scholarly journals Nanoparticle interactions with live cells: Quantitative fluorescence microscopy of nanoparticle size effects

2014 ◽  
Vol 5 ◽  
pp. 2388-2397 ◽  
Author(s):  
Li Shang ◽  
Karin Nienhaus ◽  
Xiue Jiang ◽  
Linxiao Yang ◽  
Katharina Landfester ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cellular Uptake ◽  
Live Cells ◽  
Surface Charges ◽  
Nanoparticle Interactions ◽  
Quantitative Image ◽  
Endocytic Machinery ◽  
Time Courses ◽  
Quantitative Fluorescence ◽  
Key Parameter

Engineered nanomaterials are known to enter human cells, often via active endocytosis. Mechanistic details of the interactions between nanoparticles (NPs) with cells are still not well enough understood. NP size is a key parameter that controls the endocytic mechanism and affects the cellular uptake yield. Therefore, we have systematically analyzed the cellular uptake of fluorescent NPs in the size range of 3.3–100 nm (diameter) by live cells. By using spinning disk confocal microscopy in combination with quantitative image analysis, we studied the time courses of NP association with the cell membrane and subsequent internalization. NPs with diameters of less than 10 nm were observed to accumulate at the plasma membrane before being internalized by the cells. In contrast, larger NPs (100 nm) were directly internalized without prior accumulation at the plasma membrane, regardless of their surface charges. We attribute this distinct size dependence to the requirement of a sufficiently strong local interaction of the NPs with the endocytic machinery in order to trigger the subsequent internalization.

scholarly journals Quantitative characterization of Tetraspanin 8 homointeractions in the plasma membrane

2021 ◽  
Author(s):  
Daniel Wirth ◽  
Ece Özdemir ◽  
Christopher King ◽  
Lena Ahlswede ◽  
Dirk Schneider ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Dissociation Constant ◽  
Membrane Trafficking ◽  
Dissociation Constants ◽  
Cell Communication ◽  
Live Cells ◽  
Functional Protein ◽  
Quantitative Fluorescence ◽  
Dissociation Constant Kd

The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies thus far have utilized mainly co-immunoprecipitation methods that cannot distinguish between direct and indirect, through common partners, interactions. Here we study Tetraspanin 8 homointeractions in living cells via quantitative fluorescence microscopy. We demonstrate that Tetraspanin 8 exists in a monomer-dimer equilibrium in the plasma membrane. Tetraspanin 8 dimerization is described by a high dissociation constant (Kd = 14700 ± 1100 Tspan/μm2), one of the highest dissociation constants measured for membrane proteins in live cells. We propose that this high dissociation constant, and thus the short lifetime of the Tetraspanin 8 dimer, is critical for Tetraspanin 8 functioning as a master regulator of cell signaling.

Role of plasma membrane surface charges in dictating the feasibility of membrane-nanoparticle interactions

2017 ◽  
Vol 111 (26) ◽  
pp. 263702 ◽  
Author(s):  
Shayandev Sinha ◽  
Haoyuan Jing ◽  
Harnoor Singh Sachar ◽  
Siddhartha Das
Keyword(s):  
Plasma Membrane ◽  
Membrane Surface ◽  
Surface Charges ◽  
Nanoparticle Interactions ◽  
Plasma Membrane Surface

scholarly journals β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

2021 ◽  
Vol 118 (17) ◽  
pp. e2011023118
Author(s):  
Seung-Ryoung Jung ◽  
Yifei Jiang ◽  
Jong Bae Seo ◽  
Daniel T. Chiu ◽  
Bertil Hille ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Live Cells ◽  
Cellular Functions ◽  
Coated Pits ◽  
Endocytic Machinery ◽  
S 100 ◽  
Phosphatidylinositol 4 ◽  
Protease Activated Receptor 2 ◽  
G Protein Coupled

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein–coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that β-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

scholarly journals Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

2005 ◽  
Vol 16 (9) ◽  
pp. 4231-4242 ◽  
Author(s):  
Katy Janvier ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Adaptor Protein ◽  
The Other ◽  
Sorting Signals ◽  
Efficient Delivery ◽  
Endocytic Machinery

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

Coupled sterol synthesis and transport machineries at ER–endocytic contact sites

2021 ◽  
Vol 220 (10) ◽  
Author(s):  
Javier Encinar del Dedo ◽  
Isabel María Fernández-Golbano ◽  
Laura Pastor ◽  
Paula Meler ◽  
Cristina Ferrer-Orta ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Actin Polymerization ◽  
Sterol Synthesis ◽  
Biosynthetic Enzymes ◽  
Cellular Membranes ◽  
Contact Sites ◽  
Endocytic Machinery ◽  
Mother Cells

Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.

scholarly journals Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method

1984 ◽  
Vol 224 (3) ◽  
pp. 1001-1008 ◽  
Author(s):  
A R P Paterson ◽  
E R Harley ◽  
C E Cass
Keyword(s):  
Cellular Uptake ◽  
Cultured Cells ◽  
Flow System ◽  
Kinetic Constants ◽  
Cell Water ◽  
Short Intervals ◽  
Adenosine Uptake ◽  
Uptake Rates ◽  
Time Courses ◽  
Initial Uptake

Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water.

scholarly journals A method for imaging single molecules at the plasma membrane of live cells within tissue slices

2020 ◽  
Vol 153 (1) ◽  
Author(s):  
Gregory I. Mashanov ◽  
Tatiana A. Nenasheva ◽  
Tatiana Mashanova ◽  
Catherine Maclachlan ◽  
Nigel J.M. Birdsall ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Single Molecule ◽  
Cardiac Tissue ◽  
Single Molecules ◽  
Live Cells ◽  
Biological Macromolecules ◽  
Tissue Slices ◽  
Tissue Samples ◽  
Mammalian Cell Lines

Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques.

Lipoprotein Complex of Equine Lysozyme with Oleic Acid (ELOA) Interactions with the Plasma Membrane of Live Cells

Langmuir ◽  
2010 ◽  
Vol 26 (18) ◽  
pp. 14782-14787 ◽  
Author(s):  
Vladana Vukojević ◽  
Alice M. Bowen ◽  
Kristina Wilhelm ◽  
Yu Ming ◽  
Zhang Ce ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Oleic Acid ◽  
Live Cells ◽  
Lipoprotein Complex

scholarly journals Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

2014 ◽  
Vol 207 (3) ◽  
pp. 407-418 ◽  
Author(s):  
Sara Löchte ◽  
Sharon Waichman ◽  
Oliver Beutel ◽  
Changjiang You ◽  
Jacob Piehler
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Polymer Brush ◽  
Effector Proteins ◽  
Live Cells ◽  
Type I ◽  
Rgd Peptides ◽  
Single Molecule Level ◽  
Cell Micropatterning

Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.

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