The observation that over-expression of the EH domain or of the carboxy-terminal RalBP1 binding region of POB1/REPS2 results in a 30-40% reduction of EGF or insulin internalization has linked POB1 to the molecular machinery modulating regulated endocytosis [4]. More recently, it was reported that loss of POB1 expression during human prostate cancer progression, from androgen-dependent to growth factors dependent, results in loss of control of cell growth signaling while induced expression of POB1 causes a reduction of several EGF-responsive genes (e.g., Fos and Jun). In accordance, we find that an increase of POB1 isoform 2 expression correlates with a decrease of EGF-induced phosphorylation of Erk1-2 and Shc (Additional file 6). Moreover, POB1 isoform 2 down-regulation was observed during the progression of prostate cancer [7-9].. In our study we confirm the involvement of POB1 in EGF receptor endocytosis and we provide evidence for a functional role of the central proline-rich region. In fact, ...
A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity ...
In mitosis, a dramatic decrease in endocytosis rate has been reported (Berlin et al., 1978). We repeated those observations (Fig. 1) using either fluorescent dextran or FM1-43 uptake as a measure of endocytosis rate by measuring the number of vesicles per cell and the total fluorescence intensity. With fluorescent dextran, the endocytosis rate normally decreased to ∼10% of interphase level in metaphase. FM1-43 is a water-soluble dye that is membrane impermeable, but it is readily incorporated into endocytic vesicles and is retained after cell fixation (Betz and Bewick, 1992; Terasaki, 1995). Fig. 1, b-e shows FM1-43 labeling of HeLa cells in various stages of mitosis in parallel with the DAPI staining patterns that enable rapid identification of mitotic phases in HeLa cells (Fig. 1, b-e, bottom). While interphase cells were brightly stained with FM1-43-labeled endocytic vesicles spread throughout the cytoplasm (low numerical aperture objectives were used to detect even out of focus vesicles), ...
Endocytosis is an essential and well‐regulated process in higher eukaryotes [35], [51], [52]. Sustained neurotransmission imposes a greater demand on the precision and efficiency of endocytic pathways as vesicles are recycled locally to balance continued exocytosis [1], [2], [3]. While numerous players and regulators of endocytosis have been characterized in neurons [3], [13], [14], [15], inhibitors are rare. This work reveals the cellular function of Syt11 as a novel inhibitor in the vesicle retrieval pathways.. Using an RNAi approach, we revealed the function of Syt11 in neuronal endocytosis. Membrane capacitance recording to monitor the somatic exocytosis and endocytosis in DRG neurons showed that Syt11 KD greatly accelerated exo‐endocytosis, and this was often accompanied by an excessive membrane retrieval as evidenced by Cm overshoot, indicating an unbalanced coupling of endocytosis to exocytosis (Figs 1B-G and 3A-F). Furthermore, FM1‐43, dextran, and HRP uptake assays demonstrated an ...
TY - JOUR. T1 - Distinct endocytic responses of heteromeric and homomeric transforming growth factor β receptors. AU - Anders, Robert A.. AU - Arline, Sandra L.. AU - Doré, Jules J.E.. AU - Leof, Edward B.. PY - 1997/1/1. Y1 - 1997/1/1. N2 - Transforming growth factor β (TGFβ) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGFβ receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to down- stream effectors. There is presently little data concerning the fate of TGFβ receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand- activated receptors, we have used our previously characterized chimeric receptors ...
Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed versus normal counterparts. However, when we screened 29 independently isolated non-small cell lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we ...
Clathrin-mediated endocytosis is a critical process through which a wide variety of extracellular material is internalized. The primary component, clathrin, forms a cargo-selective lattice at the plasma membrane, as well as on endosomes and the TGN, though the cargo-selective components are incompletely defined. An ideal tool for understanding the spatio-temporal dynamics of both the clathrin coat and the cargo selected is total internal reflection fluorescence microscopy (TIR-FM), which permits selective imaging of events closely apposed to the ventral plasma membrane. Previously, observation of the clathrin coat has shown both static and dynamic populations, with some dynamic structures undergoing microtubule-dependent motion; the 70-110 nm decay constant of the TIR-FM field has led to the assumption that these are all representative of coated pits. Here, I demonstrate that the dynamic population of clathrin is primarily endosomal, as it lacks colocalization with the plasma membrane-specific ...
