Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components

Oyola-Cintrón, Jessica; Caballero-Rivera, Daniel; Ballester, Leomar Y.; Baéz-Pagán, Carlos A.; Martinez, Hernan L.; Vélez-Arroyo, Karla P.; Quesada, Orestes; Lasalde‐Dominicci, José A.

doi:10.1074/jbc.m115.678573

Cited by 6 publications

(3 citation statements)

References 72 publications

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“…Similar discrepancies in diffusion coefficients calculated by FRAP or TIR FM have been reported previously for other plasma membrane proteins and may be explained by the fact that FRAP measurements can overestimate lateral diffusion by not considering that part of the fluorescence recovery is because of secretion (Ghosh et al, 2014). Regardless of this discrepancy, the diffusion coefficients obtained for fluorescently tagged PIN2 are within the range of what has been reported for other multispanning membrane proteins in animal and plant cells (Martinière et al, 2012;Oyola-Cintrón et al, 2015;Wang et al, 2016) and are useful estimates of the lateral diffusion of a potentially wide range of ESC RT cargo proteins.…”

Section: Simulation Of Esc Rt Cargo Diffusion During Ilv Formationsupporting

confidence: 84%

ESCRT-mediated vesicle concatenation in plant endosomes

Buono

Leier

Páez-Valencia

et al. 2017

Journal of Cell Biology

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Section: Simulation Of Esc Rt Cargo Diffusion During Ilv Formationsupporting

confidence: 84%

ESCRT-mediated vesicle concatenation in plant endosomes

Buono

Leier

Páez-Valencia

et al. 2017

Journal of Cell Biology

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“…The diffusion coefficient D was calculated using the equation described by Padilla-Morales [4] . Where γ is a constant with a value of 0.88 corresponds to circular beams described by Oyola and Axelrod [ 11 , 12 ]. R is the radius of ROI and t 1/2 is the half-time of fluorescence recovery.…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Biophysical characterization dataset of native nicotinic acetylcholine receptor in lipid-like detergent complexes

2020

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For a long time, traditional purification and extraction methods for the native Torpedo californica nicotinic acetylcholine receptor in lipid-like detergent complex (nAChR-DC) have compromised its purity, functionality and X-ray structural studies possibility. The dataset presented in this article provide a characterization of the Torpedo californica nAChR-DC purified using a sequential purification processes developed in our laboratory [1] . This purification takes in consideration all of the physicochemical and functional requirements stablished by several researchers for the past three decades for the nAChR. These requirements were addressed in order to preserve the stability and functionality of nAChR-DC while ensuring the highest degree of protein purity. We focused on the effect of cholesteryl hemisuccinate (CHS) supplementation on nAChR conformational changes during the purification process. Data from the size exclusion chromatography of the nAChR-DC supplemented with CHS in concentrations ranging from 0.01 mM, 0.1 mM, 0.2 mM and 0.5 mM consistently demonstrated that 0.5 mM CHS affects receptor stability via disassemble of the pentameric oligomer. However, 0.2 mM CHS produced negligible nAChR-DC subunit disruption. The purified nAChR-DC has been characterized by circular dichroism (CD) and fluorescence recovery after photobleaching (FRAP) , in order to assess its stability. The CD data was recorded in the wavelength range of 190 - 250 nm, showed that CHS induce a ⍺-helix to β-sheet transition of the nAChR-DC. The nAChR-LFC-16 delipidation with Methyl-β-Cyclodextrin decreased the percentage of α-helix and increased the β-sheet antiparallel secondary structure and levels the percentage of turns to that of the nAChR-DC without CHS treatment. Additionally, the stability of the nAChR-DC supplemented with CHS and incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of FRAP. The LCP-FRAP data allowed to establish possible optimal crystallization conditions for the development of crystals from purified nAChR-conjugated to α-Bungarotoxin, Alexa Fluor ™ 488 (α-BTX) in order to obtain a high-resolution atomic structure by X-ray diffraction.

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“…FRAP has been widely applied for studies on membrane nano-domains, by measuring the diffusion coefficients and immobile fractions of nano-domain related proteins. This has been achieved by manipulating the cholesterol levels in the cellular plasma membrane using methyl-β-cyclodextrin (MBCD) and then observing the changes in the diffusion coefficients via FRAP [83]. An increase in diffusion coefficient would mean that there are fewer or smaller nano-domains to slow down molecules resulting in faster recovery, while a measured slower diffusion coefficient would indicate the opposite result.…”

Section: Fluorescence Recovery After Photobleachingmentioning

confidence: 99%

Revealing Plasma Membrane Nano-Domains with Diffusion Analysis Methods

et al. 2020

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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.

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Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components

Cited by 6 publications

References 72 publications

ESCRT-mediated vesicle concatenation in plant endosomes

ESCRT-mediated vesicle concatenation in plant endosomes

Biophysical characterization dataset of native nicotinic acetylcholine receptor in lipid-like detergent complexes

Revealing Plasma Membrane Nano-Domains with Diffusion Analysis Methods

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