Fluid-phase Endocytosis Research Articles

Gelsolin regulates receptor-mediated and fluid-phase endocytosis in platelets

BackgroundEndocytosis is the process by which platelets incorporate extracellular molecules into their secretory granules. Endocytosis is mediated by the actin cytoskeleton in nucleated cells; however, the endocytic mechanisms in platelets are undefined. ObjectivesTo better understand platelet endocytosis, we studied gelsolin (Gsn), an actin-severing protein that promotes actin assembly. MethodsMouse platelets from Gsn-null (Gsn−/−) and wild-type (WT) controls were used. The uptake of fluorescent cargo molecules was compared as a measure of their endocytic efficiency. Receptor-mediated endocytosis was measured by the uptake of fibrinogen and transferrin; fluid-phase endocytosis was monitored by the uptake of fluorescent dextrans. ResultsAdenosine diphosphate (ADP)–stimulated WT platelets readily internalized both receptor-mediated and fluid-phase cargoes. In contrast, Gsn−/− platelets showed a severe defect in the endocytosis of both types of cargo. The treatment of WT platelets with the actin-disrupting drugs cytochalasin D and jasplakinolide also reduced endocytosis. Notably, the individual and combined effects of Gsn deletion and drug treatment were similar for both receptor-mediated and fluid-phase endocytosis, indicating that Gsn mediates endocytosis via its action on the actin cytoskeleton. ConclusionOur study demonstrates that Gsn plays a key role in the uptake of bioactive mediators by platelets.

Hypoxia modulates cellular endocytic pathways and organelles with enhanced cell migration and 3D cell invasion.

Hypoxia is characteristic of most tumors and has been shown to drive cancer progression by altering multiple subcellular processes. We hypothesized that cancer cells in hypoxic environment might have slower proliferation rates and increased invasion and migration rates with altered endocytosis compared to the cancer cells in periphery of tumor mass that experiences normoxic conditions.We measured effect of chemically induced hypoxia on cell proliferation, migration, and invasion. Uptake of fluorescently labeled transferrin, galectin3, and dextran that undergo endocytosis through major endocytic pathways (Clathrin-mediated pathway (CME), Clathrin-independent pathway (CIE), Fluid phase endocytosis (FPE)) was analyzed during hypoxia. Also, the organelle changes associated with hypoxia were studied with organelle trackers. We found that the proliferation rate decreased, and the migration and invasion rate increased in cancer cells in hypoxic conditions compared to normoxic cancer cells. A short hypoxic exposure increased endocytosis in hypoxic cancer cells, but a prolonged hypoxic exposure decreased clathrin-independent endocytic uptake. Subcellular organelles, such as mitochondria, increased to withstand the hypoxic stress, while other organelles, such as Endoplasmic reticulum (ER), were significantly decreased. These data suggest that hypoxia modulates cellular endocytic pathways with reduced proliferation and enhanced cell migration and invasion.

Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles.

Poor understanding of intracellular delivery and targeting hinders development of nucleic acid-based therapeutics transported by nanoparticles. Utilizing a siRNA-targeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3-LNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE-ID). A cell-based imaging assay and perturbation of 178 targets relevant to intracellular trafficking is used to identify corresponding effects on functional mRNA delivery. Targets improving delivery are analyzed by extracting data-rich phenotypic fingerprints from images using advanced image analysis algorithms. Machine learning is used to determine key features correlating with enhanced delivery, identifying fluid-phase endocytosis as a productive cellular entry route. With this new knowledge, MC3-LNP is re-engineered to target macropinocytosis, and this significantly improves mRNA delivery in vitro and in vivo. The ACE-ID approach can be broadly applicable for optimizing nanomedicine-based intracellular delivery systems and has the potential to accelerate the development of delivery systems for nucleic acid-based therapeutics.

