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.

scholarly journals CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

2014 ◽  
Vol 25 (4) ◽  
pp. 508-521 ◽  
Author(s):  
Greg Kabachinski ◽  
Masaki Yamaga ◽  
D. Michelle Kielar-Grevstad ◽  
Stephen Bruinsma ◽  
Thomas F. J. Martin
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Total Internal Reflection ◽  
Vesicle Fusion ◽  
Live Cells ◽  
Membrane Domains ◽  
Direct View ◽  
Vesicle Exocytosis ◽  
Dependent Protein

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca2+-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca2+-triggered fusion were unknown. Ca2+-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca2+-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca2+-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca2+ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca2+-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.

scholarly journals Characterization of TSET, an ancient and widespread membrane trafficking complex

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jennifer Hirst ◽  
Alexander Schlacht ◽  
John P Norcott ◽  
David Traynor ◽  
Gareth Bloomfield ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Scaffolding Proteins ◽  
Eukaryotic Evolution ◽  
Bioinformatics Tool ◽  
Membrane Turnover ◽  
Evolutionary Path ◽  
The Core ◽  
Eukaryotic Supergroup

The heterotetrameric AP and F-COPI complexes help to define the cellular map of modern eukaryotes. To search for related machinery, we developed a structure-based bioinformatics tool, and identified the core subunits of TSET, a 'missing link' between the APs and COPI. Studies in Dictyostelium indicate that TSET is a heterohexamer, with two associated scaffolding proteins. TSET is non-essential in Dictyostelium, but may act in plasma membrane turnover, and is essentially identical to the recently described TPLATE complex, TPC. However, whereas TPC was reported to be plant-specific, we can identify a full or partial complex in every eukaryotic supergroup. An evolutionary path can be deduced from the earliest origins of the heterotetramer/scaffold coat to its multiple manifestations in modern organisms, including the mammalian muniscins, descendants of the TSET medium subunits. Thus, we have uncovered the machinery for an ancient and widespread pathway, which provides new insights into early eukaryotic evolution.

scholarly journals Imaging FCS Delineates Subtle Heterogeneity in Plasma Membranes of Resting Mast Cells

2019 ◽  
Author(s):  
Nirmalya Bag ◽  
David A. Holowka ◽  
Barbara A. Baird
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Mast Cells ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Resting Cells ◽  
Functional Protein ◽  
Diffusion Properties

ABSTRACTA myriad of transient, nanoscopic lipid- and protein-based interactions confer a steady-state organization of plasma membrane in resting cells that is poised to orchestrate assembly of key signaling components upon reception of an extracellular stimulus. Although difficult to observe directly in live cells, these subtle interactions can be discerned by their impact on the diffusion of membrane constituents. Herein, we quantified the diffusion properties of a panel of structurally distinct lipid-anchored and transmembrane (TM) probes in RBL mast cells by multiplexed Imaging Fluorescence Correlation Spectroscopy. We developed a statistical analysis of data combined from many pixels over multiple cells to characterize differences as small as 10% in diffusion coefficients, which reflect differences in underlying interactions. We found that the distinctive diffusion properties of lipid-anchored probes can be explained by their dynamic partitioning into ordered proteo-lipid nanodomains, which encompass a major fraction of the membrane and whose physical properties are influenced by actin polymerization. Effects on diffusion by functional protein modules in both lipid-anchored and TM probes reflect additional complexity in steady-state membrane organization. The contrast we observe between different probes diffusing through the same membrane milieu represent the dynamic resting steady-state, which serves as a baseline for monitoring plasma membrane remodeling that occurs upon stimulation.

scholarly journals Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties

2021 ◽  
Author(s):  
Daniel Wirth ◽  
Michael Paul ◽  
Elena B Pasquale ◽  
Kalina Hristova
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Receptor Tyrosine Kinase ◽  
Mammalian Cells ◽  
Dissociation Constants ◽  
Live Cells ◽  
Downstream Effector ◽  
Plasma Membrane Heterogeneity ◽  
Signaling Components ◽  
Insight Into

Lipid rafts are known as highly ordered lipid domains that are enriched in saturated lipids such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have never been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here we report that microscopic GM1-enriched domains can form, without lipid crosslinking, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains, but only partially co-localize with them. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that EphA2 oligomerization is the same in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biology of lipid rafts.

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 Revealing Plasma Membrane Nano-Domains with Diffusion Analysis Methods

Membranes ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 314
Author(s):  
Jakob L. Kure ◽  
Camilla B. Andersen ◽  
Kim I. Mortensen ◽  
Paul W. Wiseman ◽  
Eva C. Arnspang
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Signalling ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Protein Oligomerization ◽  
Diffusion Analysis ◽  
Lipid Diffusion

Nano-domains are sub-light-diffraction-sized heterogeneous areas in the plasma membrane of cells, which are involved in cell signalling and membrane trafficking. Throughout the last thirty years, these nano-domains have been researched extensively and have been the subject of multiple theories and models: the lipid raft theory, the fence model, and the protein oligomerization theory. Strong evidence exists for all of these, and consequently they were combined into a hierarchal model. Measurements of protein and lipid diffusion coefficients and patterns have been instrumental in plasma membrane research and by extension in nano-domain research. This has led to the development of multiple methodologies that can measure diffusion and confinement parameters including single particle tracking, fluorescence correlation spectroscopy, image correlation spectroscopy and fluorescence recovery after photobleaching. Here we review the performance and strengths of these methods in the context of their use in identification and characterization of plasma membrane nano-domains.

Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

1980 ◽  
Vol 38 ◽  
pp. 552-553
Author(s):  
K.I. Pagh ◽  
M.R. Adelman
Keyword(s):  
Cell Shape ◽  
Physarum Polycephalum ◽  
Slime Mold ◽  
Live Cells ◽  
Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

Preparation and Characterization of β-glucosidase Films for Stabilization and Handling in Dry Configurations

2020 ◽  
Vol 21 (8) ◽  
pp. 741-747
Author(s):  
Liguang Zhang ◽  
Yanan Shen ◽  
Wenjing Lu ◽  
Lengqiu Guo ◽  
Min Xiang ◽  
...  
Keyword(s):  
Tensile Strength ◽  
Protein Function ◽  
Protein Stabilization ◽  
Functional Protein ◽  
Elongation At Break ◽  
Gelatin Films ◽  
The Stability ◽  
And Function ◽  
General Method

Background: Although the stability of proteins is of significance to maintain protein function for therapeutical applications, this remains a challenge. Herein, a general method of preserving protein stability and function was developed using gelatin films. Method: Enzymes immobilized onto films composed of gelatin and Ethylene Glycol (EG) were developed to study their ability to stabilize proteins. As a model functional protein, β-glucosidase was selected. The tensile properties, microstructure, and crystallization behavior of the gelatin films were assessed. Result: Our results indicated that film configurations can preserve the activity of β-glucosidase under rigorous conditions (75% relative humidity and 37°C for 47 days). In both control films and films containing 1.8 % β-glucosidase, tensile strength increased with increased EG content, whilst the elongation at break increased initially, then decreased over time. The presence of β-glucosidase had a negligible influence on tensile strength and elongation at break. Scanning electron-microscopy (SEM) revealed that with increasing EG content or decreasing enzyme concentrations, a denser microstructure was observed. Conclusion: In conclusion, the dry film is a promising candidate to maintain protein stabilization and handling. The configuration is convenient and cheap, and thus applicable to protein storage and transportation processes in the future.

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