Purified MC3R Total GPCR Internalization Assay Kit from Creative Biomart. MC3R Total GPCR Internalization Assay Kit can be used for research.
Purified GLP1R Activated GPCR Internalization Assay Kit from Creative Biomart. GLP1R Activated GPCR Internalization Assay Kit can be used for research.
Endocytosis is a mechanism for cells to remove ligands, nutrients, and plasma membrane (PM) proteins, and lipids from the cell surface, bringing them into the cell interior. Transmembrane proteins entering through clathrin-dependent endocytosis (CDE) have sequences in their cytoplasmic domains that bind to the APs (adaptor-related protein complexes) and enable their rapid removal from the PM. In addition to APs and clathrin, there are numerous accessory proteins including dynamin. Depending on the various proteins that enter the endosome membrane, these cargoes are sorted to distinct destinations. Some cargoes, such as nutrient receptors, are recycled back to the PM. Ubiquitylated membrane proteins, such as activated growth-factor receptors, are sorted into intraluminal vesicles and eventually end up in the lysosome lumen via multivesicular endosomes (MVEs). There are distinct mechanisms of clathrin-independent endocytosis (CIE) depending upon the cargo and the cell type ...
TY - JOUR. T1 - LRP1-Dependent endocytic mechanism governs the signaling output of the bmp system in endothelial cells and in angiogenesis. AU - Pi, Xinchun. AU - Schmitt, Christopher E.. AU - Xie, Liang. AU - Portbury, Andrea L.. AU - Wu, Yaxu. AU - Lockyer, Pamela. AU - Dyer, Laura A.. AU - Moser, Martin. AU - Bu, Guojun D. AU - Flynn, Edward J.. AU - Jin, Suk Won. AU - Patterson, Cam. PY - 2012/8/17. Y1 - 2012/8/17. N2 - Rationale: Among the extracellular modulators of Bmp (bone morphogenetic protein) signaling, Bmper (Bmp endothelial cell precursor-derived regulator) both enhances and inhibits Bmp signaling. Recently we found that Bmper modulates Bmp4 activity via a concentration-dependent, endocytic trap-and-sink mechanism. Objective: To investigate the molecular mechanisms required for endocytosis of the Bmper/Bmp4 and signaling complex and determine the mechanism of Bmpers differential effects on Bmp4 signaling. Methods and Results: Using an array of biochemical and cell biology ...
There exist three kinds of endocytosis; receptor mediated endocytosis, pinocytosis and phagocytosis. First, receptor-mediated endocytosis is essential for targeted gene/drug delivery to a specific cell type. Second, pinocytosis is the engulfing and digestion of dissolved substances and phagocytosis is the engulfing and digestion of microscopically visible particles. In this study, we developed a capacitance sensor to monitor and distinguish three kinds of endocytosis in real time. The capacitance sensor is fabricated simple photolithography process on glass substrate and target cells are positioned between gap electrodes. The capacitance sensor was able to detect a capacitance peak in different cell lines during the internalization of adenoviruses or antibodies via receptor-mediated endocytosis. In contrast, the capacitance declined without a capacitance peak when nanoparticles were taken up via non-specific pinocytosis. In case of phagocytosis, the capacitance shows a peak during the engulfing of
Nature, 417(6888): 555-559. Vesicle pools Vesicle mobilization, docking, priming and fusion Three endocytic pathways The dynamic time course of endocytosis The time course of endoc3l;osis is regulated by stimulation Mechanisms that may regulate the time course of endocytosis Clathrin-dependent versus clathrin-independent endocytosis Regulation of endocytosis by single rate-limiting factors The effects of calcium on endocytosis Hypotheses that may account for calcium-mediated facilitation of endocytosis The functional significance of regulation of endoc3l;osis The contribution of slower endocytosis to short-term synaptic depression The maintenance of transmitter release by retrieving vesicles into the reserve pool and/or the readily releasable pool Is endocytosis fast enough to limit transmitter release from the fusion pore? Waldeck RF, Pereda A, Faber DS. 2000. Properties and plasticity of paired-pulse depression at a central synapse. J Neurosci, 20: 5312-5320. Wang LY, Kaczmarek LK. 1998. ...