ODP130 Zoledronate-Induced Acute Tubular Necrosis With Baseline Normal Renal Function: A Rare Phenomenon

Abstract Introduction Zoledronate is an intravenous bisphosphonate that is commonly used to decrease bone resorption in patients with osteoporosis. The incidence of acute tubular necrosis (ATN) using zoledronate in patients with normal renal function is rare. Herein, we report the occurrence of severe ATN following treatment with a single zoledronate infusion in a patient with osteoporosis and normal renal function. Case Presentation A 51-year-old female with hypertension, well-controlled type 2 diabetes c/b gastroparesis without nephropathy(HbA1c6.8%), status-post hysterectomy, and bilateral oophorectomy, who was recently diagnosed with osteoporosis. Bone densitometry with Hologic revealed T scores of -2.8 at the spine and a T score of - 0.9 at the left hip. Her medications included lisinopril, metformin, and empagliflozin. Due to a history of gastroparesis, zoledronate was chosen as the preferred treatment. Pre-infusion blood work showed: a creatinine of 0.92 (0.5-1.1 mg/dl), 25-hydroxyvitamin-D 37.8 (30-100 NG/ml), calcium 8.9 (8.5-10.5mg/dl) and urine microalbumin 14 (<30 mg/g). Two days after the infusion, the patient developed emesis, diarrhea, oliguria, and fevers. She was evaluated in the emergency room and found to have acute renal failure (creatinine 10 mg/dL). She was given three liters of intravenous fluids with no improvement in urine output. Microscopic evaluation of spun urine revealed non-dysmorphic RBCs and numerous muddy brown granular casts consistent with ATN. Despite treatment with intravenous fluids, her creatinine did not improve and peaked on hospital days 11 to 11.65mg/dl. A 24-hour urine collection revealed proteinuria of 3 grams/24 hours. The patient was then empirically started on methylprednisolone 500mg for suspected glomerulonephritis. A renal biopsy was subsequently performed, which confirmed ATN. The patient's creatinine improved steadily to 5.49mg/dl by discharge. A subsequent creatinine one-month post-discharge was 1.36mg/dL. Discussion Zoledronate is widely used at low doses in the treatment of osteoporosis. ATN due to zoledronate infusion has been described in patients with pre-existing renal dysfunction and those receiving large and frequent doses for malignancy but rarely reported with the lower doses used to treat patients with osteoporosis and normal renal function. Zoledronate is believed to cause ATN by internalization in the renal tubular cells via the process of fluid-phase endocytosis. The intracellular accumulation of zoledronate may act as an epithelial toxin inducing tubular cytotoxicity. Interestingly, our patient had normal renal function without diabetic nephropathy and did not use nephrotoxic medications prior to the infusion. Conclusion We report a case of severe ATN due to a single dose of zoledronate in a patient with osteoporosis and previously normal renal function. This case highlights the need to be aware of the potential nephrotoxicity of zoledronate even in patients with normal renal function at baseline. Presentation: No date and time listed

Dynamic glucose uptake, storage, and release by human microvascular endothelial cells.

Endothelia determine blood-to-tissue solute delivery, yet glucose transit is poorly understood. To illuminate mechanisms, we tracked [3H]-2-deoxyglucose (2-DG) in human adipose-tissue microvascular endothelial cells. 2-DG uptake was largely facilitated by the glucose transporters GLUT1 and GLUT3. Once in the cytosol, >80% of 2-DG became phosphorylated and ∼20% incorporated into glycogen, suggesting that transported glucose is readily accessible to cytosolic enzymes. Interestingly, a fraction of intracellular 2-DG was released over time (15-20% over 30 min) with slower kinetics than for uptake, involving GLUT3. In contrast to intracellular 2-DG, the released 2-DG was largely unphosphorylated. Glucose release involved endoplasmic reticulum-resident translocases/phosphatases and was stimulated by adrenaline, consistent with participation of glycogenolysis and glucose dephosphorylation. Surprisingly, the fluorescent glucose derivative 2-NBD-glucose (2-NBDG) entered cells largely via fluid phase endocytosis and exited by recycling. 2-NBDG uptake was insensitive to GLUT1/GLUT3 inhibition, suggesting poor influx across membranes. 2-NBDG recycling, but not 2-DG efflux, was sensitive to N-ethyl maleimide. In sum, by utilizing radioactive and fluorescent glucose derivatives, we identified two parallel routes of entry: uptake into the cytosol through dedicated glucose transporters and endocytosis. This reveals the complex glucose handling by endothelial cells that may contribute to glucose delivery to tissues.

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium.

