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), ...

The Slit diaphragm (SD) is the key filtration structure in human glomerular kidney that is affected in many types of renal diseases. SD proteins are known to undergo endocytosis and recycling to maintain the integrity of the filtration structure. However, the key components of this pathway remain unclear. Using the Drosophila nephrocyte as a genetic screen platform, we screened most genes involved in endocytosis and cell trafficking, and identified the key components of the cell trafficking pathway required for SD protein endocytosis and recycling. We discovered that the SD protein endocytosis and recycling pathway contains clathrin, dynamin, AP-2 complex, like-AP180 (Lap), auxilin and Hsc70-4 (the endocytosis part) followed by Rab11 and the exocyst complex (the recycling part). Disrupting any component in this pathway led to disrupted SD on the cell surface and intracellular accumulation of mislocalized SD proteins. We also showed the first in vivo evidence of trapped SD proteins in clathrin-coated

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 ...

A targeted variation of endocytosis employs receptor proteins in the plasma membrane that have a specific binding affinity for certain substances (Figure 3).. In receptor-mediated endocytosis, as in phagocytosis, clathrin is attached to the cytoplasmic side of the plasma membrane. If uptake of a compound is dependent on receptor-mediated endocytosis and the process is ineffective, the material will not be removed from the tissue fluids or blood. Instead, it will stay in those fluids and increase in concentration.. Some human diseases are caused by the failure of receptor-mediated endocytosis. For example, the form of cholesterol termed low-density lipoprotein or LDL (also referred to as bad cholesterol) is removed from the blood by receptor-mediated endocytosis. In the human genetic disease familial hypercholesterolemia, the LDL receptors are defective or missing entirely. People with this condition have life-threatening levels of cholesterol in their blood, because their cells cannot clear ...

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. ...

Recent reports have suggested a role for microtubules in the endocytic process and pointed out the role for actin filaments for the early steps of apical endocytosis in polarized epithelial cells. However, these studies do not address the respective contribution of these two types of filaments along the endocytic pathway. In addition, several studies failed to demonstrate the role for actin filaments in clathrin dependent endocytosis in non polarized cells. In the present study we have investigated the role for both filaments in the endocytosis of two ligands internalized via clathrin coated pits (transferrin and alpha 2-macroglobulin) in a mouse hepatoma cell line. By immunocytochemical analysis with confocal microscopy, and biochemical analysis using a temperature sensitive step at 18 degrees C, we have shown that actin filaments are involved in two steps of the degradative pathway, and that microtubules are required at a stage in between. Actin filaments increase first, the uptake of ligands ...

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 ...

article{69117807-3dd2-46c9-a208-5f5373157a52, abstract = {,p,High-content real-time imaging of hormone secretion in tissues or cell populations is a challenging task, which is unlikely to be resolved directly, despite immense translational value. We approach this problem indirectly, using compensatory endocytosis, a process that closely follows exocytosis in the cell, as a surrogate read-out for secretion. The tissue is immobilized in an open-air perifusion chamber and imaged using a two-photon microscope. A fluorescent polar tracer, perifused through the experimental circuit, gets trapped into the cells via endocytosis, and is quantified using a feature-detection algorithm. The signal of the tracer that accumulates into the endocytotic system reliably reflects stimulated exocytosis, which is demonstrated via co-imaging of the latter using existing reporters. A high signal-to-noise ratio and compatibility with multisensor imaging affords the real-time quantification of the secretion at the ...

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 ...

Supplementary MaterialsFigure S1: Internalization assay. endosomes associated with dynamic actin assemblies. 3T6 cells stably expressing EGFP-fused -actin (green) were infected with Alexa Fluor 546-labeled MPyV (reddish) (MOI of 102 to 103 computer virus particles per cell) at 37C and scanned with T?=?4 s. Selected frames of cell at 45 min p.i. with corresponding transmission light images illustrate short-distance movement of virus-carrying endosomes associated with dynamic assemblies of EGFP-actin (observe Movie S3). White colored arrowheads point to MPyV virions. Arrows point to endosome-associated actin assemblies. Black arrowheads show MPyV-containing endosomes. Bars, 5 m. Cells were examined using a Leica TCS SP2 AOBS confocal microscope.(TIF) pone.0096922.s002.tif (4.6M) GUID:?75128633-6C0E-4733-829A-F64BA5BC7B02 Number S3: Intracellular distribution of fluorescently tagged transferrin during expression of Rab11 GTPase mutants. 3T6 cells expressing EGFP-fused wt, DN or CA version of Rab11 ...