The function of receptor-mediated endocytosis is diverse. It is widely used for the specific uptake of certain substances required by the cell (examples include LDL via the LDL receptor or iron via transferrin). The role of receptor-mediated endocytosis is well recognized up take downregulation of transmembrane signal transduction but can also promote sustained signal transduction.[3] The activated receptor becomes internalised and is transported to late endosomes and lysosomes for degradation. However, receptor-mediated endocytosis is also actively implicated in transducing signals from the cell periphery to the nucleus. This became apparent when it was found that the association and formation of specific signaling complexes is required for the effective signaling of hormones (e.g. EGF). Additionally it has been proposed that the directed transport of active signaling complexes to the nucleus might be required to enable signaling as random diffusion is too slow[4] and mechanisms permanently ...
Amphiphysin recruits dynamin to sites of endocytosis. Links to research on the mechanism of dynamin in vesicle scission and endocytosis. Research on proteins involved in clathrin-coated vesicle formation. Research on membrane bending. Research on protein-lipid interactions. Research on vesicle trafficking pathways. As a group we investigate vesicle budding using a variety of structural and functional approaches.
Cytosis is a transport mechanism for the movement of large quantities of molecules into and out of cells. There are three main types of cytosis: endocytosis (into the cell), exocytosis (out of the cell), and transcytosis (through the cell, in and out). The word cytosis (/saɪˈtoʊsɪs/) uses combining forms of cyto- and -osis, reflecting a cellular process. The term was coined by Novikoff in 1961. Endocytosis is when a cell absorbs molecules, such as proteins, from outside the cell by engulfing it with the cell membrane. It is used by most cells, because many critical substances are large polar molecules that cannot pass through the cell membrane. The two major types of endocytosis are pinocytosis and phagocytosis. Pinocytosis Pinocytosis, also known as cell drinking, is the absorption of small aqueous particles along with the membrane receptors that recognize them. It is an example of fluid phase endocytosis and is usually a continuous process within the cell. The particles are absorbed ...
In this study, we report the interaction of amphiphysin 2 with SNX4, a partnership that might be biologically relevant between endocytosis and endosomal trafficking. On one hand, amphiphysin 2 has been shown to be important for the early steps of endocytosis in mammalian cells (Wigge and McMahon, 1998). It is now well established that amphiphysin 2 helps to recruit dynamin via its C-terminal SH3 domain, at the site of clathrin-dependent endocytosis. It may also control membrane curvature either directly or indirectly through endophilin and the uncoating of clathrin-coated vesicles through synaptojanins. On the other hand, SNX4 belongs to a family of molecules initially characterized for their ability to bind membrane receptors such as EGF, PDGF, insulin or leptin receptors (Kurten et al., 1996; Haft et al., 1998). SNX molecules have been conserved throughout evolution and exist in yeast where Vps5p is the ortholog of human SNX1 (Horazdovsky, 1997). In yeast cells, Vps5p associates with other ...
Purpose: Myocilin has two distinct cellular distributions, cytoplasmic and membrane-associated, yet appears extracellularly. We have previously shown that myocilin is released from cells on exosomes, which are a component of the endosomal pathway. In this study we tested the hypothesis that myocilin enters the exosome pathway during plasma membrane receptor endocytosis.. Methods: Retinal pigment epithelia (RPE) in human eye-cups was stimulated with L-DOPA (1uM) to monitor endocytosis of GPR143 (OA1), an endogenous G-protein coupled receptor (GPCR) on the surface of RPE. Recombinant myocilin (WT, P370L and T377M) was heterologously co-expressed in transformed cells with GPR143 to test whether myocilin participates in receptor endocytosis and to determine the kinetics of binding. Cell surface proteins in eye-cups and transformed cells were biotinylated and protein trafficking and myocilin association were monitored over time using streptavidin beads. Finally, we tested myocilin binding to the ...