Endocytosis allows cells to internalise a wide range of molecules from their environment and to maintain their plasma membrane composition. It is vital during development and for maintenance of tissue homeostasis. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualise endocytosis in the neuroepithelium of living zebrafish embryos. Injection of fluorescent tracers into the brain ventricles followed by live imaging was used to study fluid-phase or receptor-mediated endocytosis, for which we used receptor-associated protein (RAP, encoded by Lrpap1) as a ligand for low-density lipoprotein receptor-related protein (LRP) receptors. Using dual-colour imaging combined with expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also found that the RAP-binding receptor Lrp2 (encoded by lrp2a) appears to contribute only partially to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo and support a role for Ocrl in this process. This article has an associated First Person interview with the first author of the paper.

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.

PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica.

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite.

Fluid-Phase Endocytosis and Lysosomal Degradation of Bovine Lactoferrin in Lung Cells

The iron-binding protein lactoferrin and the cell-penetrating peptides derived from its sequence utilise endocytosis to enter different cell types. The full-length protein has been extensively investigated as a potential therapeutic against a range of pathogenic bacteria, fungi, and viruses, including SARS-CoV-2. As a respiratory antiviral agent, several activity mechanisms have been demonstrated for lactoferrin, at the extracellular and plasma membrane levels, but as a protein that enters cells it may also have intracellular antiviral activity. Characterisation of lactoferrin’s binding, endocytic traffic to lysosomes, or recycling endosomes for exocytosis is lacking, especially in lung cell models. Here, we use confocal microscopy, flow cytometry, and degradation assays to evaluate binding, internalisation, endocytic trafficking, and the intracellular fate of bovine lactoferrin in human lung A549 cells. In comparative studies with endocytic probes transferrin and dextran, we show that lactoferrin binds to negative charges on the cell surface and actively enters cells via fluid-phase endocytosis, in a receptor-independent manner. Once inside the cell, we show that it is trafficked to lysosomes where it undergoes degradation within two hours. These findings provide opportunities for investigating both lactoferrin and derived cell-penetrating peptides activities of targeting intracellular pathogens.

Low-Dose Fluvoxamine Modulates Endocytic Trafficking of SARS-CoV-2 Spike Protein: A Potential Mechanism for Anti-COVID-19 Protection by Antidepressants.

Commonly prescribed antidepressants may be associated with protection against severe COVID-19. The mechanism of their action in this context, however, remains unknown. Here, I investigated the effect of an antidepressant drug fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system.

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. They have recently been found to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, making it extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed cell lines with conditional KO (CKO) of either Rab1 (Rab1A and Rab1B) or Rab5 (Rab5A, Rab5B and Rab5C) by using the auxin-inducible protein degradation system. Rab1 CKO and Rab5 CKO led to eventual cell death from 18 h and 48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and endoplasmic reticulum (ER)-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

NRF3 upregulates gene expression in SREBP2-dependent mevalonate pathway with cholesterol uptake and lipogenesis inhibition

SummaryLipids, such as cholesterol and fatty acids, influence cell signaling, energy storage, and membrane formation. Cholesterol is biosynthesized through the mevalonate pathway, and aberrant metabolism causes metabolic diseases. The genetic association of a transcription factor NRF3 with obesity has been suggested, although the molecular mechanisms remain unknown. Here, we show that NRF3 upregulates gene expression in SREBP2-dependent mevalonate pathway. We further reveal that NRF3 overexpression not only reduces lanosterol, a cholesterol precursor, but also induces the expression of the GGPS1 gene encoding an enzyme in the production of GGPP from farnesyl pyrophosphate (FPP), a lanosterol precursor. NRF3 overexpression also enhances cholesterol uptake through RAB5-mediated macropinocytosis process, a bulk and fluid-phase endocytosis pathway. Moreover, we find that GGPP treatment abolishes NRF3 knockdown-mediated increase of neutral lipids. These results reveal the potential roles of NRF3 in the SREBP2-dependent mevalonate pathway for cholesterol uptake through macropinocytosis induction and for lipogenesis inhibition through GGPP production.

Essential role of the endocytic site-associated protein Ecm25 in stress-induced cell elongation.

