Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Tham, Daniel Kai Long; Moukhles, Hakima

doi:10.3791/55974

Cited by 5 publications

(5 citation statements)

References 16 publications

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“…Biotinylation was performed using the method previously described (Tham and Moukhles, 2017 ). Briefly, confluent astrocyte and MDA-435 cultures were treated with H 2 O 2 for 1 h, washed with cold DPBS (Gibco, Invitrogen, Burlington, ON, Canada), and then labeled for 30 min at 4°C using 0.5 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce Biotechnology, Rockford, IL, USA).…”

Section: Methodsmentioning

confidence: 99%

The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

Tham

Perronnet

et al. 2017

Front. Cell. Neurosci.

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The reperfusion of ischemic brain tissue following a cerebral stroke causes oxidative stress, and leads to the generation of reactive oxygen species (ROS). Apart from inflicting oxidative damage, the latter may also trigger the upregulation of aquaporin 4 (AQP4), a water-permeable channel expressed by astroglial cells of the blood-brain barrier (BBB), and contribute to edema formation, the severity of which is known to be the primary determinant of mortality and morbidity. The mechanism through which this occurs remains unknown. In the present study, we have attempted to address this question using primary astrocyte cultures treated with hydrogen peroxide (H2O2) as a model system. First, we showed that H2O2 induces a significant increase in AQP4 protein levels and that this is inhibited by the antioxidant N-acetylcysteine (NAC). Second, we demonstrated using cell surface biotinylation that H2O2 increases AQP4 cell-surface expression independently of it’s increased synthesis. In parallel, we found that caveolin-1 (Cav1) is phosphorylated in response to H2O2 and that this is reversed by the Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). PP2 also abrogated the H2O2-induced increase in AQP4 surface levels, suggesting that the phosphorylation of tyrosine-14 of Cav1 regulates this process. We further showed that dominant-negative Y14F and phosphomimetic Y14D mutants caused a decrease and increase in AQP4 membrane expression respectively, and that the knockdown of Cav1 inhibits the increase in AQP4 cell-surface, expression following H2O2 treatment. Together, these findings suggest that oxidative stress-induced Cav1 phosphorylation modulates AQP4 subcellular distribution and therefore may indirectly regulate AQP4-mediated water transport.

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Section: Methodsmentioning

confidence: 99%

The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

Tham

Perronnet

et al. 2017

Front. Cell. Neurosci.

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“…Examples of biochemical isolation of surface expressed receptors include biotinylation protocols in cultured cells or brain slices and subcellular fractionation of cultured cells or tissue 11 . Surface biotinylation is based on the labeling of surface-expressed receptors with biotin by incubating the cultures with ester-activated biotin.…”

Section: Discussionmentioning

confidence: 99%

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Chiu¹,

Barse²,

Hubálková

et al. 2019

JoVE

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Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An "antibody-feeding" approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

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“…Cell surface biotinylation on live cells is commonly used to study mechanisms of endocytosis in cells ( Leser et al, 1996 ; Meulendyke et al, 2005 ; Bitsikas et al, 2014 ; Ito et al, 2014 ). For instance, similar approaches were used to determine the cell-surface expression and endocytic rate of proteins in primary astrocytes, to study endocytosis and recycling of membrane proteins, and also to compare the endocytic flux of different uptake mechanisms ( Cihil and Swiatecka-Urban, 2013 ; Bitsikas et al, 2014 ; Tham and Moukhles, 2017 ). Cell surface biotinylation has also been performed on brain tissue slices to measure plasma membrane protein trafficking in neurons ( Gabriel et al, 2014 ).…”

Section: Methods To Elucidate Nanoparticle Biomolecular Corona-receptmentioning

confidence: 99%

Disentangling Biomolecular Corona Interactions With Cell Receptors and Implications for Targeting of Nanomedicines

Aliyandi

Zuhorn

Salvati

2020

Front. Bioeng. Biotechnol.

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Nanoparticles are promising tools for nanomedicine in a wide array of therapeutic and diagnostic applications. Yet, despite the advances in the biomedical applications of nanomaterials, relatively few nanomedicines made it to the clinics. The formation of the biomolecular corona on the surface of nanoparticles has been known as one of the challenges toward successful targeting of nanomedicines. This adsorbed protein layer can mask targeting moieties and creates a new biological identity that critically affects the subsequent biological interactions of nanomedicines with cells. Extensive studies have been directed toward understanding the characteristics of this layer of biomolecules and its implications for nanomedicine outcomes at cell and organism levels, yet several aspects are still poorly understood. One aspect that still requires further insights is how the biomolecular corona interacts with and is “read” by the cellular machinery. Within this context, this review is focused on the current understanding of the interactions of the biomolecular corona with cell receptors. First, we address the importance and the role of receptors in the uptake of nanoparticles. Second, we discuss the recent advances and techniques in characterizing and identifying biomolecular corona-receptor interactions. Additionally, we present how we can exploit the knowledge of corona-cell receptor interactions to discover novel receptors for targeting of nanocarriers. Finally, we conclude this review with an outlook on possible future perspectives in the field. A better understanding of the first interactions of nanomaterials with cells, and -in particular -the receptors interacting with the biomolecular corona and involved in nanoparticle uptake, will help for the successful design of nanomedicines for targeted delivery.

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Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cited by 5 publications

References 16 publications

The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Disentangling Biomolecular Corona Interactions With Cell Receptors and Implications for Targeting of Nanomedicines

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