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 ...

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

2014 Project discussion}} ,br> --[[User:Z8600021,Mark Hill]] ([[User talk:Z8600021,talk]]) 16:49, 20 March 2014 (EST) --[[User:Z3399239,Z3399239]] ([[User talk:Z3399239,talk]]) 16:49, 20 March 2014 (EST) --[[User:Z3420257,Z3420257]] ([[User talk:Z3420257,talk]]) 16:49, 20 March 2014 (EST) --[[User:Z3373930,Z3373930]] ([[User talk:Z3373930,talk]]) 16:50, 20 March 2014 (EST) Hello dear colleagues. Is anyone else interested in doing into the cell from the plasma membrane (Endocytosis)? --[[User:Z3399239,Z3399239]] ([[User talk:Z3399239,talk]]) 16:52, 20 March 2014 (EST) And if not Endocytosis would anyone be interested in doing from the trans Golgi network to the cell exterior (Exocytosis) ? --[[User:Z3399239,Z3399239]] ([[User talk:Z3399239,talk]]) 12:58, 27 March 2014 (EST) So would z3373930 research: CLIC/GEEC endocytic pathway and arf6-dependent endocytosis and z3420257 research: flotillin-dependent endocytosis and macropinocytosis and z3375490 research: circular doral ruffles and ...

The life cycle of an adenovirus is divided into early and late phases, separated by the DNA replication process. In the early phase, the virus attaches to a cell with its fibers. The penton base protein interacts with the host cell integrins, and the penton is internalized by the host cell through receptor-mediated endocytosis. The penton is disassembled as it is transported to the nucleus, where the viral particle releases its DNA. The viral DNA takes over as terminal protein attached to the end of the DNA strand initiates transcription. The early genes are responsible for making regulatory proteins, which alter the host proteins to prepare for DNA synthesis, activate other virus genes, and provide protection from the hosts immune system. Viral DNA replication now occurs. The late phase begins when the late genes are expressed during DNA replication. These genes produce proteins that are involved in virus particle assembly. The hosts cellular processes are shut down as transport of mRNA to ...

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 ...

Coronaviruses, including SARS-CoV-2, are proposed to use either the endocytic or non-endosomal pathway for cell entry (46). Our study provides biophysical evidence for the functionality of SLiMs in the cytoplasmic tails of ACE2 and integrin β3, which provide possible molecular links between the established (ACE2) and putative (integrin beta3) SARS-CoV-2 receptors and mediators of endocytosis and autophagy. Our finding that an endocytic AP2 μ2 binding motif exists in the ACE2 C-terminus and that such binding is negatively regulated by Tyr781 phosphorylation supports the possibility of clathrin-dependent endocytosis of SARS-CoV-2 upon receptor binding. It will, of course, be necessary to assess the biological relevance of the ACE2-AP2 μ2 interaction, particularly in light of its low affinity. Concerning the latter, we note that while affinities of AP2 core complexes for endocytic sorting signals are reported in the nanomolar range, measured KD values of the AP2 μ2 subunit alone vary between 10 ...

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 ...

What is endocytosis? Here is a definite meaning, types , and process of endocytosis that is welll explained by our biology professors

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

Studies of membrane traffic between the cytoplasm and surface of a cell suggest that membrane internalization is tightly coupled to secretion. Studies using the capacitance technique show that endocytosis can follow evoked exocytosis within a second or less. The capacitance technique, however, measu …

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.. ...

Sapreme, a biotechnology company focused on improving the delivery and efficacy of macromolecule therapeutics, today announced positive preclinical data on its proprietary endosomal escape platform in two presentations at the 16th Annual Meeting of the Oligonucleotide Therapeutics Society (OTS), held virtually from September 27th to 30th, 2020.. Sapreme is developing macromolecule delivery methods based on compounds that release therapeutic cargo from the endo-lysosome, improving access to intracellular targets and enhancing the therapeutic window for these therapeutics. Current macromolecular biologics rely on receptor-mediated endocytic uptake into the endosome and inefficient passive release from these vesicles into the cell to achieve therapeutic efficacy. The companys presentations demonstrate that Sapremes SPT001 compound improves intracellular release of targeted antisense oligonucleotides (ASOs) and thereby also their therapeutic efficacy. In addition to ASOs, SPT001 has also been ...

