Surface chemistry of nanoparticles play significant role in their cellular interaction. Along with other group, we previously demonstrated that dynamic alteration of cell membrane during uptake of gold nanoparticles can be thoroughly probed by nanomechanical properties of cell membrane. Additionally, endocytosis influences intracellular cytokines expression that also impact membrane stiffness. Hence, we have hypothesized that surface chemistry of gold nanoparticles influences intracellular cytokines which in turn imparts dynamic alteration of nanomechanical properties of cellular membrane of pancreatic cancer cells. Various gold nanoparticles decorated with targeting peptide, polyethylene glycol or their combinations have been used to treat two pancreatic cancer cell lines, Panc-1 and AsPC1, for 1 and 24 hours. Atomic force microscope is used to measure linear and nonlinear nanomechanical properties of cell membrane. Intracellular cytokine has been measured using real time polymeric chain reaction. We evaluated several criteria such as receptor dependent vs independent, PEGylated vs non-PEGylated and different timepoints, to deduce correlations between cytokines and nanomechanical attributes. We have identified unique relationship pro-tumorigenic cytokines with both linear and non-linear nanomechanical properties of Panc-1 and AsPC1 cell membrane during uptake of pristine gold nanoparticles or for PEGylation and for targeting peptide conjugation at the nanoparticle surface.

Uveal melanoma is an ocular tumor with a high risk of developing metastases. The endo-lysosomal system can affect the melanoma progression by accelerating and facilitating invasion or metastasis. This study aims to conduct comparative analysis of normal choroidal melanocytes and uveal melanoma cells ultrastructure with a focus on intracellular transport system, and to examine the patterns of autophagy- and vesicular trafficking-related proteins expression in a case series of uveal melanomas. Transmission electron microscopy was used to assess the ultrastructure of normal choroidal melanocytes and uveal melanoma cells. The expression levels of autophagy- and vesicular trafficking-related proteins in three histological types of uveal melanoma were analyzed by immunofluorescence staining. Electron microscopy results showed that the autophagic vacuoles were more abundant in normal choroidal melanocytes, than in uveal melanoma cells. The normal choroidal melanocytes were characterized by active intracellular vesicular trafficking; however, the proportion of caveolae was higher in uveal melanoma cells. The spindle type of tumor was characterized by a high expression levels of LC3 beta, while Rab7 and Rab11 proteins expression was significantly up-regulated in the mixed-type tumor cells. The results indicate that uveal melanoma cells probably have lower basal levels of autophagy and higher receptor-mediated endocytic trafficking-associated with caveolae than normal choroidal melanocytes. RESEARCH HIGHLIGHTS: The autophagic vacuoles are abundant in normal choroidal melanocytes. Uveal melanoma cells are characterized by a high proportion of caveolae. The high expression levels of LC3 beta were revealed in a spindle type of tumor, while Rab7 and Rab11 proteins expression was up-regulated in the mixed-type tumor cells.

The clearance of brain interstitial fluid (ISF) is important in maintaining brain homeostasis. ISF clearance impairment leads to toxic material accumulation in the brain, and ischemic stroke could impair ISF clearance. The present study investigates ISF clearance under normal and ischemic conditions. The carboxylate-modified FluoSpheres beads (0.04 μm in diameter) were injected into the striatum. Sham or transient middle cerebral artery occlusion surgeries were performed on the mice. The brain sections were immunostained with cell markers, and bead distribution at various time points was examined with a confocal microscope. Primary mouse neuronal cultures were incubated with the beads to explore in vitro endocytosis.  Two physiological routes for ISF clearance were identified. The main one was to the lateral ventricle (LV) through the cleft between the striatum and the corpus callosum (CC)/external capsule (EC), where some beads were captured by the ependymal macrophages and choroid plexus. An alternative and minor route was to the subarachnoid space through the CC/EC and the cortex, where some of the beads were endocytosed by neurons. After ischemic stroke, a significant decrease in the main route and an increase in the minor route were observed. Additionally, microglia/macrophages engulfed the beads in the infarction. In conclusion, we report that the physiological clearance of ISF and beads mainly passes through the cleft between the CC/EC and striatum into the LV, or alternatively through the cortex into the subarachnoid space. Stroke delays the main route but enhances the minor route, and microglia/macrophages engulf the beads in the infarction. Ischemic stroke impairs the clearance of brain interstitial fluid/beads. Under physiological conditions, the main route ( ① ) of interstitial fluid clearance is to the lateral ventricle, and the minor one ( ② ) is to the subarachnoid space. Ischemic stroke weakens the main route ( ① ), enhances the minor one ( ② ), and leads to microglial/macrophage phagocytosis within the infarction ( ③ ).

