Cell Biology


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0 Q&A 441 Views May 5, 2024

Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell–cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry–based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level.

0 Q&A 2032 Views Dec 20, 2023

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Graphical overview

0 Q&A 636 Views Sep 5, 2023

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling modality mediated by Orai Ca2+ channels at the plasma membrane (PM) and the endoplasmic reticulum (ER) Ca2+ sensors STIM1/2. At steady state, Orai1 constitutively cycles between an intracellular compartment and the PM. Orai1 PM residency is modulated by its endocytosis and exocytosis rates. Therefore, Orai1 trafficking represents an important regulatory mechanism to define the levels of Ca2+ influx. Here, we present a protocol using the dually tagged YFP-HA-Orai1 with a cytosolic YFP and extracellular hemagglutinin (HA) tag to quantify Orai1 cycling rates. For measuring Orai1 endocytosis, cells expressing YFP-HA-Orai1 are incubated with mouse anti-HA antibody for various periods of time before being fixed and stained for surface Orai1 with Cy5-labeled anti-mouse IgG. The cells are fixed again, permeabilized, and stained with Cy3-labeled anti-mouse IgG to reveal anti-HA that has been internalized. To quantify Orai1 exocytosis rate, cells are incubated with anti-HA antibody for various incubation periods before being fixed, permeabilized, and then stained with Cy5-labeled anti-mouse IgG. The Cy5/YFP ratio is plotted over time and fitted with a mono-exponential growth curve to determine exocytosis rate. Although the described assays were developed to measure Orai1 trafficking, they are readily adaptable to other PM channels.

Key features

• Detailed protocols to quantify endocytosis and exocytosis rates of Orai1 at the plasma membrane that can be used in various cell lines.

• The endocytosis and exocytosis assays are readily adaptable to study the trafficking of other plasma membrane channels.

Graphical overview

0 Q&A 3530 Views Aug 5, 2020
The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited. Here, we describe a novel quantitative and effectively real-time single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with split-fluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. This approach will greatly extend the ability to study the underlying biology of intracellular membrane trafficking, and how trafficking systems can adapt to the physiologic needs of different cell types and cell states.
0 Q&A 3973 Views Dec 20, 2019
Functional activities of many transmembrane proteins are controlled by their endocytosis. One of the most studied experimental models is the epidermal growth factor (EGF) receptor (EGFR). However, endocytic trafficking of EGFR has been predominantly analyzed using labeled EGF, whereas quantitative analyses of the endocytosis of the receptor itself have been sparse. The fluorescence microscopy methods described here are designed to directly quantify EGFR internalization in living cells without labeled EGFR ligands or antibodies. These methods utilize an engineered EGFR chimera in which the fluorogen activating protein (FAP) is fused to the receptor extracellular domain (FAP-EGFR). Binding of malachite green (MG) based dyes to FAP results in a strong far-red fluorescence of MG, thus efficiently labeling FAP-EGFR. In particular, binding of the cell impermeant MG-Bis-SA dye to FAP produces the pH-sensitive dual-excitation fluorescence, which allows differentiation of the cell-surface and internalized pools of FAP-EGFR. Two modifications of the methodology are described: 1) single-cell three-dimensional confocal imaging; and 2) high-throughput assay in multi-well plates. These methodologies can be adopted to study endocytosis of any other transmembrane protein extracellularly tagged with FAP.
0 Q&A 4107 Views Nov 20, 2019
Merocytophagy (“mero”, Greek for partial; “cytophagy” for cell eating) is a process by which cells acquire microbes and cytosolic material through phagocytosis of a small portion of neighboring cells upon cell-cell contact. Cell-cell contact dependent transfer events can be assessed through co-incubation of differently labeled cells. With these assays, it is difficult to analyze the recipient cells by microscopy or bacterial burden within only recipient cells. Therefore, we established a synchronized transfer assay that allows for recipient cells to be isolated from donor cells following transfer events at a high purity. Here, we present this assay in context of bacterial infections and cytosolic cellular staining. With this protocol, mechanisms of cell-cell contact dependent transfer events and the events following merocytophagy can easily be investigated.
0 Q&A 9300 Views Feb 20, 2019
Efficiency of drug and gene delivery via nonviral vehicles is contingent on proper cellular uptake and intracellular release. Further, various cargos, such as nucleases for gene editing or inhibitors for endosomal receptors, require transport to specific compartments of the cell. Hence, characterization of cellular uptake and endocytic pathways is crucial for the optimization of any nanoparticle-mediated intracellular delivery system. Previous work on endocytic pathways looks at the effect of various pathway inhibitors on the uptake efficiency of nanoparticles carrying fluorescently-labeled cargo. While this helps attribute particle uptake to specific pathways like caveolae-mediated or clathrin-mediated endocytosis, this does not provide a holistic picture of the delivery process. Here, we provide a general protocol that combines systematic studies of inhibitor effects on efficiency with quantification of particle-induced cell membrane permeability. By applying this methodology to a nucleic acid delivery system, for example a helical polypeptide-based nanoparticle for plasmid and guide RNA delivery, we gain understanding of the endocytic mechanisms and cell uptake for intelligent design of intracellular delivery.
0 Q&A 5331 Views Sep 5, 2018
Microglia are professional phagocytes in the brain and deficiency in their phagocytic activity plays an important role in Parkinson’s disease. This protocol mainly describes the phagocytosis assay for uptake of α-synuclein preformed fibrils, a pathologic form of α-synuclein, by primary microglia.

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