TY - JOUR. T1 - The SNARE proteins SNAP25 and synaptobrevin are involved in endocytosis at hippocampal synapses. AU - Zhang, Zhen. AU - Wang, Dongsheng. AU - Sun, Tao. AU - Xu, Jianhua. AU - Chiang, Hsueh Cheng. AU - Shin, Wonchul. AU - Wu, Ling Gang. PY - 2013/5/22. Y1 - 2013/5/22. N2 - SNAP25, an essential component of the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex that mediates exocytosis, is not considered to play a role in endocytosis, which couples to exocytosis by retrieving a similar amount of exocytosed vesicles. By knocking down SNAP25 and imaging slow endocytosis at a conventional synapse, the rat cultured hippocampal synapse, we found that SNAP25 is involved in slow, clathrin-dependent endocytosis. With similar techniques, we found that not only SNAP25, but also synaptobrevin is involved in slow endocytosis. These results provide the first evidence showing the dual role of SNAP25 and synaptobrevin in both exocytosis and slow ...

UNC-51 overexpression can inhibit the transferrin endocytosis in the transfected COS-7 cells. We think that this inhibition of transferrin endocytosis should be caused by UNC-51-dependent...

article{1864472, abstract = {Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of ...

The heterotetrameric adaptor protein complex AP2 is one of the best-studied components of the endocytic machinery. The AP2 complex consists of four different subunits, α, β2, σ2, and μ2, which assemble into a core domain with two appendages (Fig. 2; Collins et al., 2002; Jackson et al., 2010). AP2 has multiple binding partners, including phosphatidylinositol 4,5-bisphosphate (PIP2), clathrin, several endocytic accessory proteins, and two signaling motifs present on some cargo receptors (see Traub, 2009 for a detailed review). The AP2 complex has classically been considered to be the master initiator of clathrin-mediated endocytosis through its role in recruiting clathrin molecules to the membrane. However, several lines of evidence question this idea.. If the AP2 complex has an essential role in initiation then its presence would be required for the formation of endocytic sites. However, in yeast the endocytosis of mating pheromone α-factor is unaffected in strains lacking functional AP2 ...

The potential for transcytosis (endocytosis → intracellular transport → exocytosis) of protein and membrane events associated with fluid phase and adsorptive endocytic processes within epithelia of the choroid plexus [blood-cerebrospinal fluid (CSF) barrier] were investigated in mice injected intravenously or into the lateral cerebral ventricle with native horseradish peroxidase (HRP) or the lectin wheatgerm agglutinin (WGA) conjugated to HRP. WGA binds to specific cell surface oligosaccharides and enters cells by the process of adsorptive endocytosis; native HRP is taken into cells non-specifically by fluid phase endocytosis. The lysosomal system of organelles and the endoplasmic reticulum, identified by enzyme cytochemical markers applied to choroid epithelia, were analysed for possible participation in transcytosis and compared to epithelial organelles harbouring the exogenous tracer proteins. Blood-borne native HRP was endocytosed readily by choroid epithelia whereas WGA-HRP was not, perhaps

article{f63aac35-6350-4bf7-866a-e224429f2246, abstract = {BACKGROUND: Galectin-3 (the Mac-2 antigen) is abundantly expressed in both macrophage like cells and certain non-macrophage cells. We have studied endocytosis of galectin-3 as one important step relevant for its function, and compared it between variants of a macrophage like cell line, and non-macrophage cells. ,br/,,br, ,br/,,br, METHODS: Endocytosis of galectin-3 was observed by fluorescence microscopy and measured by flow cytometry. The endocytosis mechanism was analysed using galectin-3 mutants, galectin-3 inhibitors and endocytic pathways inhibitors in the human leukaemia THP-1 cell line differentiated into naïve (M0), classical (M1) or alternatively activated (M2) macrophage like cells, and the non-macrophage cell lines HFL-1 fibroblasts and SKBR3 breast carcinoma. ,br/,,br, ,br/,,br, RESULTS: Galectin-3 endocytosis in non-macrophage cells and M2 cells was blocked by lactose and a potent galectin-3 inhibitor TD139, and also by the ...

