endocytic Vesicles in Cell Lines and Human Primary CD4+ T-Cells.

Despite extensive efforts, the principal sites of productive HIV-1 entry in different target cells─plasma membrane (PM) vs endosomes─remain controversial. To delineate the site(s) of HIV-1 fusion, we implemented a triple labeling approach that involves tagging pseudoviruses with the fluid-phase viral content marker, iCherry, the viral membrane marker, DiD, and the extraviral pH sensor, ecliptic pHluorin. The viral content marker iCherry is released into the cytoplasm upon virus-cell fusion irrespective of the sites of fusion. In contrast, the extent of dilution of the membrane marker upon fusion with the PM (loss of signal) vs the endosomal membrane (no change in punctate DiD appearance) discriminates between the principal sites of viral fusion. Additionally, ecliptic pHluorin incorporated into the viral membrane reports whether virus fusion occurs in acidic endosomes. Real-time single virus imaging in living HeLa-derived cells, a CD4+ T-cell line, and activated primary human CD4+ T-cells revealed a strong (80-90%) HIV-1 preference for fusion with endosomes. Intriguingly, we observed HIV-1 fusion only with pH-neutral intracellular vesicles and never with acidified endosomes. These endocytic fusion events are likely culminating in productive infection since endocytic inhibitors, such as EIPA, Pitstop2, and Dynasore, as well as a dominant-negative dynamin-2 mutant, inhibited HIV-1 infection in HeLa-derived and primary CD4+ T-cells. Furthermore, the inhibition of endocytosis in HeLa-derived cells promoted hemifusion at the PM but abrogated complete fusion. Collectively, these data reveal that the primary HIV-1 entry pathway in diverse cell types is through fusion with pH-neutral intracellular vesicles.

Receptor-associated protein impairs ligand binding to megalin and megalin-dependent endocytic flux in proximal tubule cells.

Proximal tubule (PT) cells retrieve albumin and a broad array of other ligands from the glomerular ultrafiltrate. Efficient uptake of albumin requires PT expression of both megalin and cubilin receptors. Although most proteins engage cubilin selectively, megalin is required to maintain robust flux through the apical endocytic pathway. Receptor-associated protein (RAP) is a chaperone that directs megalin to the cell surface, and recombinant RAP dramatically inhibits the uptake of numerous megalin and cubilin ligands. The mechanism by which this occurs has been suggested to involve competitive inhibition of ligand binding and/or conformational changes in megalin that prevent interaction with ligands and/or with cubilin. To discriminate between these possibilities, we determined the effect of RAP on endocytosis of albumin, which binds to cubilin and megalin receptors with high and low affinity, respectively. Uptake was quantified in opossum kidney (OK) cells and in megalin or cubilin (Cubn) knockout (KO) clones. Surprisingly, RAP inhibited fluid-phase uptake in addition to receptor-mediated uptake in OK cells and Cubn KO cells but had no effect on endocytosis when megalin was absent. The apparent Ki for RAP inhibition of albumin uptake was 10-fold higher in Cubn KO cells compared with parental OK cells. We conclude that in addition to its predicted high-affinity competition for ligand binding to megalin, the primary effect of RAP on PT cell endocytosis is to globally dampen megalin-dependent endocytic flux. Our data explain the complex effects of RAP on binding and uptake of filtered proteins and reveal a novel role in modulating endocytosis in PT cells.NEW & NOTEWORTHY Receptor-associated protein inhibits binding and uptake of all known endogenous ligands by megalin and cubilin receptors via unknown mechanism(s). Here, we took advantage of recently generated knockout cell lines to dissect the effect of this protein on megalin- and cubilin-mediated endocytosis. Our study reveals a novel role for receptor-associated protein in blocking megalin-stimulated endocytic uptake of fluid-phase markers and receptor-bound ligands in proximal tubule cells in addition to its direct effect on ligand binding to megalin receptors.

Megalin, cubilin, and Dab2 drive endocytic flux in kidney proximal tubule cells.