During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro, little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5β1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5β1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro. Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus-epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Nanoparticles have been widely used in biomedical applications such as gene/drug delivery, molecular imaging and diagnostics. Among the physicochemical properties, shape is a vital design parameter for tuning the cell uptake of nanoparticles. However, the regulatory mechanism remains elusive due to the complexity of the cell membrane and multiple pathways of cell uptake. Therefore, in this computational study, we design and clarify cell membrane wrapping on different shaped nanoparticles (sphere, rod and disk) with a clathrin assembly to model the clathrin-mediated endocytosis, which is an important pathway of nanoparticle cell uptake. Our simulations revealed that the clathrin-mediated endocytosis is shape sensitive for nanoparticles. Spherical nanoparticles are easier to be wrapped by the membrane with the self-assembly of clathrins than the other shaped nanoparticles with the same volume, and the efficiency declines with the increase in the nanoparticle shape anisotropy. Furthermore, simulation results showed clear evidence that rotation is one of the typical characteristics determining the kinetics of clathrin-mediated endocytosis of shaped nanoparticles. Especially for rod nanoparticles with high aspect ratios, nanoparticle rotation occurs in both the invagination and wrapping stages, which is different from the case without clathrins. The size and shape mismatch between the clathrin-mediated vesicle and the nanoparticle determines how the nanoparticle rotates and is wrapped by the membrane. In addition, the wrapping time of nanoparticles depends not only on the shape of the nanoparticle but also on its initial orientation and size, the rate of clathrin self-assembly and the surface tension of the membrane. These results provide insights into the interplay between cell membrane wrapping and clathrin assembly, where the nanoparticle shape matters. Understanding the dynamics mechanism of clathrin-mediated endocytosis of nanoparticles will help to design targeted nanomedicines with an improved efficacy.

The steroidogenic activity of the granulosa cells is important for the reproductive cycle, and lipoproteins are involved in this process. The clathrin-mediated endocytosis pathway for LDL transport is considered to be the main one in eukaryotic cells. However, there are no studies that elucidate LDL internalization in human granulosa cells clarifying whether the clathrin-mediated endocytic pathway is functional in this process. The aim of this study is to investigate the role of clathrin and v-SNARE proteins in the formation of vesicles in human granulosa cells. In this study, the COV434 human granulosa cells were cultured and divided into four groups where in some of the groups Dil-conjugated LDL and Icarugamycin (ICA) a clathrin-mediated endocytosis inhibitor were added. From the collected mediums pregnenolone and progesterone levels were measured using ELISA. Oil red O staining was performed to show the intracellular lipids in the cells. Clathrin-coated vesicles believed to be responsible for carrying LDL, and v-SNARE proteins that direct the vesicles to their target molecules were also labeled and investigated by histological and ultrastructural methods. Our results show that human granulosa cells as well use the LDL cholesterol for steroid biosynthesis and they may prefer the clathrin-mediated endocytotic pathway to internalize it.

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways through permeabilization of IV membranes and the formation of endosomes and lysosomes. A method named chromophore-assisted laser inactivation (CALI) is applied to study the formation of IVs and the materials in controlling IV regulation. CALI is an imaging-based photodynamic methodology to study the signaling pathway induced by membrane permeabilization. The method allows spatiotemporal manipulation of the selected organelle to be permeabilized in a cell. The CALI method has been applied to observe and monitor specific molecules through the permeabilization of endosomes and lysosomes. The membrane rupture of IVs is known to selectively recruit glycan-binding proteins, such as galectin-3. Here, the protocol describes the induction of IV rupture by AlPcS2a and the use of galectin-3 as a marker to label impaired lysosomes, which is useful in studying the downstream effects of IV membrane rupture and their downstream effects under various situations.

