Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae

BACKGROUND: A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae. RESULTS: A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked. CONCLUSIONS: Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

Phagocytosis and Endocytosis of Silver Nanoparticles Induce Interleukin-8 Production in Human Macrophages

Phagocytosis or endocytosis by macrophages is critical to the uptake of fine particles, including nanoparticles, in order to initiate toxic effects in cells. Here, our data enhance the understanding of the process of internalization of silver nanoparticles by macrophages. When macrophages were pre-treated with inhibitors to phagocytosis, caveolin-mediated endocytosis, or clathrin-mediated endocytosis, prior to exposure to silver nanoparticles, Interleukin-8 (IL-8) production was inhibited. Although cell death was not reduced, the inflammatory response by macrophages was compromised by phagocytosis and endocytosis inhibitors.

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.

Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56 percent lipids and 9 and 7 percent carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Increased endocytic activity in monocyte-derived dendritic cells in patients with psoriasis vulgaris.

BACKGROUND & OBJECTIVE: The understanding of the pathogenesis of psoriasis vulgaris has focused on T cell mediated immune disorder for many years. Recent studies provide evidence that dendritic cells may be of major importance as regulatory cells driving the psoriasis tissue reaction, and they are one of the therapeutic targets. In order to further characterize the role of dendritic cells in psoriasis, this study was designed to assess the differentiation of dendritic cells from monocytes (MoDC), the expression of phagocytosis related receptors by MoDC, their endocytic activity for fluorescent beads and lucifer yellow as well as their superoxide generation in patients with psoriasis. METHODS: Twenty eight patients with psoriasis vulgaris and 12 healthy controls were included in the study. MoDC were obtained by culturing monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days. Cell surface expression of CD1a, CD14, CD40, CD80, CD83, CD86, HLA-DR, mannose receptor (MR) and Fcg receptors by MoDC and their endocytosis of dextran and lucifer yellow were analyzed by flow cytometry. Zymosan ingestion was measured to access the phagocytosis of MoDC. RESULTS: Differentiation of monocytes to dendritic cells was upregulated in patients manifested as significantly increased expression of CD40, CD80, CD86 and HLA-DR compared with that in healthy controls (P<0.01). Expression of MR and Fcg receptor II (CD32) by MoDC was significantly increased in patients with psoriasis as well (P<0.01). Endocytosis of dextran but not lucifer yellow in patients was significantly higher than controls (P<0.01), and significantly enhanced phagocytosis by increasing zymosan ingestion was also observed (P<0.01) in patients. Taken together, endocytic and phagocytic activity of MoDC in psoriasis was increased than normal persons. INTERPRETATION & CONCLUSION: Enhanced activity of dendritic cells binding and capturing foreign antigens for subsequent antigen presentation and the initiation of immune responses in psoriasis may contribute to the pathogenesis of the disease. The upregulated expression of MR and the enhanced endocytic activity of DC might be an explanation for the absence of skin infection observed in psoriasis.

Cellular uptake of magnetic nanoparticle is mediated through energydependent endocytosis in A549 cells

Biocompatible silica-overcoated magnetic nanoparticles containing an organic fluorescence dye, rhodamine B isothiocyanate (RITC), within a silica shell [50 nm size, MNP@SiO2(RITC)s] were synthesized. For future application of the MNP@SiO2(RITC)s into diverse areas of research such as drug or gene delivery, bioimaging, and biosensors, detailed information of the cellular uptake process of the nanoparticles is essential. Thus, this study was performed to elucidate the precise mechanism by which the lung cancer cells uptake the magnetic nanoparticles. Lung cells were chosen for this study because inhalation is the most likely route of exposure and lung cancer cells were also found to uptake magnetic nanoparticles rapidly in preliminary experiments. The lung cells were pretreated with different metabolic inhibitors. Our results revealed that low temperature disturbed the uptake of magnetic nanoparticles into the cells. Metabolic inhibitors also prevented the delivery of the materials into cells. Use of TEM clearly demonstrated that uptake of the nanoparticles was mediated through endosomes. Taken together, our results demonstrate that magnetic nanoparticles can be internalized into the cells through an energy-dependent endosomal-lysosomal mechanism.

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization

Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells

Calcium oxalate (CaOx) crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively) to the appearance of urinary calculi. The present study analyzes a series of mechanisms possibly involved in CaOx uptake by Madin-Darby canine kidney (MDCK) cells. CaOx crystals were added to MDCK cell cultures and endocytosis was evaluated by polarized light microscopy. This process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nM; N = 6; 43.9 percent inhibition; P<0.001) or thapsigargin (1 µM; N = 6; 33.3 percent inhibition; P<0.005) administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 µM; N = 8; 46.1 percent inhibition; P<0.001) or cytochalasin B (10 µM; N = 8; 34.2 percent inhibition; P<0.001). Furthermore, CaOx uptake was reduced when the activity of protein kinase C was inhibited by staurosporine (10 nM; N = 6; 44 percent inhibition; P<0.01), or that of cyclo-oxygenase by indomethacin (3 µM; N = 12; 17.2 percent inhibition; P<0.05); however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 µM; N = 6), tetraethylammonium (1 mM; N = 6) or cromakalim (1 µM; N = 6). Taken together, these data indicate that the process of CaOx internalization by renal tubular cells is similar to the endocytosis reported for other systems. These findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones

Vesicular transport as a new paradigm in short-term regulation of transepithelial transport

The vectorial transepithelial transport of water and electrolytes in the renal epithelium is achieved by the polarized distribution of various transport proteins in the apical and basolateral membrane. The short-term regulation of transepithelial transport has been traditionally thought to be mediated by kinetic alterations of transporter without changing the number of transporters. However, a growing body of recent evidence supports the possibility that the stimulus-dependent recycling of transporter-carrying vesicles can alter the abundance of transporters in the plasma membrane in parallel changes in transepithelial transport functions. The abundance of transporters in the plasma membrane is determined by net balance between stimulus-dependent exocytic insertion of transporters into and endocytic retrieval of them from the plasma membrane. The vesicular recycling occurs along the tracts of the actin microfilaments and microtubules with associated motors. This review is to highlight the importance of vesicular transport in the short-term regulatory process of transepithelial transport in the renal epithelium. In the short-term regulation of many other renal transporters, vesicular transport is likely to be also involved. Thus, vesicular transport is now emerged as a wide-spread general regulatory mechanism involved in short-term regulation of renal functions.