The kidney proximal tubule (PT) elaborates a uniquely high-capacity apical endocytic pathway to retrieve albumin and other proteins that escape the glomerular filtration barrier. Megalin and cubilin/amnionless (CUBAM) receptors engage Dab2 in these cells to mediate clathrin-dependent uptake of filtered ligands. Knockout of megalin or Dab2 profoundly inhibits apical endocytosis and is believed to atrophy the endocytic pathway. We generated CRISPR/Cas9 knockout (KO) clones lacking cubilin, megalin, or Dab2 expression in highly differentiated PT cells and determined the impact on albumin internalization and endocytic pathway function. KO of each component had different effects on the concentration dependence of albumin uptake as well its distribution within PT cells. Reduced uptake of a fluid phase marker was also observed, with megalin KO cells having the most dramatic decline. Surprisingly, protein levels and distribution of key endocytic proteins were preserved in KO PT cell lines and in megalin KO mice, despite the reduced endocytic activity. Our data highlight specific functions of megalin, cubilin, and Dab2 in apical endocytosis and demonstrate that these proteins drive endocytic flux without compromising the physical integrity of the apical endocytic pathway. Our studies suggest a novel model to explain how these components coordinate endocytic uptake in PT cells.

Entry of the Varicellovirus Canid herpesvirus 1 into Madin-Darby canine kidney epithelial cells is pH-independent and occurs via a macropinocytosis-like mechanism but without increase in fluid uptake.

Canid herpesvirus 1 (CHV-1) is a Varicellovirus that causes self-limiting infections in adult dogs but morbidity and mortality in puppies. Using a multipronged approach, we discovered the CHV-1 entry pathway into Madin-Darby canine kidney (MDCK) epithelial cells. We found that CHV-1 triggered extensive host cell membrane lamellipodial ruffling and rapid internalisation of virions in large, uncoated vacuoles, suggestive of macropinocytosis. Treatment with inhibitors targeting key macropinocytosis factors, including inhibitors of Na+ /H+ exchangers, F-actin, myosin light-chain kinase, protein kinase C, p21-activated kinase, phosphatidylinositol-3-kinase and focal adhesion kinase, significantly reduced viral replication. Moreover, the effect was restricted to exposure to the inhibitors early in infection, confirming a role for the macropinocytic machinery during entry. The profile of inhibitors also suggested a role for signalling via integrins and receptor tyrosine kinases in viral entry. In contrast, inhibitors of clathrin, caveolin, microtubules and endosomal acidification did not affect CHV-1 entry into MDCK cells. We found that the virus colocalised with the fluid-phase uptake marker dextran; however, surprisingly, CHV-1 infection did not enhance the uptake of dextran. Thus, our results indicate that CHV-1 uses a macropinocytosis-like, pH-independent entry pathway into MDCK cells, which nevertheless is not based on stimulation of fluid uptake. TAKE AWAYS: CHV-1 enters epithelial cells via a macropinocytosis-like mechanism. CHV-1 induces extensive lamellipodial ruffling. CHV-1 entry into MDCK cells is pH-independent.

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line.

Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule

The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid–base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats.

Maduramicin induces cardiotoxicity via Rac1 signaling-independent methuosis in H9c2 cells.

Maduramicin frequently induces severe cardiotoxicity in target and nontarget animals in clinic. Apoptotic and non-apoptotic cell death mediate its cardiotoxicity; however, the underlying non-apoptotic cell death induced by maduramicin remains unclear. In current study, a recently described non-apoptotic cell death "methuosis" caused by maduramicin was defined in mammalian cells. Rat myocardial cell H9c2 was used as an in vitro model, showing excessively cytoplasmic vacuolization upon maduramicin (0.0625-5μg/mL) exposure for 24 h. Maduramicin-induced reversible cytoplasmic vacuolization of H9c2 cells in a time- and concentration-dependent manner. The vacuoles induced by maduramicin were phase lucent with single membrane and were not derived from the swelling of organelles such as mitochondria, endoplasmic reticulum, lysosome, and Golgi apparatus. Furthermore, maduramicin-induced cytoplasmic vacuoles are generated from micropinocytosis, which was demonstrated by internalization of extracellular fluid-phase marker Dextran-Alexa Fluor 488 into H9c2 cells. Intriguingly, these cytoplasmic vacuoles acquired some characteristics of late endosomes and lysosomes rather than early endosomes and autophagosomes. Vacuolar H+ -ATPase inhibitor bafilomycin A1 efficiently prevented the generation of cytoplasmic vacuoles and decreased the cytotoxicity of H9c2 cells triggered by maduramicin. Mechanism studying indicated that maduramicin activated H-Ras-Rac1 signaling pathway at both mRNA and protein levels. However, the pharmacological inhibition and siRNA knockdown of Rac1 rescued maduramicin-induced cytotoxicity of H9c2 cells but did not alleviate cytoplasmic vacuolization. Based on these findings, maduramicin induces methuosis in H9c2 cells via Rac-1 signaling-independent seriously cytoplasmic vacuolization.

