scholarly journals Single-molecule live-cell imaging of clathrin-based endocytosis.

2005 ◽  
Vol 72 ◽  
pp. 71-76 ◽  
Author(s):  
Tomas Kirchhausen ◽  
Werner Boll ◽  
Antoine van Oijen ◽  
Marcelo Ehrlich
Keyword(s):  
Single Molecule ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Coated Pits ◽  
Constant Growth Rate ◽  
Nucleation Sites ◽  
Real Time Visualization ◽  
Time Required ◽  
Constant Growth

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.

scholarly journals The Adaptor Protein ARH Escorts Megalin to and through Endosomes

2003 ◽  
Vol 14 (12) ◽  
pp. 4984-4996 ◽  
Author(s):  
Masaaki Nagai ◽  
Timo Meerloo ◽  
Tetsuro Takeda ◽  
Marilyn Gist Farquhar
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Coated Pits ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Autosomal Recessive Hypercholesterolemia

Megalin is an endocytic receptor that binds multiple ligands and is essential for many physiological processes such as brain development and uptake of proteins by the kidney tubule, yolk sac, and thyroid. The cytoplasmic tail of megalin contains two FXNPXY motifs. Autosomal recessive hypercholesterolemia (ARH) is an adaptor protein that binds to the FXNPXY motif of the low-density lipoprotein receptor as well as clathrin and AP-2. We found that ARH also binds to the first FXNPXY motif of megalin in two-hybrid, pull-down and coimmunoprecipitation assays. ARH colocalizes with megalin in clathrin coated pits and in recycling endosomes in the Golgi region. When cells are treated with nocodazole, the recycling endosomes containing megalin and ARH disperse. On internalization of megalin, ARH and megalin are first seen in clathrin coated pits followed by sequential localization in early endosomes and tubular recycling endosomes in the pericentriolar region followed by their reappearance at the cell surface. Expression of ARH in Madin-Darby canine kidney cells expressing megalin mini-receptors enhances megalin-mediated uptake of125I-lactoferrin, a megalin ligand. These results show that ARH facilitates endocytosis of megalin, escorts megalin along its endocytic route and raise the possibility that transport through the endosomal system is selective and requires interaction with specific adaptor proteins.

scholarly journals Receptor Clustering Is Involved in Reelin Signaling

2004 ◽  
Vol 24 (3) ◽  
pp. 1378-1386 ◽  
Author(s):  
Vera Strasser ◽  
Daniela Fasching ◽  
Christoph Hauser ◽  
Harald Mayer ◽  
Hans H. Bock ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Low Density Lipoprotein ◽  
Hippocampal Slices ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Long Term Potentiation ◽  
Hek293 Cells ◽  
Akt Phosphorylation ◽  
Reelin Signaling

ABSTRACT The Reelin signaling cascade plays a crucial role in the correct positioning of neurons during embryonic brain development. Reelin binding to apolipoprotein E receptor 2 (ApoER2) and very-low-density-lipoprotein receptor (VLDLR) leads to phosphorylation of disabled 1 (Dab1), an adaptor protein which associates with the intracellular domains of both receptors. Coreceptors for Reelin have been postulated to be necessary for Dab1 phosphorylation. We show that bivalent agents specifically binding to ApoER2 or VLDLR are sufficient to mimic the Reelin signal. These agents induce Dab1 phosphorylation, activate members of the Src family of nonreceptor tyrosine kinases, modulate protein kinase B/Akt phosphorylation, and increase long-term potentiation in hippocampal slices. Induced dimerization of Dab1 in HEK293 cells leads to its phosphorylation even in the absence of Reelin receptors. The mechanism for and the sites of these phosphorylations are identical to those effected by Reelin in primary neurons. These results suggest that binding of Reelin, which exists as a homodimer in vivo, to ApoER2 and VLDLR induces clustering of ApoER2 and VLDLR. As a consequence, Dab1 becomes dimerized or oligomerized on the cytosolic side of the plasma membrane, constituting the active substrate for the kinase; this process seems to be sufficient to transmit the signal and does not appear to require any coreceptor.

scholarly journals The low-density lipoprotein receptor-related protein 1 (LRP1) mediates the endocytosis of the cellular prion protein

2007 ◽  
Vol 402 (1) ◽  
pp. 17-23 ◽  
Author(s):  
David R. Taylor ◽  
Nigel M. Hooper
Keyword(s):  
Prion Protein ◽  
Low Density Lipoprotein ◽  
Neuronal Cells ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Cellular Prion Protein ◽  
Low Density ◽  
Related Protein ◽  
Density Lipoprotein Receptor

PrPC (cellular prion protein) is located at the surface of neuronal cells in detergent-insoluble lipid rafts, yet is internalized by clathrin-dependent endocytosis. As PrPC is glycosyl-phosphatidylinositol-anchored, it requires a transmembrane adaptor protein to connect it to the clathrin endocytosis machinery. Using receptor-associated protein and small interfering RNA against particular LDL (low-density lipoprotein) family members, in combination with immunofluorescence microscopy and surface biotinylation assays, we show that the transmembrane LRP1 (LDL receptor-related protein 1) is required for the Cu2+-mediated endocytosis of PrPC in neuronal cells. We show also that another LRP1 ligand that can cause neurodegenerative disease, the Alzheimer's amyloid precursor protein, does not modulate the endocytosis of PrPC.

scholarly journals Single molecule imaging of assembly of adaptor protein AP2 into clathrin-coated pits

