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Functional Study of Lysosomal Nutrient Transporters
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Xavier Leray, Corinne Sagné, Bruno Gasnier
Designing a whole-cell assay of a given transporter thus consists in identifying, and mutating, candidate sorting motives in its primary sequence and assessing the impact of such mutations on its intracellular localization. Diverse classes of lysosomal sorting motives have been described, including the tyrosine-based motives YXXØ and NPXY, where X is any amino acid and Ø is a bulky amino acid, and the dileucine-based motives [D/E]XXXL[L/I] and DXXLL (Braulke and Bonifacino, 2009). YXXØ, NPXY and [D/E]XXXL[L/I] motives interact with heterotetrameric adaptor protein complexes (AP-1, AP-2 or AP-3), whereas DXXLL motives bind Golgi localized, γ-adaptin ear-containing, ARF-binding protein (GGA) components of the clathrin coat. The tyrosine residue and the pair of leucine/isoleucine are critical for these interactions. Mutating these residues to alanine or glycine is sufficient to disrupt recognition by the trafficking machinery. Lipid modification, phosphorylation or even unconventional sorting motifs can also affect intracellular sorting of lysosomal membrane proteins (Storch et al., 2004, 2007).
Internalization of Lipopolysaccharide by Phagocytes
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Richard L. Kitchens, Robert S. Munford
Wang et al. (36) reported that LPS binds to CD14 in low-density plasma membrane regions (fragments), which have biochemical similarities to the structures known as caveolae (37). Caveolae are small, often flask-shaped, non-clathrin-coated membrane invagi-nations found in many cell types (38–40). They (or the membrane regions in which they reside) are enriched in sphingomyelin and glycolipids, facilitating their isolation on various density gradient media (37,41). Caveolae typically are organized around one or more structural proteins called caveolins (VIP21) (42,43), and they may function as signaling centers for various extracellular ligands (44). In addition, caveolae may be portals for the internalization of ligands such as folate (potocytosis) (45) or the transcytosis of molecules such as albumin (46). Many GPI-anchored proteins seem to concentrate in (38), or near (44), caveolae. Like caveolae, the CD14-enriched membrane microdomains of THP-1 cells are also relatively enriched in several potential signaling molecules but depleted in β-adaptin, a coated pit protein (36). Moreover, some of the non-clathrin-coated invaginations (NCCI) of the monocyte plasma membrane morphologically resemble caveolae (30), although the NCCI are more often tubular than flask-shaped. Although caveolin has not been found in these cells, there is thus indirect evidence from cell fractionation studies as well as direct observations using electron microscopy (discussed above) that CD 14-dependent LPS uptake can proceed via caveolae-like membrane invaginations. Structures that resemble caveolae but lack caveolin have also been described in human lymphocytes (47); these NCCI internalize another GPI-anchored protein, CD59, after it is cross-linked with antibodies.
Clathrin-mediated integrin αIIbβ3 trafficking controls platelet spreading
Published in Platelets, 2018
Wen Gao, Panlai Shi, Xue Chen, Lin Zhang, Junling Liu, Xuemei Fan, Xinping Luo
Integrin endocytosis can be triggered by intracellular signaling at the extracellular surface of the cell through clathrin-dependent and clathrin-independent manners [3]. In clathrin-mediated integrin endocytosis, the integrin budding-in process is initiated by a pit coated with clathrin assisted by a set of cytoplasmic proteins including integrin cytoplasmic domains [5], dynamin [6], and adaptors [7] such as adaptin [8]. The pit is further converted into a short-life clathrin-coated vesicle [9]. Then, the coat can be shed, and the remaining vesicle fuses with endosomes and proceeds down the endocytic pathway [10]. Tubular actin-dependent recycling endosomes [11] and ARF6 pathways [12] have been described in integrin recycling back to the plasma membrane, but whether clathrin-mediated trafficking functions in this process has not been determined.
Mechanisms for cellular uptake of nanosized clinical MRI contrast agents
Published in Nanotoxicology, 2020
Emily J. Guggenheim, Joshua Z. Rappoport, Iseult Lynch
The Rab5-mRFP construct was a gift from Ari Helenius (Institute of Biochemistry, ETH, Zurich). The Rab11-mRFP construct was a gift from Prof. Thomas Kirchausen (Harvard Medical School, Boston, USA). A non-silencing control (NSC) siRNA (ON-TARGETplus NON-targeting siRNA; Dharmacon) designed for use with rat, mouse or human cell lines was employed as a negative control. The α-adaptin (AP2 inhibition) siRNA was custom made with target sequence: 5′-AAGAGCAUGUGCACGCUGGCCA-3′ as used in previous studies (Rappoport and Simon 2009). The caveolin-1 siRNA was a SMARTpool of 4 siRNAs (ON-TARGETplus Human Cav1 siRNA; Dharmacon) targeted to caveolin-1. The P21-protein activated kinase (PAK-1) siRNA was custom made with target sequence: 5′-UGAAUGUCUAGGCCGUUAU-3′.
Maternal hypercaloric diet affects factors involved in lipid metabolism and the endogenous cannabinoid systems in the hypothalamus of adult offspring: sex-specific response of astrocytes to palmitic acid and anandamide
Published in Nutritional Neuroscience, 2022
Patricia Rivera, Santiago Guerra-Cantera, Antonio Vargas, Francisca Díaz, Rocío García-Úbeda, Rubén Tovar, María Teresa Ramírez-López, Jesús Argente, Fernando Rodríguez de Fonseca, Juan Suárez, Julie A. Chowen
The hypothalamus or astrocyte cultures (n = 3–4) were homogenized in 500 µL of ice-cold lysis buffer containing Triton X-100, 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1 M ethylenediaminetetraacetic acid (EDTA), sodium pyrophosphate, sodium fluoride (NaF), sodium orthovanadate (NaOV) and protease inhibitors using a tissue-lyser system (Qiagen). After centrifuging at 26,000x g for 30 min at 4°C, the supernatant was transferred to a new tube. The Bradford method was used to measure the protein concentration of the samples. A quantity of 30 µg of each total protein sample was separated on 4–12% polyacrylamide gradient gels. The gels were then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) and stained with Ponceau red. Membranes were blocked in TBS-T (50mMTris-HCl pH 7.6, 200 mM NaCl, and 0.1% Tween 20) with 2% albumin fraction V from BSA (Roche, Mannheim, Germany) for 1 h at room temperature. The membranes were incubated overnight at 4°C with the primary antibodies to the proteins of interest (Supplementary Table 2). Mouse βactin or adaptin were used as the reference protein. After several washes in TBS-T containing 1% Tween 20, an HRP-conjugated anti-rabbit or anti-mouse IgG (H+L) secondary antibody (Promega, Madison, WI, USA) diluted 1 : 10,000 was added followed by incubation for 1 h at room temperature. After extensive washing in TBS-T, the membranes were incubated for 1 min with the Western Blotting Luminol Reagent kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the specific protein bands were visualized and quantified by chemiluminescence using a ChemiDocTM MP Imaging System (Bio-Rad, Barcelona, Spain). The results are expressed as the target protein/βactin or adaptin ratios.