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The blood-urine barrier is the tightest and most impermeable barrier in the body and as such represents a problem for intravesical drug delivery applications. Differentiation-dependent low endocytotic rate of urothelial cells has already been noted; however, the differences in endocytosis of normal and cancer urothelial cells have not been exploited yet. Here we analysed the endocytosis of rhodamine B isothiocyanate-labelled polyacrylic acid-coated cobalt ferrite nanoparticles (NPs) in biomimetic urothelial in vitro models, i.e., in highly and partially differentiated normal urothelial cells, and in cancer cells of the papillary and invasive urothelial neoplasm. We demonstrated that NPs enter papillary and invasive urothelial neoplasm cells by ruffling of the plasma membrane and engulfment of NP aggregates by macropinocytotic mechanism. Transmission electron microscopy (TEM) and spectrophotometric analyses showed that the efficacy of NPs delivery into normal urothelial cells and intercellular space is largely restricted, while it is significantly higher in cancer urothelial cells. Moreover, we showed that the quantification of fluorescent NP internalization in cells or tissues based on fluorescence detection could be misleading and overestimated without TEM analysis. Our findings contribute to the understanding of endocytosis-mediated cellular uptake of NPs in cancer urothelial cells and reveal a highly selective mechanism to distinguish cancer and normal urothelial cells.

During differentiation, superficial urothelial cells (UCs) of the urinary bladder form the apical surface, which is almost entirely covered by urothelial plaques containing densely packed uroplakin particles. These urothelial plaques are the main structural components of the blood-urine permeability barrier in the urinary bladder. We have shown previously that endocytosis from the apical plasma membrane decreases during urothelial cell differentiation. Here, we investigated the role of actin filament and microtubule rearrangements in apical endocytosis of differentiating UCs cells using hyperplastic and normoplastic porcine urothelial models. Partially differentiated normal porcine UCs contained actin filaments in the subapical cytoplasm, while microtubules had a net-like appearance. In highly differentiated UCs, actin filaments mostly disappeared from the subapical cytoplasm and microtubules remained as a thin layer close to the apical plasma membrane. Inhibition of actin filament formation with cytochalasin-D in partially differentiated UCs caused a decrease in apical endocytosis. Depolymerisation of microtubules with nocodazole did not prevent endocytosis of the endocytotic marker WGA into the subapical cytoplasm; however, it abolished WGA transport to endolysosomal compartments in the central cytoplasm. Cytochalasin-D or nocodazole treatment did not significantly change apical endocytosis in highly differentiated UCs. In conclusion, we showed that the physiological differentiation-dependent or chemically induced redistribution and reorganization of actin filaments and microtubules impair apical endocytosis in UCs. Importantly, reduced apical endocytosis due to cytoskeletal rearrangements in highly differentiated UCs, together with the formation of rigid urothelial plaques, reinforces the barrier function of the urothelium.

Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours' exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours' exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP-cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo.

The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5-15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.

The Golgi complex undergoes considerable structural remodeling during differentiation of urothelial cells in vivo and in vitro. It is known that in a healthy bladder the differentiation from the basal to the superficial cell layer leads to the formation of the tightest barrier in our body, i.e., the blood–urine barrier. In this process, urothelial cells start expressing tight junctional proteins, apical membrane lipids, surface glycans, and integral membrane proteins, the uroplakins (UPs). The latter are the most abundant membrane proteins in the apical plasma membrane of differentiated superficial urothelial cells (UCs) and, in addition to well-developed tight junctions, contribute to the permeability barrier by their structural organization and by hindering endocytosis from the apical plasma membrane. By studying the transport of UPs, we were able to demonstrate their differentiation-dependent effect on the Golgi architecture. Although fragmentation of the Golgi complex is known to be associated with mitosis and apoptosis, we found that the process of Golgi fragmentation is required for delivery of certain specific urothelial differentiation cargoes to the plasma membrane as well as for cell–cell communication. In this review, we will discuss the currently known contribution of the Golgi complex to the formation of the blood–urine barrier in normal UCs and how it may be involved in the loss of the blood–urine barrier in cancer. Some open questions related to the Golgi complex in the urothelium will be highlighted. [ABSTRACT FROM AUTHOR]

Superficial urothelial cells that line the urinary bladder accommodate cyclical changes in organ volume while maintaining a permeability barrier between urine and tissue fluids. The specific apical plasma membrane traffic is necessary for their proper function. The composition of the apical plasma membrane is dramatically modified during differentiation of bladder urothelial cells, most notably by assembly of urothelial plaques containing uroplakins. However, the assembly of uroplakins into plaques, their insertion and removal from the apical surface, and the regulation of these processes are still poorly understood. This review examines the traffic (exocytosis/endocytosis) of the apical plasma membrane during differentiation of urothelial cells and focuses on the physiological and clinical significance of the apical plasma membrane traffic in bladder superficial urothelial cells. [ABSTRACT FROM AUTHOR]

Normal porcine urothelial cells have been shown to have a much lower rate of endocytosis than urothelial papillary neoplasm cells. This could be used as a mechanism for selective delivery of toxic compounds, such as polyethyleneimine coated nanoparticles (NPs). However, these NPs induce nonselective toxicity through direct membrane disruption. This toxicity can be reduced by functionalization of NPs with L-glutathione reduced or bovine serum albumin by reducing their surface charge. Functionalization was confirmed with Fourier Transform Infrared Spectroscopy, Dynamic Light Scattering and zeta potential measurements. Viability assays showed that bovine serum albumin coating reduced NPs cytotoxicity immediately after 3 h exposure and that such NPs were more toxic to urothelial papillary neoplasm cells compared to normal porcine urothelial cells at 50 µg/ml NPs concentration. However, 24 h after exposure, bovine serum albumin functionalized NPs had similar effect on viability of both cell lines. NPs showed some selective toxicity towards urothelial papillary neoplasm cells compared to normal cells after 3 h, however this was not confirmed after 24 h. [ABSTRACT FROM AUTHOR]

Abstract: Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca2+-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca2+-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles. [Copyright &y& Elsevier]