Endocytic Uptake Pathways Research Articles

Effective Cell Transfection in An Ultrasonically Levitated Droplet for Sustainable Technology.

The levitation methodology, which enables us to operate a contactless reaction without a container, is likely to be a revolutionary technology in the fields of chemistry and biology to reduce the plastic waste in life science laboratories. Here, the authors show that plasmid DNA can be effectively transfected into animal cells in a floating droplet of culture medium levitated using ultrasonic standing waves. The data indicate that there is no significant damage to the plasmid and cells during the levitating transfection time, and the transgene expression efficiency and cellular uptake in the droplet are significantly higher than those in the conventional tube, with and without shaking. These results suggest the consolidation of the endocytic uptake pathway into macropinocytosis, indicating that ultrasonic levitation induced a change in cell characteristics. This study suggests that transfection methodology using ultrasonic levitation has the potential to advance the current experimental procedures in the field of cell engineering, in addition to presenting a revolutionary containerless reactor for sustainable technology.

Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons.

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors.

Intracellular trafficking and endocytic uptake pathway of Pepper vein banding virus-like particles in epithelial cells.

Aim: Plant virus-like particles (VLPs) have emerged as a novel platform for delivery of drugs/antibodies. The aim of the present investigation is to establish the entry mechanism of flexuous rod-shaped virus particles into mammalian cells. Methods: Far-Western blot analysis, pull-down and ELISA were used to characterize vimentin and Hsp60 interaction with VLPs. The mode/kinetics of internalization of VLPs was deciphered using pharmacological inhibitors/endosomal markers. Results & discussion: The flexuous rod-shaped VLPs of Pepper vein banding virus (PVBV) enter HeLa and HepG2 cells via cell-surface proteins: vimentin and Hsp60, respectively. VLPs internalize via different modes of endocytosis in HeLa, HepG2 cells and are biodegradable. Vimentin and Hsp60 could be potential epithelial ligands that facilitate targeting of nanoparticles to tumor cells.

The gene transfection and endocytic uptake pathways mediated by PEGylated PEI-entrapped gold nanoparticles

The development of gene vectors is the vital step in gene therapy. The cationic polymer polyethylenimine (PEI) is widely applied as an efficient and low cost nonviral gene vehicle. However, its further practical applications in gene therapy are limited due to its high cytotoxicity. To obtain safe and efficient gene vectors, hydrophilic polyethylene glycol (PEG) has been conjugated onto its surface due to its good biocompatibility, and the PEGylated PEI was used as a template to entrap gold nanoparticles (Au NPs) with different Au atom/PEI molar ratios (25:1, 50:1, 100:1, and 200:1, respectively). The formed PEGylated PEI-entrapped Au NPs (PEG-Au PENPs) and their cytotoxic effects as well as ability to transfect plasmid DNA (pDNAs) to HeLa cells were analyzed using Cell Counting Kit-8 (CCK-8) assay, flow cytometry and confocal microscopic imaging. To further understand cell internalization pathway of PEG-Au PENPs, several pharmacologic inhibitions of endocytic pathways were conducted. Our results revealed that the PEG-Au PENPs were not only able to transfect pDNAs into cells with decreased cytotoxicity, but also showed high transfection efficiency compared with PEI alone. The cellular uptake data indicated that the clathrin-mediated endocytosis is the main pathway in the internalization of the formed polyplexes. These findings suggested that the developed PEG-Au PENPs may serve as a safe gene carrier with non-compromised DNA transfection efficiency and promote the further development of efficient and safe gene delivery strategies based on nanoparticles.

Chitosan nanoparticles for the oral delivery of tenofovir disoproxil fumarate: formulation optimization, characterization and ex vivo and in vivo evaluation for uptake mechanism in rats

Objective: Design chitosan based nanoparticles for tenofovir disoproxil fumarate (TDF) with the purpose of enhancing its oral absorption.Significance: TDF is a prodrug that has limited intestinal absorption because of its susceptibility to gut wall esterases. Hence, design of chitosan based polymeric novel nanocarrier systems can protect TDF from getting metabolized and also enhance the oral absorption.Methods: The nanoparticles were prepared using the ionic gelation technique. The factors impacting the particle size and entrapment efficiency of the nanoparticles were evaluated using design of experiments approach. The optimized nanoparticles were characterized and evaluated for their ability to protect TDF from esterase metabolism. The nanoparticles were then studied for the involvement of active transport in their uptake during the oral absorption process. Further, in vivo pharmacokinetic studies were carried out for the designed nanoparticles.Results: The application of design of experiments in the optimization process was useful to determine the critical parameters and evaluate their interaction effects. The optimized nanoparticles had a particle size of 156 ± 5 nm with an entrapment efficiency of 48.2 ± 1%. The nanoparticles were well characterized and provided metabolic protection for TDF in the presence of intestinal esterases. The nanoparticles were able to increase the AUC of tenofovir by 380%. The active uptake mechanisms mainly involving clathrin-mediated uptake played a key role in increasing the oral absorption of tenofovir.Conclusions: These results show the ability of the designed chitosan based nanoparticles in enhancing the oral absorption of TDF along the oral route by utilizing the active endocytic uptake pathways.