BACKGROUND: Blood-brain barrier (BBB) disruption is a major adverse event after ischemic stroke (IS). Caveolin-1 (Cav-1), a scaffolding protein, played multiple roles in BBB permeability after IS, while the pros and cons of Cav-1 on BBB permeability remain controversial. Numerous studies revealed that extracellular vesicles (EVs), especially stem cells derived EVs, exerted therapeutic efficacy on IS; however, the mechanisms of BBB permeability needed to be clearly illustrated. Herein, we compared the protective efficacy on BBB integrity between bone marrow mesenchymal stem cells derived extracellular vesicles (BMSC-EVs) and EVs from brain endothelial cells (BEC-EVs) after acute IS and investigated whether the mechanism was associated with EVs antagonizing Cav-1-dependent tight junction proteins endocytosis.
METHODS: BMSC-EVs and BEC-EVs were isolated and characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscope. Oxygen and glucose deprivation (OGD) treated b. End3 cells were utilized to evaluate brain endothelial cell leakage. CCK-8 and TRITC-dextran leakage assays were used to measure cell viability and transwell monolayer permeability. Permanent middle cerebral artery occlusion (pMCAo) model was established, and EVs were intravenously administered in rats. Animal neurological function tests were applied, and microvessels were isolated from the ischemic cortex. BBB leakage and tight junction proteins were analyzed by Evans Blue (EB) staining and western blotting, respectively. Co-IP assay and Cav-1 siRNA/pcDNA 3.1 vector transfection were employed to verify the endocytosis efficacy of Cav-1 on tight junction proteins.
RESULTS: Both kinds of EVs exerted similar efficacies in reducing the cerebral infarction volume and BBB leakage and enhancing the expressions of ZO-1 and Claudin-5 after 24 h pMCAo in rats. At the same time, BMSC-EVs were outstanding in ameliorating neurological function. Simultaneously, both EVs treatments suppressed the highly expressed Cav-1 in OGD-exposed b. End3 cells and ischemic cerebral microvessels, and this efficacy was more prominent after BMSC-EVs administration. Cav-1 knockdown reduced OGD-treated b. End3 cells monolayer permeability and recovered ZO-1 and Claudin-5 expressions, whereas Cav-1 overexpression aggravated permeability and enhanced the colocalization of Cav-1 with ZO-1 and Claudin-5. Furthermore, Cav-1 overexpression partly reversed the lower cell leakage by BMSC-EVs and BEC-EVs administrations in OGD-treated b. End3 cells.
CONCLUSIONS: Our results demonstrated that Cav-1 aggravated BBB permeability in acute ischemic stroke, and BMSC-EVs exerted similar antagonistic efficacy to BEC-EVs on Cav-1-dependent ZO-1 and Claudin-5 endocytosis. BMSC-EVs treatment was superior in Cav-1 suppression and neurological function amelioration.

As the most common cancer in women, efforts have been made to develop novel nanomedicine-based therapeutics for breast cancer. In the present study, the in silico curcumin (Cur) properties were investigated, and we found some important drawbacks of Cur. To enhance cancer therapeutics of Cur, three different nonionic surfactants (span 20, 60, and 80) were used to prepare various Cur-loaded niosomes (Nio-Cur). Then, fabricated Nio-Cur were decorated with folic acid (FA) and polyethylene glycol (PEG) for breast cancer suppression. For PEG-FA@Nio-Cur, the gene expression levels of Bax and p53 were higher compared to free drug and Nio-Cur. With PEG-FA-decorated Nio-Cur, levels of Bcl2 were lower than the free drug and Nio-Cur. When MCF7 and 4T1 cell uptake tests of PEG-FA@Nio-Cur and Nio-Cur were investigated, the results showed that the PEG-FA-modified niosomes exhibited the most preponderant endocytosis. In vitro experiments demonstrate that PEG-FA@Nio-Cur is a promising strategy for the delivery of Cur in breast cancer therapy. Breast cancer cells absorbed the prepared nanoformulations and exhibited sustained drug release characteristics.