SUMMARYHow cells adopt a different morphology to cope with stress is not well understood. Here, we show that budding yeast Ecm25 associates with polarized endocytic sites and interacts with the polarity regulator Cdc42 and several late-stage endocytic proteins via distinct regions, including an actin filament-binding motif. Deletion of ECM25 does not affect Cdc42 activity or cause any strong defects in fluid-phase and clathrin-mediated endocytosis but completely abolishes hydroxyurea-induced cell elongation. This phenotype is accompanied by depolarization of the spatiotemporally coupled exo-endocytosis in the bud cortex while maintaining the overall mother-bud polarity. These data suggest that Ecm25 provides an essential link between the polarization signal and the endocytic machinery to enable adaptive morphogenesis under stress conditions.

Macropinocytosis: the big drinker behind cancer cell self-consumption

ABSTRACT 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. Abbreviations 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.

Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani.

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Å resolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed β-sheet surrounded by five α-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the γ-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors.

Rab5b function is essential to acquire heme from hemoglobin endocytosis for survival of Leishmania

Previously, we showed that Rab5a and Rab5b differentially regulate fluid-phase and receptor-mediated endocytosis in Leishmania, respectively. To unequivocally demonstrate the role of Rab5b in hemoglobin endocytosis in Leishmania, we generated null-mutants of Rab5b parasites by sequentially replacing both copies of LdRab5b with the hygromycin and neomycin resistance gene cassettes. LdRab5b−/− null-mutant parasite was confirmed by qPCR analysis of genomic DNA using LdRab5b specific primers. LdRab5b−/− cells showed severe growth defect indicating essential function of LdRab5b in parasite. To characterize the role of Rab5b in Hb endocytosis in parasites, LdRab5b−/− cells were rescued by exogenous addition of hemin in growth medium. Our results showed that LdRab5b−/− cells are relatively smaller in size. Ultrastructural analysis revealed the presence of relatively enlarged flagellar pocket and bigger intracellular vesicles in these cells in comparison to control cells. Both promastigotes and amastigotes of Rab5b null-mutant parasites were unable to internalize Hb but fluid phase endocytosis of different markers was not affected. However, complementation of LdRab5b:WT in LdRab5b−/− cells (LdRab5b−/−:pRab5b:WT) rescued Hb internalization in these cells. Interestingly, LdRab5b−/− cells showed significantly less Hb-receptor on cell surface in comparison to control cells indicating a block in HbR trafficking. Finally, we showed that LdRab5b−/− parasites can infect the macrophages but are unable to survive after 96 h of infection in comparison to control cells. However, supplementation of hemin in the growth medium significantly rescued LdRab5b−/−Leishmania survival in macrophage indicating that LdRab5b function is essential for the acquisition of heme from internalized Hb for the survival of Leishmania.

On the structural organization of the bacillary band of Trichuris muris under cryopreparation protocols and three-dimensional electron microscopy

Whipworms of the genus Trichuris are nematode parasites that infect mammals and can lead to various intestinal diseases of human and veterinary interest. The most intimate interaction between the parasite and the host intestine occurs through the anterior region of the nematode body, inserted into the intestinal mucosa during infection. One of the most prominent structures of the nematode surface found at the infection site is the bacillary band, a surface domain formed by a number of cells, mostly stichocytes and bacillary glands, whose structure and function are still under debate. Here, we used confocal microscopy, field emission scanning electron microscopy, helium ion microscopy, transmission electron microscopy and FIB-SEM tomography to unveil the functional role of the bacillary gland cell. We analyzed the surface organization as well as the intracellular milieu of the bacillary glands of Trichuris muris in high pressure frozen/freeze-substituted samples. Results showed that the secretory content is preserved in all gland openings, presenting a projected pattern. FIB-SEM analysis showed that the lamellar zone within the bacillary gland chamber is formed by a set of lacunar structures that may exhibit secretory or absorptive functions. In addition, incubation of parasites with the fluid phase endocytosis marker sulforhodamine B showed a time-dependent uptake by the parasite mouth, followed by perfusion through different tissues with ultimate secretion through the bacillary gland. Taken together, the results show that the bacillary gland possess structural characteristics of secretory and absorptive cells and unequivocally demonstrate that the bacillary gland cell functions as a secretory structure.