Tor2 is an activator of the Rom2/Rho1 pathway that regulates α-factor internalization. Since the recruitment of endocytic proteins such as actin binding proteins and the amphiphysins precedes the internalization of α-factor, I hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. I report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, I found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1∆tor2ts double mutant cells. I also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, I found that in tor1∆tor2ts double mutant cells, as well as other tor mutant cells, the overall PIP2 level was dramatically increased. Finally, the cell wall chitin content in tor2ts and tor1∆tor2ts mutant cells was also
In this study, we have described a new biochemical assay that efficiently reconstitutes the endocytosis of E-cadherin using the AJ plasma membranes. We have found here that non-trans-interacting E-cadherin is constitutively endocytosed like integrin (ligand-independent endocytosis), that the formation of endocytosed vesicles of E-cadherin is clathrin dependent, and that E-cadherin, but not other CAMs at AJs and TJs including nectins, claudins, and occludin, is selectively sorted into the endocytosed vesicles. Recent cell-level studies using chemical inhibitors have shown that E-cadherin might be internalized by clathrin- or caveolin-dependent endocytosis (Le et al., 1999; Akhtar and Hotchin, 2001; Palacios et al., 2002; Thomsen et al., 2002; Paterson et al., 2003; Ivanov et al., 2003). However, the results obtained in this way are indirect and not substantial because the low resolution indirect immunofluorescence staining could only follow the appearance of E-cadherin in large vesicular ...
Proteins internalized at the cell surface by clathrin-mediated endocytosis contain specific sorting sequences that bind to the internalization machinery. The best characterized of these is the tyrosine-based YXXΦ motif (in which X is any residue and Φ is a bulky, hydrophobic residue). This binds to a specific region in the μ2 subunit of the AP2 clathrin adaptor protein, and structural studies have shown that the spacing between the Y and Φ residues is crucial. Ruth Murrell and co-workers now unveil a novel type of tyrosine-based endocytic motif (p. 3073). They have used site-directed mutagenesis and CD8-based chimeras to analyse endocytosis of P2X4 receptors, ATP-gated cation channels that rapidly cycle off the plasma membrane. These receptors possess consensus YXXΦ motifs, but surprisingly these are inaccessible to AP2 and not needed for endocytosis. Instead, the authors show, a downstream YXXGΦ motif is required. Determining the structure of a YXXGΦ-μ2 complex, they demonstrate that ...
TY - JOUR. T1 - Derailed endocytosis. T2 - An emerging feature of cancer. AU - Mosesson, Yaron. AU - Mills, Gordon. AU - Yarden, Yosef. PY - 2008/11/1. Y1 - 2008/11/1. N2 - Once engaged by soluble or matrix-anchored ligands, cell surface proteins are commonly sorted to lysosomal degradation through several endocytic pathways. Defective vesicular trafficking of growth factor receptors, as well as unbalanced recycling of integrin- and cadherin-based adhesion complexes, has emerged in the past 5 years as a multifaceted hallmark of malignant cells. In line with the cooperative nature of endocytic machineries, multiple oncogenic alterations underlie defective endocytosis, such as altered ubiquitylation (Cbl and Nedd4 ubiquitin ligases, for example), altered cytoskeletal interactions and alterations to Rab family members. Pharmaceutical interception of the propensity of tumour cells to derail their signalling and their adhesion receptors may constitute a novel target for cancer therapy.. AB - Once ...
Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called top and side sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated
It is known for decades, and most recently seen in dynamin knockout mice, that endocytosis continues when classical mechanisms are blocked or deleted. With the help of ubiquitin, yeast live without clathrin and fungi reveal still other alternatives in the absence of clathrin. We discovered a few years ago that 50 percent of the cell surface of many cells can be removed in seconds by Ca-dependent endocytosis that requires no classical endocytic player. This response is unrelated to either apoptosis or autophagy; cell cultures survive and even flourish in the wake of this remodeling. This form of endocytosis, relying on no known adapter or cytoskeleton, can quantitatively remove PD-1 receptors from the T-cell surface membrane, receptors whose inactivation is a key to activating immune responses. Thus, domain-dependent endocytosis can be concentrative as well as contributing to overall fluid-phase endocytosis. As for exocytosis, Ca stress-dependent exocytosis can rapidly increase membrane area by ...
Zwiewka, M; Nodzynski, T; Robert, S; Vanneste, S; Friml, J, 2015: Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana. MOLECULAR PLANT 8(8), p. 1175 - 1187, doi: 10.1016/j.molp.2015.03.007. Research Groups:. ...
Receptor-based endocytosis. Illustration of receptor-mediated endocytosis, where receptors on cells allow them to engulf target molecules (red). The target molecules are first shown outside the cell (centre left). They then bind (upper right) to receptor proteins (orange) on a cell membrane pit coated by the protein clathrin (yellow). The membrane pit forms a vesicle (lower right) around the molecules within the cell cytoplasm (blue). For three forms of endocytosis, see images C023/8787, C023/8789 and C023/8793. For exocytosis (the reverse process), see image C023/8796. For this artwork without labels, see image C023/8795. - Stock Image C023/8793
To obtain insights into the mechanism by which FCHo2 couples CCP growth and lifetime in CME, we analyzed the nanoscale localization of FCHo2 at CCPs. Dual-color SD-dSTORM (spectral demixing direct stochastic optical reconstruction microscopy) analysis of the distribution of endogenous FCHo2 within CCPs followed by quantitative averaging of ,250 images revealed a marked concentration of FCHo2 in ring-like structures (about 225 nm in diameter) at the outer rim of CCPs [consistent with (27)] while being largely absent from the CCP center (Fig. 3, D and E) that eventually gives rise to the dome as CCPs invaginate. Hence, FCHo2 selectively accumulates at the rim of CCPs, consistent with its early role in coupling CCP growth and dynamics.. Different models have been proposed regarding the early endocytic function of FCHo2. According to one model, FCHo2 nucleates CCPs by acting as a plasma membrane-associated recruitment hub for early-acting endocytic proteins bound to its μ-homology domain (20). This ...
Background and Hypothesis: Recently, a study conducted by the Hundley lab revealed that mutation of a specific RNA editing protein, ADR2, in the model organism C. elegans increased receptor-mediated endocytosis of yolk protein vit-2 and oocyte maturation. Of interest to us was what additional RNA binding proteins were involved in the expression of vit-2. To this end, RNA interference (RNAi) was used to knock out relevant RNA binding proteins. We hypothesized we would find candidates that both enhanced and reduced the expression of vit-2.. Project Methods: Previously, adr-2(-) C. elegans strain expressing a yolk protein (vit-2) fused to GFP and a library of E. coli strains expressing RNAi directed to specific RNA binding proteins were engineered. vit-2:GFP reporter worms were grown on each RNAi bacterial strain. The COPAS Biosort, a large particle sorter which detects fluorescent intensity, was used to visualize and quantify vit-2 endocytosis.. Results: Our screen yielded ten candidate proteins. ...
Enlarged early endosomes in the neurons of young Down syndrome (DS) and pre-Alzheimers disease (AD) brains suggest that a disturbance in endocytosis is one of the earliest hallmarks of AD pathogenesis in both conditions. We identified a chromosome 21 gene, Intersectin-1 (ITSN1) that is up-regulated in DS brains and has a putative function in endocytosis and vesicle trafficking. To elucidate the function of ITSN1 and assess its contribution to endocytic defects associated with DS and AD, we generated Itsn1 null mice. In knockout mice we found alterations in a number of parameters associated with endocyic and vesicle trafficking events. We found a reduced number of exocytosis events in chromaffin cells and a slowing of endocytosis in neurons. Endosome size was increased in neurons and NGF levels were reduced in the septal region of the brain. Our data is the first indication that Itsn1 has a role in endocytosis in an in vivo mammalian model, and that a disruption in Itsn1 expression causes a ...
