Cell Biology

Endocytosis is an essential membrane transport mechanism that is indispensable for the maintenance of life. It is responsible for the selective internalization and subsequent degradation or recycling of specific extracellular proteins and nutrients, thereby facilitating cellular nutrient supply, modulation of receptor signaling, and clearance of foreign substances. However, methods for the quantitative analysis of lysosomal degradation of extracellular proteins via endocytosis remain limited. This protocol describes a method for purifying the protein-of-interest (POI)–red fluorescent protein (RFP)–green fluorescent protein (GFP) fusion protein, which is modified with specific mammalian cell glycans or other modifications, from the conditioned medium of mammalian cell cultures. Subsequently, the protocol details a quantitative approach for evaluating its internalization and lysosomal degradation within cells using the RFP–GFP tandem fluorescent reporter. Following the addition of POI-RFP-GFP to the medium, cells can be subjected to cell biological assays, such as flow cytometry, as well as biochemical analyses, such as immunoblotting. This protocol is broadly applicable to studies of the internalization of extracellular proteins.

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.

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

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