Fluid-phase and membrane markers reveal spatio-temporal dynamics of membrane traffic and repair in the green alga Chara australis

We investigated the mechanisms and the spatio-temporal dynamics of fluid-phase and membrane internalization in the green alga Chara australis using fluorescent hydrazides markers alone, or in conjunction with styryl dyes. Using live-cell imaging, immunofluorescence and inhibitor studies we revealed that both fluid-phase and membrane dyes were actively taken up into the cytoplasm by clathrin-mediated endocytosis and stained various classes of endosomes including brefeldin A- and wortmannin-sensitive organelles (trans-Golgi network and multivesicular bodies). Uptake of fluorescent hydrazides was poorly sensitive to cytochalasin D, suggesting that actin plays a minor role in constitutive endocytosis in Chara internodal cells. Sequential pulse-labelling experiments revealed novel aspects of the temporal progression of endosomes in Chara internodal cells. The internalized fluid-phase marker distributed to early compartments within 10 min from dye exposure and after about 30 min, it was found almost exclusively in late endocytic compartments. Notably, fluid cargo consecutively internalized at time intervals of more than 1h, was not targeted to the same vesicular structures, but was sorted into distinct late compartments. We further found that fluorescent hydrazide dyes distributed not only to rapidly recycling endosomes but also to long-lived compartments that participated in plasma membrane repair after local laser injury. Our approach highlights the benefits of combining different fluid-phase markers in conjunction with membrane dyes in simultaneous and sequential application modus for investigating vesicle traffic, especially in organisms, which are still refractory to genetic transformation like characean algae.

Monitoring Activity-Dependent Bulk Endocytosis in Primary Neuronal Culture Using Large Fluorescent Dextrans.

The efficient recycling of synaptic vesicles (SVs) during neuronal activity is central for sustaining brain function. During intense neuronal activity, the dominant mechanism of SV retrieval is activity-dependent bulk endocytosis (ADBE). Here, we describe a method to monitor ADBE in isolation from other SV endocytosis modes, via the uptake of large fluorescent fluid-phase markers in primary neuronal culture. Furthermore, we outline how to monitor ADBE using this approach across a field of neurons or in individual neurons.

Loss of cell-cell and cell-substrate contacts in single pancreatic β-cells divert insulin release to intracellular vesicular compartments.

Cell-cell or cell-substrate interactions are lost when cells are dissociated in culture, or during pathophysiological breakdowns, therefore impairing their structure and polarity, and affecting their function. We show that single rat β-cells, cultured under non-adhesive conditions, form intracytoplasmic vacuoles increasing in number and size over time. We characterized these structures and their implication in β-cell function. Ultrastructurally, the vacuoles resemble vesicular apical compartments and are delimited by a membrane, containing microvilli and expressing markers of the plasma membrane, including glucose transporter 2 and actin. When insulin secretion is stimulated, insulin accumulates in the lumen of the vacuoles. By contrast, when the cells are incubated under low calcium levels, the hormone is undetectable in vesicular compartments. Insulin release studies from single cells revealed that vacuole-containing cells release less insulin as compared to control cells. When added to the medium, a non-permeant fluid phase marker becomes trapped within vacuoles. Inhibition of vesicular trafficking and exocytosis as well as dynamin-dependent endocytosis changed the percentage of vacuole-containing cells, suggesting that both endocytic and exocytic track contribute to their formation. These results suggest that loss of cell-cell and cell-substrate contacts in isolated β-cells affect normal vesicular trafficking and redirects insulin secretion to intracellular vesicular compartments. Our study reveals for the first time that single β-cells develop vacuolar compartments when cultured in suspension and redirect their insulin secretion to these vacuoles. This may underlie a compensatory process for cultured cells who lost their interactions with adhesive substrates or neighbouring cells.