The animation shows calcium-stimulated exocytosis of synaptic vesicles followed by clathrin-mediated vesicle recycling. Many of the molecular components that are involved in synaptic vesicle priming, docking, fusion, and endocytosis are shown. Although the process in the animation describes synaptic vesicle cycling, similar cellular processes occur for most calcium-coupled secretory and clathrin-mediated endocytotic events. The animation could be used to help illustrate the sequence of events associated with both exocytosis and endocytosis, as well as aid in understanding the processes involved in neurotransmitter release in response to nerve stimulation.. [Resource Details] ...

The animation shows calcium-stimulated exocytosis of synaptic vesicles followed by clathrin-mediated vesicle recycling. Many of the molecular components that are involved in synaptic vesicle priming, docking, fusion, and endocytosis are shown. Although the process in the animation describes synaptic vesicle cycling, similar cellular processes occur for most calcium-coupled secretory and clathrin-mediated endocytotic events. The animation could be used to help illustrate the sequence of events as. Published by Learning Registry #GoOpen. 4 Views, 0 Likes on Docs.com. #synapse #neuron #NSDL #NSDL_SetSpec_BEN #movie #signal transduction #endocytosis #Life Science

The central finding of the present study is that Mt3 plays a key role in the clathrin-dependent endocytosis of Aβ in astrocytes. In Mt3 −/− astrocytes, clathrin-mediated endocytosis, the mechanism responsible for Aβ endocytosis, was markedly decreased, whereas caveolin-mediated endocytosis was not altered. Astrocytes are likely key players in the clearance of extracellular Aβ; thus, our results suggest that changes in the Mt3 expression in astrocytes may have clinical relevance in AD. Taken together with our previous findings that Mt3 helps to maintain lysosomal degradation in astrocytes, the reduction in Mt3 in astrocytes may aggravate Aβ accumulation in the extracellular space.. Early studies showed that AD brain extracts induce more neurite outgrowth in cell cultures than do control brain extracts [27], suggesting upregulation of a growth-inducing factor or downregulation of a growth-inhibitory factor (GIF) in AD brains. The latter was shown to be the case, and a subsequent study ...

Link to Pubmed [PMID] - 20486136. Bioessays 2010 Jun;32(6):496-504. Clathrin and the endocytosis machinery has recently been described as being required in mammalian cells for the internalization of large particles including pathogenic bacteria, fungi, and large viruses. These apparently unexpected observations, within the framework of the classical mechanisms for the formation of clathrin-coated vesicles, are now considered as examples of a new non-classical function of clathrin, which can promote the internalization of membrane domains associated to planar clathrin lattices. The role of actin downstream of clathrin seems to be critical for this still poorly characterized process. The historical frontier between endocytosis and phagocytosis is vanishing in the light of this new role for clathrin.. http://www.ncbi.nlm.nih.gov/pubmed/20486136 ...

Looking for endocytic vacuole? Find out information about endocytic vacuole. A membrane-bound cellular organelle containing extracellular particles engulfed by the mechanisms of endocytosis Explanation of endocytic vacuole

PathHunter® Receptor Tyrosine Kinase (RTK) Internalization Assays are nonimaging, non-antibody based chemiluminescent detection assays that provide a direct and quantitative measurement of internalized RTKs localized to early endosomes. Your PathHunter Receptor Tyrosine Kinase Internalization Cell Line, when used in conjunction with a PathHunter Detection Kit (DiscoveRx, Cat. #93-0001 Series), provides a cell-based functional assay for receptor tyrosine kinase internalization. ...

Efficient non-viral gene delivery often involves the conjugation of a cell-specific ligand to the vector, which directs the vector to its intended target through binding to a cellular receptor followed by internalization via endocytosis. However, little is known in terms of how the various endocytosis pathways affect the performance of the delivery vehicle. Previously, the Pack lab has demonstrated that caveolin-mediated endocytosis is important to in vitro polyethylenimine (PEI)- and polyamidoamine (PAMAM)-mediated gene delivery in HeLa cells through the use of small molecule drugs and small-interfering RNA [1-2]. The goal of this thesis is to further elucidate the effects of cellular uptake mechanism on non-viral gene delivery in vitro and in vivo utilizing a small hairpin RNA (shRNA) Tet-on system. Specifically, we have investigated the effects of clathrin-dependent endocytosis and caveolin-dependent endocytosis on the efficacy of PEI and PEI-derivative gene delivery in HeLa and MDA-MB-231 ...