hCTR1 is important not only because it is the major copper influx transporter but also because it mediates a significant component of the cellular accumulation of DDP. As one step toward identifying how hCTR1 transports DDP, we sought to determine the mechanism by which endogenously expressed hCTR1 is degraded following brief exposure to DDP using several different strategies for inhibiting endocytotic pathways and proteasomal degradation. The results support the conclusion that the initial step of DDP-induced loss of hCTR1 from human ovarian carcinoma cells is mediated by a process that is inhibited by amiloride but not by nystatin or methyl-β-cyclodextrin and is not dependent on either dynamin I or the Rac1 GTPase. Among the various endocytotic pathways currently defined, this pattern of inhibition most closely matches the process of macropinocytosis.. As currently understood, macropinocytosis occurs through the formation of large primary endocytotic vesicles in regions of membrane ruffling. ...
AP2-associated protein kinase 1 (Aak1) is a member of the Ark1/Prk1 subfamily of Ser/Thr protein kinases that are thought to regulate endocytosis by phosphorylating the accessory endocytic components. Aak1 interacts with and phosphorylates the mu2 subunit of the AP-2 complex, which promotes binding of the AP-2 to tyrosine based (Yxxf) internalization motif-containing receptors and subsequent receptor endocytosis. At least two isoforms of Aak1 are known to exist; the longer isoform contains an extended carboxy-terminus that contains an additional clathrin-binding domain. Overexpression of this long isoform or Aak1 depletion by RNA interference impairs transferrin recycling from the early/sorting endosome, suggesting that Aak1 functions at multiple steps of the endosomal pathway by regulating transferrin internalization and its recycling back to the plasma membrane. ...
Video articles in JoVE about clathrin coated vesicles include Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast, Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy, Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons, The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins, Pulling Membrane Nanotubes from Giant Unilamellar Vesicles, In vivo and in vitro Studies of Adaptor-clathrin Interaction, Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers, Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell, Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes, Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the
Developmental regulation of apical endocytosis controls epithelial patterning in vertebrate tubular organs.s profile, publications, research topics, and co-authors
The molecular composition of plasma membranes is constantly remodeled by endocytosis and exocytosis. Eisosomes are large cytoplasmic protein assemblies that localize to specialized domains on the yeast plasma membrane. They are of uniform size and immobile, and their disruption leads to large aberrant plasma membrane invaginations and endocytic defects. It is unknown how eisosomes are formed or inherited and what governs their size, distribution, and location. Here we show that eisosomes are formed de novo in the bud of dividing cells. They colonize newly formed membrane at a fixed density in a polarized wave proceeding from the bud neck to the bud tip and become anchored at the site of their formation. Pil1, one of the two main eisosome subunits, emerges as the central regulator of eisosome biogenesis that determines both size and location of eisosomes. Lowering Pil1 expression leads to normal-sized eisosomes at a reduced density, suggesting that eisosomes must be of a minimal size. Conversely, ...
When sending chemical messages, cells release transmitters or hormones by Ca-triggered exocytosis. During exocytosis, the membrane of a secretory vesicle fuses...
Specific receptor-mediated endocytosis pathways can be investigated with fluorescent ligands including pHrodo™ dyes targeted to common receptors, including, EGF, LDL, and transferrin.. The most convenient, no-wash assay format uses pHrodo™ Red or pHrodo™ Green to allow discrimination of stages in the endocytic pathway from early endosome to lysosome formation with no quench or wash required.. pHrodo™ dyes are essentially non-fluorescent at neutral pH and exhibit increasing signal with a red or green readout respectively as the pH decreases. The increase in fluorescent signal can be used to monitor progression in the endocytic pathway.. ...
In endocytosis, substances are internalized by a cell through the formation of vesicles. Types of endocytosis include phagocytosis and pinocytosis.