2003 ◽  
Vol 43 (supplement) ◽  
pp. S102
Author(s):  
T. Fujiwara ◽  
T. Kobayashi ◽  
James Keen ◽  
A. Kusumi
Keyword(s):  
Single Molecule ◽  
Adaptor Protein ◽  
Single Molecule Imaging ◽  
Coated Pits

scholarly journals Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes

2013 ◽  
Vol 200 (4) ◽  
pp. 419-428 ◽  
Author(s):  
Ingyu Kim ◽  
Weijun Pan ◽  
Sara A. Jones ◽  
Youxin Zhang ◽  
Xiaowei Zhuang ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Super Resolution ◽  
Adaptor Protein ◽  
Related Protein ◽  
Cell Surfaces ◽  
Wnt Proteins ◽  
Canonical Wnt ◽  
Wnt3a Stimulation

Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P2) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P2 regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P2, and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P2 hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P2 promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to β-catenin stabilization.

scholarly journals Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin

2000 ◽  
Vol 347 (3) ◽  
pp. 613-621 ◽  
Author(s):  
Andrew V. OLEINIKOV ◽  
Jun ZHAO ◽  
Sudesh P. MAKKER
Keyword(s):  
Signal Transduction ◽  
Hybrid System ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Human Kidney ◽  
Negative Regulator ◽  
Interaction Domain ◽  
Two Hybrid

gp600/megalin, an endocytic receptor, belongs to the low-density lipoprotein receptor family. It is most abundant in the renal proximal tubular cells, where it is implicated in the reabsorption of a number of molecules filtered through the glomerulus. The cytoplasmic tail (CT) of gp600/megalin contains a number of sequence similarities, which indicate that gp600/megalin might be involved in signal transduction. To find intracellular proteins that would interact with the gp600/megalin CT, a human kidney cDNA library was screened by using the yeast two-hybrid system. The phosphotyrosine interaction domain (PID) of the Disabled protein 2 (Dab2), a mammalian structural analogue of Drosophila Disabled, was found to bind to the gp600/megalin CT in this system. The interaction between these two proteins was confirmed by a binding assay in vitro and by the co-immunoprecipitation of both proteins from renal cell lysates. The gp600/megalin CT contains three ΨXNPXY motifs (in which Ψ represents a hydrophobic residue) that are potentially able to interact with PID. Analysis of the CT deletion and point-mutation variants of gp600/megalin by the two-hybrid system revealed that the third ΨXNPXY motif is most probably involved in this interaction. Dab2 is a mitogen-responsive phosphoprotein thought to be an adaptor molecule involved in signal transduction, and a suggested negative regulator of cell growth. Dab2 is the first intracellular ligand identified for gp600/megalin; gp600/megalin is the first known transmembrane receptor that interacts with the cytosolic protein Dab2. We speculate that their interaction might involve gp600/megalin in signal transduction pathways or might mediate the intracellular trafficking of this receptor.

scholarly journals Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis

2017 ◽  
Vol 37 (11) ◽  
pp. 2043-2052 ◽  
Author(s):  
Paola M. Marcovecchio ◽  
Graham D. Thomas ◽  
Zbigniew Mikulski ◽  
Erik Ehinger ◽  
Karin A.L. Mueller ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Fold Increase ◽  
Vascular Wall ◽  
Western Diet ◽  
Oxidized Lipoprotein

Objective— Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature. Approach and Results— To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE −/− (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet–fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36 −/− mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein. Conclusions— Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.

scholarly journals Molecular Characterization of Familial Hypercholesterolemia in a North American Cohort

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Abhimanyu Garg ◽  
Sergio Fazio ◽  
P Barton Duell ◽  
Alexis Baass ◽  
Chandrasekhar Udata ◽  
...  
Keyword(s):  
Familial Hypercholesterolemia ◽  
North American ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
The United States ◽  
Serum Triglycerides ◽  
Premature Cardiovascular Disease ◽  
Density Lipoprotein Receptor ◽  
North American Cohort

Abstract Background Familial hypercholesterolemia (FH) confers a very high risk of premature cardiovascular disease and is commonly caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9) and very rarely in LDLR adaptor protein 1 (LDLRAP1) genes. Objective To determine the prevalence of pathogenic mutations in the LDLR, APOB, and PCSK9 in a cohort of subjects who met Simon Broome criteria for FH and compare the clinical characteristics of mutation-positive and mutation-negative subjects. Methods Ninety-three men and 107 women aged 19 to 80 years from lipid clinics in the United States and Canada participated. Demographic and historical data were collected, physical examination performed, and serum lipids/lipoproteins analyzed. Targeted sequencing analyses of LDLR and PCSK9 coding regions and exon 26 of APOB were performed followed by detection of LDLR deletions and duplications. Results Disease-causing LDLR and APOB variants were identified in 114 and 6 subjects, respectively. Of the 58 LDLR variants, 8 were novel mutations. Compared with mutation-positive subjects, mutation-negative subjects were older (mean 49 years vs 57 years, respectively) and had a higher proportion of African Americans (1% vs 12.5%), higher prevalence of hypertension (21% vs 46%), and higher serum triglycerides (median 86 mg/dL vs 122 mg/dL) levels. Conclusions LDLR mutations were the most common cause of heterozygous FH in this North American cohort. A strikingly high proportion of FH subjects (40%) lacked mutations in known culprit genes. Identification of underlying genetic and environmental factors in mutation-negative patients is important to further our understanding of the metabolic basis of FH and other forms of severe hypercholesterolemia.

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