Adapter protein that functions as clathrin-associated sorting protein (CLASP) required for clathrin-mediated endocytosis of selected cargo proteins. Can bind and assemble clathrin, and binds simultaneously to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and cargos containing non-phosphorylated NPXY internalization motifs, such as the LDL receptor, to recruit them to clathrin-coated pits. Can function in clathrin-mediated endocytosis independently of the AP-2 complex. Involved in endocytosis of integrin beta-1; this function seems to redundant with the AP-2 complex and seems to require DAB2 binding to endocytosis accessory EH domain-containing proteins such as EPS15, EPS15L1 and ITSN1. Involved in endocytosis of cystic fibrosis transmembrane conductance regulator/CFTR. Involved in endocytosis of megalin/LRP2 lipoprotein receptor during embryonal development. Required for recycling of the TGF-beta receptor. Involved in CFTR trafficking to the late endosome. Involved in several receptor-mediated

Membrane vesicle traffic to and from the plasma membrane is essential for cellular homeostasis in all eukaryotes. In plants, constitutive traffic to and from the plasma membrane has been implicated in maintaining the population of integral plasma-membrane proteins and its adjustment to a variety of hormonal and environmental stimuli. However, direct evidence for evoked and selective traffic has been lacking. Here, we report that the hormone abscisic acid (ABA), which controls ion transport and transpiration in plants under water stress, triggers the selective endocytosis of the KAT1 K+ channel protein in epidermal and guard cells. Endocytosis of the K+ channel from the plasma membrane initiates in concert with changes in K+ channel activities evoked by ABA and leads to sequestration of the K+ channel within an endosomal membrane pool that recycles back to the plasma membrane over a period of hours. Selective K+ channel endocytosis, sequestration, and recycling demonstrates a tight and dynamic ...

Recently, several theoretical and computational studies have also been performed to investigate the membrane wrapping efficiency of elastic NPs during endocytosis, which has been shown regulated synergistically by NP size, shape, and elasticity (22, 23, 33). However, despite the fact that the effects of NP elasticity on different endocytic uptake mechanisms have been experimentally explored in this work, the theoretical modeling of these uptake mechanisms remains challenging. More thorough and realistic modelings with explicit consideration of the actin network and adaptor proteins underneath the cell membrane are called for. Clathrin- and caveolin-independent endocytosis can be modeled using a membrane wrapping based on the Helfrich-Canham membrane theory (34), suggesting that stiff NPs are energetically more prone to full wrapping than soft ones (21). This elasticity effect on the simple membrane wrapping of an NP gradually becomes less significant as the NP elasticity increases. For isotropic ...

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Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia Daniel Gündel,1 Mareli Allmeroth,2 Sarah Reime,1 Rudolf Zentel,2 Oliver Thews1 1Institute of Physiology, Martin Luther University Halle-Wittenberg, Halle (Saale), 2Institute of Organic Chemistry, Johannes Gutenberg-University, Mainz, Germany Background: Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis.Materials and methods: Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence

The ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dents disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. Here we identify a carboxyl-terminal internalization motif in ClC-5. It resembles the PY motif, which is crucial for the endocytosis and degradation of epithelial Na+ channels. Mutating this motif increases surface expression and currents about 2-fold. This is probably because of interactions with WW domains, because dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents but did not affect ...

All corticosteroid hormones share cholesterol as a common precursor. Therefore, the first step in steroidogenesis is cholesterol uptake or synthesis. Cells that produce steroid hormones can acquire cholesterol through two paths. The main source is through dietary cholesterol transported via the blood as cholesterol esters within low density lipoproteins (LDL). LDL enters the cells through receptor-mediated endocytosis. The other source of cholesterol is synthesis in the cells endoplasmic reticulum. Synthesis can compensate when LDL levels are abnormally low.[4] In the lysosome, cholesterol esters are converted to free cholesterol, which is then used for steroidogenesis or stored in the cell.[29]. The initial part of conversion of cholesterol into steroid hormones involves a number of enzymes of the cytochrome P450 family that are located in the inner membrane of mitochondria. Transport of cholesterol from the outer to the inner membrane is facilitated by steroidogenic acute regulatory protein ...