Effect of endothelial cell heterogeneity on nanoparticle uptake

Endothelial cells exhibit distinct properties in morphology and functions in different organs that can be exploited for nanomedicine targeting. In this work, endothelial cells from different organs, i.e. brain, lung, liver, and kidney, were exposed to plain, carboxylated, and amino-modified silica. As expected, different protein coronas were formed on the different nanoparticle types and these changed when foetal bovine serum (FBS) or human serum were used. Uptake efficiencies differed strongly in the different endothelia, confirming that the cells retained some of their organ-specific differences. However, all endothelia showed higher uptake for the amino-modified silica in FBS, but, interestingly, this changed to the carboxylated silica when human serum was used, confirming that differences in the protein corona affect uptake preferences by cells. Thus, uptake rates of fluid phase markers and transferrin were determined in liver and brain endothelium to compare their endocytic activity. Overall, our results showed that endothelial cells of different organs have very different nanoparticle uptake efficiency, likely due to differences in receptor expression, affinity, and activity. A thorough characterization of phenotypic differences in the endothelia lining different organs is key to the development of targeted nanomedicine.

Distinct functions of megalin and cubilin receptors in recovery of normal and nephrotic levels of filtered albumin.

Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.

A Novel Bivalent Mannosylated Targeting Ligand Displayed on Nanoparticles Selectively Targets Anti-Inflammatory M2 Macrophages.

Persistent activation of macrophages (MP)s into a proinflammatory M1 or anti-inflammatory M2 phenotype plays a role in several pathological conditions, including autoimmune diseases, fibrosis, infections, atherosclerosis and tumor development. The mannose receptor (MR, CD206), expressed at low levels on resting MPs and absent on M1 MPs, is highly expressed on M2 MPs, making it a potential target and drug delivery portal. Recently, we developed a novel, highly selective MR targeting ligand (MRTL), consisting of two mannose molecules separated by a monodisperse 12 unit poly(ethylene glycol) linker, to enhance the cellular uptake of polymeric nanocarriers. The feasibility of using the MRTL ligand for selectively targeting M2 MPs for intracellular delivery of nanoparticles (NPs) was investigated. Rat peritoneal MPs were differentiated into an M1 or M2 phenotype using IFN-γ and IL-4/IL-13, respectively. Expression of the M1 marker, inducible nitric oxide synthase (iNOS), and the M2 markers arginase (Arg)-1 and MR (at both the mRNA and protein levels) confirmed MP phenotypic activation. Resting, M1 and M2 MPs were treated with fluorescein isothiocyanate (FITC)-labeled MRTL or NPs displaying FITC-labeled MRTL at two surface densities (1 and 10%) and examined by confocal microscopy. Intracellular fluorescence was also quantified. Uptake of the MRTL was 2.4- and 11.8-fold higher in M2 MPs when compared to resting or M1 MPs, respectively, consistent with marker expression levels. Mannan, a competitive inhibitor of the MR, abrogated MRTL uptake. MRTL also co-localized with a fluid-phase endocytosis marker, further suggesting that uptake was mediated by MR-mediated endocytosis. Intracellular NP fluorescence was confirmed by flow cytometry and by confocal microscopy. MRTL-NPs accumulated intracellularly with no significant cell surface binding, suggesting efficient translocation. NPs displaying a low surface density (1%) of the MRTL exhibited significantly higher (2.3-fold) uptake into M2 MPs, relative to resting and M1 MPs. The 10% MRTL-NPs displayed greater uptake by M2 MPs when compared to resting and M1 MPs, but less uptake than 1% MRTL-NPs into M2 MPs. Control FITC-labeled plain NPs did not exhibit selective MP uptake. These studies demonstrate that M2 MPs are selectively targeted by NPs displaying a novel bivalent ligand that utilizes the MR as a target/portal for cell entry. This study also establishes the feasibility of the approach allowing for further investigation in vivo.