Design of nanocarriers for efficient cellular uptake and endosomal release of small molecule and nucleic acid drugs: learning from virus

There are many challenges in developing efficient and target specific delivery systems of small molecule and nucleic acid drugs. Cell membrane presents one of the major barriers for the penetration of hydrophilic macromolecules across the plasma membrane. Nanocarriers have been designed to enhance their cellular uptake via endocytosis but following their cellular uptake, endosomal escape is the rate limiting step which restricts the value associated with the enhanced uptake by nanocarriers. Viruses are an excellent model for efficient cytosolic delivery by nanocarriers. Viruses exploit intracellular cues to release the genome to cytosol. In this review, we first discuss different endocytic uptake pathways and endosomal escape mechanisms. We then summarize the existing tools for studying the intracellular trafficking of nanocarriers. Finally, we highlight the important design elements of recent virus-based nanocarriers for efficient cellular uptake and endosomal escape.

The Productive Entry Pathway of HIV-1 in Macrophages Is Dependent on Endocytosis through Lipid Rafts Containing CD4

Macrophages constitute an important reservoir of HIV-1 infection, yet HIV-1 entry into these cells is poorly understood due to the difficulty in genetically manipulating primary macrophages. We developed an effective genetic approach to manipulate the sub-cellular distribution of CD4 in macrophages, and investigated how this affects the HIV-1 entry pathway. Pluripotent Stem Cells (PSC) were transduced with lentiviral vectors designed to manipulate CD4 location and were then differentiated into genetically modified macrophages. HIV-1 infection of these cells was assessed by performing assays that measure critical steps of the HIV-1 lifecycle (fusion, reverse transcription, and expression from HIV-1 integrants). Expression of LCK (which tethers CD4 to the surface of T cells, but is not normally expressed in macrophages) in PSC-macrophages effectively tethered CD4 at the cell surface, reducing its normal endocytic recycling route, and increasing surface CD4 expression 3-fold. This led to a significant increase in HIV-1 fusion and reverse transcription, but productive HIV-1 infection efficiency (as determined by reporter expression from DNA integrants) was unaffected. This implies that surface-tethering of CD4 sequesters HIV-1 into a pathway that is unproductive in macrophages. Secondly, to investigate the importance of lipid rafts (as detergent resistant membranes - DRM) in HIV-1 infection, we generated genetically modified PSC-macrophages that express CD4 mutants known to be excluded from DRM. These macrophages were significantly less able to support HIV-1 fusion, reverse-transcription and integration than engineered controls. Overall, these results support a model in which productive infection by HIV-1 in macrophages occurs via a CD4-raft-dependent endocytic uptake pathway.

Endocytic Uptake Pathways Utilized by CPMV Nanoparticles

Cowpea mosaic virus (CPMV) has been used as a nanoparticle platform for biomedical applications including vaccine development, in vivo vascular imaging, and tissue-targeted delivery. A better understanding of the mechanisms of CPMV targeting and cell internalization would enable enhanced targeting and more effective delivery. Previous studies showed that, following binding and internalization by mammalian cells, CPMV localizes in a perinuclear late-endosome compartment where it remains for as long as several days. To further investigate endocytic trafficking of CPMV within the cell, we used multiple approaches including pharmacologic inhibition of pathways and colocalization with endocytic vesicle compartments. CPMV internalization was clathrin-independent and utilized a combination of caveolar endocytosis and macropinocytosis pathways for entry. CPMV particles colocalized with Rab5(+) early endosomes to traffic ultimately to a lysosomal compartment. These studies facilitate the further development of effective intracellular drug-delivery strategies using CPMV.

Dynamic Exchange of Myosin VI on Endocytic Structures

The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.

The Endocytic Uptake Pathways of Targeted Toxins Are Influenced by Synergistically Acting Gypsophila Saponins

The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better.

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell's waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell's waltzer fibroblasts.

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.

Selective Disruption of Early/Recycling Endosomes: Release of Disulfide-Linked Cargo Mediated by a N-Alkyl-3β-Cholesterylamine-Capped Peptide

The use of endocytic uptake pathways to deliver poorly permeable molecules into mammalian cells is often plagued by entrapment and degradation of material in late endosomes and lysosomes. As a strategy to prevent the exposure of cargo to these highly hydrolytic membrane-sealed compartments, we synthesized derivatives of the membrane anchor N-alkyl-3beta-cholesterylamine that selectively target linked compounds to less hydrolytic early/recycling endosomes. By targeting a pH-dependent membrane-lytic dodecapeptide and a disulfide-linked fluorophore to these compartments in Chinese hamster ovary cells or Jurkat lymphocytes, membranes of early/recycling endosomes were selectively disrupted, resulting in cleavage of the disulfide and escape of the fluorophore into the cytosol and nucleus with low toxicity. The ability of appropriately designed N-alkyl-3beta-cholesterylamines to deliver cargo into and release disulfide-linked cargo from relatively nonhydrolytic early/recycling endosomes may be useful for the delivery of a variety of sensitive molecules into living mammalian cells.