Nano-meter sized particles of de-aluminated zeolite Y has a high adsorption capacity of both low molecular weight bio-molecules and macromolecules. In this study we used de-aluminated zeolite Y as a novel approach to study the mechanisms of endocytosis in immature human peripheral blood dendritic cells (DCs). Probes detecting pH neutral and acidic endosomes were adsorbed to the zeolite and used as a tracer of the endosomal pathway of a cell in the form of acidification and lysosomal function. Both the myeloid (M-DCs) and the plasmacytoid (P-DCs) dendritic cell subsets showed an endocytosing capacity comparable to peripheral blood monocytes but only the M-DCs were able to form acidic endosomes after internalization of zeolite particles. Furthermore, during lipopolysaccharide (LPS) stimulation of the DCs population, an enhanced induction of acidic endosomes was only seen in the M-DC population. Proteolytic degradation of endocytosed proteins was detected using self-quenched DQ-ovalbumin adsorbed ...

Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification, Scientific Reports 6, 36938 (2016). Since their discovery twenty years ago, cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have been described as promising drug delivery systems. There are increasing numbers of successful applications of CPPs/PTDs in vivo. However, one of the limitations to their wide and diverse application is the diversity of their uptake pathways. The CPP and its cargo may end up free in the cytosol and reach their biological target only following translocation, but remain confined in intracellular vesicles after endocytosis, unless subsequent events such as endosomal rupture occur. Along with the development of CPPs/PTDs as vectors to carry various macromolecules for targeted cellular therapies, engineering new CPPs/PTDs with optimized transport and ...

Endocytosis by African trypanosomes. I. Three dimensional structure of the endocytic organelles in Trypanosoma brucei and Trypanosoma ...

Clathrin-mediated endocytosis regulates the signalling activity and turnover of multiple plasma membrane proteins. Interfering with endocytosis can therefore have complex effects on developmental processes. William Sessa and colleagues (p. 1465) investigate the role of the Dynamin 2 (Dnm2) GTPase - a key component of the endocytic machinery - during angiogenesis in mice. Using endothelial cells in culture, they find that downregulation of Dnm2 impairs angiogenesis, even though vascular endothelial growth factor (VEGF) signalling is enhanced - due to increased surface levels of the receptor. This is in contrast to previous work suggesting that endocytosis might promote VEGF signalling. The authors ascribe the Dnm2 knockdown-induced defect in vessel formation at least partially to disrupted integrin turnover: focal adhesion size is increased and inactive integrin receptors appear to accumulate on the cell surface. Importantly, these observations hold true in vivo: conditional deletion of Dnm2 in ...

TY - JOUR. T1 - Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells. AU - Sundara Rajan, Sujata. AU - Liu, Hong Yan. AU - Vu, Tania Q.. PY - 2008/6/1. Y1 - 2008/6/1. N2 - Endocytic receptor trafficking is a complex, dynamic process underlying fundamental cell function. An integrated understanding of endocytosis at the level of single or small numbers of ligand bound-receptor complexes inside live cells is currently hampered by technical limitations. Here, we develop and test ligand nerve growth factor-bound quantum dot (NGF-QD) bioconjugates for imaging discrete receptor endocytic events inside live NGF-responsive PC12 cells. Using single particle tracking, QD hybrid gel coimmunoprecipitation, and immunocolocalization, we illustrate and validate the use of QD-receptor complexes for imaging receptor trafficking at synchronized time points after QD-ligand - receptor binding and internalization (t = 15-150 min). The unique value ...

On Some Aspects of the Thermodynamic of Membrane Recycling Mediated by Fluid Phase Endocytosis: Evaluation of Published Data and ...

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was

Ligand-induced endocytosis of receptor tyrosine kinases (RTKs) is a dynamic process governed by numerous protein-protein and protein-lipid interactions. This is a major mechanism of signal termination and is also frequently impaired in cancer. The Cbl family of ubiquitin ligases has been shown to play a key role in downregulation of RTKs, by directing their ligand-induced ubiquitination and subsequent lysosomal degradation. My thesis work has led to the identification of novel, ubiquitin-ligase independent, functions of Cbl in receptor endocytosis. We demonstrated that the adaptor protein CIN85 links Cbl with epidermal growth factor receptor (EGFR) internalization. The three SH3 domains of CIN85 interact with Cbl/Cbl-b in a phosphotyrosine dependent manner, whereas its proline-rich region constitutively binds endophilins, known regulators of plasma membrane invagination. The SH3 domains of CIN85 recognize an atypical proline-arginine (PxxxPR) motif present in Cbl and Cbl-b. Moreover, we showed ...