High-efficiency unassisted transfection of platelets with naked double-stranded miRNAs modulates signal-activated translation and platelet function

We sought novel approaches to improve transfection efficiencies of microRNAs (miRNAs) in platelets, and to apply these approaches to investigate the roles of miRNAs in regulating signal-activated protein translation and functional effects. We found that ex vivo human platelets support gymnosis––-internalization of ectopic miRNAs following co-incubation in the absence of conventional transfection reagents or schemes---and subsequently incorporate transfected miRNA into ARGONAUTE2 (AGO2)-based RNA-induced silencing complexes (RISC). Thrombin/fibrinogen stimulation activated translation of miR-223-3p target SEPTIN2, which was suppressed by miR-223-3p transfection in an AGO2/RISC-dependent manner. Thrombin/fibrinogen-induced exosome and microvesicle generation was inhibited by miR-223-3p transfection, and this effect was reversed with a RISC inhibitor. Platelet gymnosis of naked miRNAs appeared to be mediated in part by endocytic pathways including clathrin-dependent and fluid-phase endocytosis and caveolae. These results demonstrate the ability of ex vivo platelets to internalize ectopic miRNAs by unassisted transfection, and utilize them to modulate signal-activated translation and platelet function. Our results identify new roles for miR-223-3p in extracellular vesicle generation in stimulated platelets. High-efficiency gymnotic transfection of miRNAs in ex vivo platelets may be a broadly useful tool for exploring molecular genetic regulation of platelet function.

Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy.

Since the discovery of the causative agent for the novel severe acute respiratory syndrome (SARS)–like pneumonia syndrome pandemic that started in China in 2019 (1,2), a coronavirus named SARS coronavirus 2 (SARS-CoV-2), electron microscopy images have populated the medical literature (2) and media outlets alike displaying the characteristic 60–140 nm round particles surrounded by a “corona” of 9–12 nm distinctive spikes (2). Although many of these images were obtained after “ in vitro ” infection of cultured cells with SARS-CoV-2 (2) and are thus likely a true representation of viral particles, we have observed morphologically indistinguishable inclusions within podocytes and tubular epithelial cells both in patients negative for coronavirus disease 2019 (COVID-19) as well as in renal biopsies from the pre–COVID-19 era. Although direct infection of the kidney is theoretically possible, given the presence of angiotensin-converting enzyme 2 (the receptor used by SARS-CoV-2 to gain access to cells) within proximal tubular epithelium (3) and podocytes (4), the virus has not been detected by real-time RT-PCR in urine samples from patients with COVID-19 (5⇓–7). Additionally, for the virus to have access to kidney parenchyma, viremia should occur, and this has only been detected in a minority of patients (6⇓–8). We would, therefore, like to issue a note of caution for inferring viral tissue infection by morphology alone using electron microscopy images from tissues obtained from biopsies or autopsy material in patients with COVID-19. Moreover, caution should be used when interpreting immunohistochemical results, especially within proximal tubules, which are prone to nonspecific staining by a variety of antibodies due to their intense reabsorptive capacity. Additionally, more specific techniques such as immunoelectron microscopy using specific viral antigens (9), or in situ hybridization for viral RNA, are likely necessary to undoubtedly confirm tissue infection in …

Investigating the role of RME‐8 as a late onset Parkinson’s Disease marker in SH‐SY5Y neuroblastoma cells

Approximately, 10 million people worldwide live with the progressive, neurological Parkinson’s Disease (PD) experiencing a wide range of symptoms from resting tremors, bradykinesia, rigidity, to postural instability. Recent research has characterized a new mutation in Receptor‐Mediated Endocytosis 8 (RME‐8)/DNAJC13 Asn855Ser conferring a toxic gain‐of‐function resulting in impairment of endosomal transport. Furthermore, in the late‐onset of idiopathic PD, subtle deficits in endosomal receptor‐sorting/recycling are evident through the discover of pathogenic mutations of VPS35, LRRK2, and now DNAJC13/RME‐8.RME‐8 is composed of 2000 amino acids, containing four IWN repeats of unknown functions and a DNAJ binding between the second and third repeat. Through the discovery of genetic screens of Caenorhabditis elegans, RME‐8 mutants exhibited defects in receptor mediated yolk endocytosis in the oocyte and fluid phase endocytosis in the coelomocyte. The continued studies of RME‐8 mutants shows that it is highly conserved. Our laboratory is interested in studying RME‐8 in the context of Parkinson’s and further investigate the effect of RME‐8 N855S in the disease relevant SH‐SY5Y neuroblastoma cell line.