ST6Gal-I Restrains CD22-Dependent Antigen Receptor Endocytosis and Shp-1 Recruitment in Normal and Pathogenic Immune Signaling†: The ST6Gal-I sialyltransferase
Overview of Interests. Dr. Fergusons research is focused on the functional regulation and activity of G protein-coupled receptors (GPCRs) as a consequence of their interactions with other proteins expressed inside and outside of the cell and how these interactions regulate both normal pathological cell signaling. His current research efforts are primarily focused on the role of metabotropic glutamate receptor signaling in Huntingtons, Alzheimers and Parkinsons disease, the regulation of serotonin receptor activity by corticotrophin releasing factor receptors in response stress with a goal of understanding the effect stress has on anxiety and depression behaviours, as well as understanding the molecular changes in GPCR signaling associated with hypertension.. Research Achievements. Dr. Ferguson was the first to identify beta-arrestins as endocytic adaptor proteins for GPCRs and to show that they contribute to the coupling of GPCRs to G protein-independent signal transduction pathways. His ...
by Elio | What if I told you that engineering a single protein into E. coli is sufficient to make it fill up with membrane-bound vesicles? Would you send me to the couch or to a padded cell? Not so fast, as this is precisely what a group of sixteen investigators from three…
Euroscreen is a private Belgian, preclinical-stage, Drug Discovery and Development Company targeting G-Protein-Coupled-Receptors (GPCRs) to provide...
This activity is headed by David Perrais (DR2, senior researcher) and currently involves, Magalie Martineau (post-doctoral fellow: Marie Curie fellowship) and Léa Claverie (PhD student). The main objective is to study the molecular and cellular mechanisms of endocytosis, exocytosis and recycling and their relevance for cellular function. We are specifically interested in these processes in neuronal dendrites, and how it organizes neuronal function, in particular synaptic transmission and plasticity. We focus on the imaging of single exo-and endocytic events in living cells, which permit us to ...
Schematics of endocytosis of a single NP.The membrane is partitioned into three regions due to the wrapping: the bound region of area , the impacted region of a
April 2011- Visual Pigment and Endocytosis - Dynamin- and Rab5-dependent endocytosis is required to prevent Drosophila photoreceptor degeneration. Pinal N, Pichaud F. J Cell Sci. ...
Most living cells maintain internal environments that are different from their extracellular environment, as well as concentration differences between the cytosol and internal compartments. In human tissues, for example, all cells have a higher concentration of Na+ outside the cell than inside, and a higher concentration of K+ inside the cell than outside. These concentration gradients of Na+ and K+ represent a form of energy storage, similar to a battery. An example of a concentration difference between the cytosol and an internal compartment is found in the lysosome, where the concentration of hydrogen ions (H+) can be 100 to 1000 times greater than the concentration outside, in the cytosol.. ...
Development of more efficacious ADCs requires improved delivery of payload to target cells. Increased understanding of ADC cellular processing (e.g., internalization kinetics) will facilitate the design of better ADCs. Furthermore, processing is largely driven by properties of the target antigen. Although enhanced expression of EGFR in many cancers makes it an attractive target for ADC approaches, ultimately the unique internalization and degradation characteristics of EGFR in different tumor types will influence whether ADC EGFR therapeutics will be successful. To improve our understanding of cellular processing of EGFR-directed ADCs, the internalization and trafficking of Ab033 were assessed in the EGFR-expressing cancer cells A431 and H441. This work was centered on the cellular processing of ADCs. Cytotoxicity data were therefore not included to keep the focus on the determination of inefficient cellular processing steps of ADCs. A431 cells have approximately 10-fold higher cell surface EGFR ...
When a receptor is overactive - because of a drug or disease - the body attempts to normalize activity by internalizing the receptor, hiding it from molecules at the cell surface. Internalization is a key homeostatic mechanism. But a receptors degree of activation doesnt perfectly parallel the subsequent internalization.
Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct signature of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3-5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous ...
The purpose of this review is to present biochemical and ultrastructural evidence which demonstrate that differences exist between the binding, internalization and intracellular processing of insulin...
MARCH2 is an E3 ubiquitin-protein ligase that may mediate ubiquitination of TFRC and CD86, and promote their subsequent endocytosis and sorting to…