The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor.: Endocytosis of the low density lipoprotein (LDL) receptor (LDL

Regulation of synaptic transmission is a widespread means for dynamic alterations in nervous system function. In several cases, this regulation targets vesicular recycling in presynaptic terminals and may result in substantial changes in efficiency of synaptic transmission. Traditionally, experimental accessibility of the synaptic vesicle cycle in central neuronal synapses has been largely limited to the exocytotic side, which can be monitored with electrophysiological responses to neurotransmitter release. Recently, physiological measurements on the endocytotic portion of the cycle have been made possible by the introduction of styryl dyes such as FM1-43 as fluorescent markers for recycling synaptic vesicles. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was greatly speeded by exposure to staurosporine or ...

Probable lipid-binding protein with higher affinity for phosphatidic acid, a lipid enriched in recycling endosome membranes. On endosome membranes, may act as a downstream effector of Rab proteins recruiting cytosolic proteins to regulate membrane tubulation. May be involved in a late step of receptor-mediated endocytosis regulating for instance endocytosed-EGF receptor trafficking. Alternatively, may regulate slow endocytic recycling of endocytosed proteins back to the plasma membrane. May indirectly play a role in neurite outgrowth.

Drug-dependent neural plasticity related to drug addiction and schizophrenia can be modeled in animals as behavioral sensitization, which is induced by repeated noncontingent or self-administration of many drugs of abuse. Molecular mechanisms that are critical for behavioral sensitization have yet to be specified. Long-term depression (LTD) of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR)-mediated synaptic transmission in the brain has been proposed as a cellular substrate for learning and memory. The expression of LTD in the nucleus accumbens (NAc) required clathrin-dependent endocytosis of postsynaptic AMPARs. NAc LTD was blocked by a dynamin-derived peptide that inhibited clathrin-mediated endocytosis or by a GluR2-derived peptide that blocked regulated AMPAR endocytosis. Systemic or intra-NAc infusion of the membrane-permeable GluR2 peptide prevented the expression of amphetamine-induced behavioral sensitization in the rat. ...

Image:exo_endo_cytosis.jpg,300px,thumb,right,Exocytosis and Endocytosis cartoon]] == Cell Import - Endocytosis == [[Image:Endocytosis types.png,thumb,300px,Endocytosis types]] This lecture introduces how substances that are imported (endocytosis) and processed by the cell. Notes in Preparation (notice removed when complete) There are a number of processes by which the cell can absorb substances. The two main forms are by endocytosis or phagocytosis. Absorbed substances include: substances required for cell growth, cell signaling toxic chemicals and drugs, bacteria and viruses. In addition, this is the main method for membrane removal and recycling. We will study this topic at the level of the cellular components and organelles involved in the process: endosomes, lysosomes, peroxisomes, transport vesicles, Golgi apparatus and endoplasmic reticulum. :Movie [[Media:JCB200109030.v2.mov,Vesicles Display Bidirectional Motility along Microtubules]] Visualization of Rab9-mediated vesicle transport ...

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is

Parkinsons disease (PD) is linked to impaired degradation and accumulation of the misfolded protein, alpha-synuclein. Therefore, accelerating the degradation of alpha-synuclein is of therapeutic interest. Our lab has tested the hypothesis that α-synuclein uses endocytosis as a route to the lysosome for its degradation. We have found evidence that several endocytosis genes regulate α-synucleins PD related properties. Most importantly, the absence of vps28 increases α-synuclein aggregation and cellular accumulation, and confers toxicity in a yeast PD model. I, specifically, found that increasing the concentration of α-synuclein in yeast that lack vps28 further enhances each of these pathological characteristics.

Here, we describe an approach to directly visualize the activity of proteases that are incorporated into the phagosome at different stages of maturation. Only small numbers of phagocytes are required and there is no need to isolate individual endosomal compartments before analysis. This method is sensitive enough to allow an examination of primary cultures of professional APC, including DCs. Furthermore, the use of covalent active site-directed probes in conjunction with electrophoresis ensures specificity. Methods that employ fluorogenic substrates to detect protease activity suffer from the drawback that more than one enzyme can usually cleave a given peptide substrate. Analysis of the delivery of active hydrolases to the phagosome helped clarify both the distribution of cysteine protease activities among the different endocytic organelles and the dynamics of phagosomal maturation in primary cultures of professional APCs.. We validated our method of in vivo labeling of phagosomal proteolytic ...

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Failure of trabecular myocytes to undergo appropriate cell cycle withdrawal leads to ventricular noncompaction and heart failure. Signaling of growth factor receptor ERBB2 is critical for myocyte proliferation and trabeculation. However, the mechanisms underlying appropriate downregulation of trabecular ERBB2 signaling are little understood. Here, we have found that the endocytic adaptor proteins NUMB and NUMBL were required for downregulation of ERBB2 signaling in maturing trabeculae. Loss of NUMB and NUMBL resulted in a partial block of late endosome formation, resulting in sustained ERBB2 signaling and STAT5 activation. Unexpectedly, activated STAT5 overrode Hippo-mediated inhibition and drove YAP1 to the nucleus. Consequent aberrant cardiomyocyte proliferation resulted in ventricular noncompaction that was markedly rescued by heterozygous loss of function of either ERBB2 or YAP1. Further investigations revealed that NUMB and NUMBL interacted with small GTPase Rab7 to transition ERBB2 from ...

One unique aspect of VEGFR2 endocytosis is its regulation by several transmembrane and cytosolic interacting proteins, which play a role in either VEGFR2 internalization or degradation. A group of such proteins involved in VEGFR2 internalization is Dab2, ephrin-B2, and PAR-3. Dab2,46 a clathrin-associated sorting protein, and the cell polarity regulator PAR-3 interact with the transmembrane protein ephrin-B2 and VEGFR2. Disruption of this interaction by silencing of Dab2 or PAR-3 causes reduced VEGFR2 internalization and impaired VEGF-induced angiogenesis. After RTKs are internalized into early endosomes, a proportion of the receptors is modified by ubiquitin and then sorted for lysosomal degradation. CCM347 and myoferlin,48 respectively, associate with VEGFR2 in ECs and serve to enhance VEGFR2 stability by preventing receptor degradation.. Apart from internalization and degradation, VEGFR2 signaling is regulated by protein tyrosine phosphatases (PTPs), such as VE-PTP and PTP1b. VE-PTP is a ...

set by micropipette aspiration to be less than 1 over a wide range of spontaneous curvatures, indicating a high membrane-tension regime in their set up. Thus, our model is consistent with their observations of shallow buds observed in isotonic conditions. One result that our model cannot explain is the lack of any clathrin assembly observed under hypotonic conditions. It is possible that at extremely high membrane tensions, the coat is simply unable to stay bound to the membrane at the extremely flat morphology that would be expected.. Avinoam et al. (46) found that the size of the clathrin coat does not change substantially during membrane deformation in CME in human skin melanoma (SK-MEL-2) cells. This observation is in contrast to the canonical view that the clathrin coat should directly impose its preferred curvature on the underlying membrane (8). There are two possible explanations for this observation in the context of our study. One is that the membrane tension is too high for the coat ...

The early events in the infection of normal B lymphocytes and B lymphoblastoid cells by Epstein-Barr virus (EBV) were examined by electron and immunoelectron microscopy and by infectivity and inhibition studies. Purified EBV remained on the cell surface at 4 degrees and appeared as 250-nm ovoid particles in contact with the cell membrane through 50-nm envelope projections. Internalization of EBV in normal B lymphocytes into large (300-500 nm) uncoated vacuoles was initiated within 2 to 5 min at 37 degrees. At this stage approximately 1/3 of cell-associated virus was located in cellular invaginations while another 1/3 was in cell vacuoles. Direct fusion of EBV with the outer cell membrane was not observed. Instead, viral deenvelopment and nucleocapsid transit into the cytoplasm occurred from the large endocytic vesicles within 15 to 30 min at 37 degrees and did not involve lysosomal enzymes. During this time, the viral envelope became amorphous and its separation from the nucleocapsid was ...