Endocytosis

 

 

Endocytosis refers to the process of internalization of substances into the cell.

There are two main types of endocytosis: phagocytosis and pinocytosis. While during phagocytosis, large particles and bacteria are engulfed, in pinocytosis fluid and molecules contained in it are brought into the cell.

Endocytosis involves cytoskeletal and structural modifications. The 3D Cell Explorer allows visualizing of fine membrane deformations and posterior vesicle formation that occur in endocytic processes.

Phagocytosis

Macrophages are present in almost all tissues. They are contributing to various processes in the healthy organism, such as development, wound healing, infection and tissue homeostasis. They can rapidly change their phenotype in response to variations in their environment. Macrophages are known for their classical function as antimicrobial phagocytes but support immune function as well by the presentation of antigens. Their research applications are vast, and in vitro assays are increasingly used in a wide range of research areas, including immunology, bacteriology and parasitology, as well as in biomedical and transplantation studies. Two advantages of macrophages in cell culture are that they are relatively easy to generate and to cultivate.

Marker-free Imaging of Cryopreserved Human M1 Macrophages

In these videos – obtained with Nanolive’s 3D Cell Explorer – we present cryopreserved human M1 macrophages from PromoCell in cell culture (video 1). The 3D Cell Explorer allows to image these living macrophages in a novel, marker-free fashion. A special note goes to the visualization of membrane ruffling as waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body. Macrophages were imaged for over 24h at a frequency of 1 image every 10 seconds.


Video 1

Phagocytosis Assay Kits by PromoCell were used to test the viability and cellular functioning of the macrophages (video 2 & 3). E.coli particles, visible as small ellipsoid particles, are trapped by the cells, transported and lysed. This system can be used to provide a robust screening system for activators and/or inhibitors of phagocytosis and Toll-like Receptor (TLR) ligands.

Video 2: Macrophages were imaged with Nanolive’s 3D Cell Explorer for over 24h at a frequency of 1 image every 10 seconds. Green circles indicate E.coli being engulfed through phagocytosis

Video 3: Macrophages were imaged with Nanolive’s 3D Cell Explorer for over 4h at a frequency of 1 image every 10 seconds. Green circles indicate E.coli being engulfed through phagocytosis

The Perfect Murder – Macrophage Cell Killed by T-cells

In this movie, we can observe macrophages and T-cells interacting. Naive T-cells are being presented with antigens by the macrophages which “instruct” T-cells on what type of cells to target (such as cancer cells) and kill. During this interaction, T-cells can play a role in immune system homeostasis [1] by killing the macrophage presenting the antigen. It was documented that the event is triggered by the presence of specific markers on the macrophage surface (called TRAIL and TWEAK [2]) telling T-cells to induce apoptosis of their fellow macrophage. The dead macrophage is then seen to be recycled by other macrophages, making space for new macrophages to be produced while keeping the same overall macrophage population.

In this video, pre-stimulated antigen presenting cells (APCs, namely dendritic cells and macrophages, obtained after isolation and in vitro differentiation of bone marrow cells from C57BL/6 mice, pre-stimulation of the APCs has been done with IFN-gamma and LPS) are cultured with freshly isolated “naïve T cells” from the spleen of OT-I mice and observed at a frequency of 1 image every 10 sec for 16 hours.*

*All primary cells were freshly isolated from mice (C57BL/6 background). Naïve t-cells were also obtained from OT-I mice.

[1] Andersen, M. H. (2018). The Balance Players of the Adaptive Immune System, (15), 1–5. https://doi.org/10.1158/0008-5472.CAN-17-3607

[2] Kaplan, M. J., Ray, D., Mo, R., Raymond, L., Richardson, B. C., & Richardson, B. C. (2018). TRAIL (Apo2 Ligand) and TWEAK (Apo3 Ligand) Mediate CD4 + T Cell Killing of Antigen-Presenting Macrophages. https://doi.org/10.4049/jimmunol.164.6.2897

Macropinocytosis

Vesicle Transport

Vesicle transport is an active and thus energy consuming process that the cell uses in order to capture or release macromolecules into or out of the cell.

While exocytosis helps release content like proteins, waste products or toxins to the outside of the cell via vesicles that are formed in the Golgi apparatus and then fused with the plasma membrane, endocytosis aims to capture substances by enclosing them in vesicles resulting from cell membrane folding.

Endocytosis

Depending on the content to engulf, endocytosis can be divided in phagocytosis, receptor-mediated endocytosis, and pinocytosis.

As we showed in our Macrophages – the big eater’s post, phagocytosis is a crucial process in macrophages, as it allows them to get rid of microbes, thus playing a role in cell immunity. This cell eating of solid material results in the formation of phagosomes, which are vesicles coming from the evagination of the cell membrane that will fuse with lysosomes carrying the enzymes required to break the engulfed substances.

When instead of solid particles it is liquid material encapsulated and internalized by the cell we can differentiate between receptor-mediated endocytosis and pinocytosis… but what is the difference between them? While receptor-mediated endocytosis has specificity in the captured substances, pinocytosis is a non-specific cell drinking.

Pinocytosis

Hence, pinocytosis is the process where fluid matter coming from the outside of the cell is obtained via invaginations of the cell membrane. Its biological meaning is mainly to absorb extracellular fluids (ECF) containing solutes like sugars or proteins, once triggered by the presence of certain substances outside the cell (amino acids or certain ions) but it is also involved in cell immunity.

Two types of pinocytosis can be characterized. Micropinocytosis is observed in cells such as the microvilli of the digestive tract and involves the intake of small vesicles. Vesicles 5 to 50 times bigger than those formed during pinocytosis are seen in macropinocytosis, usually as a result of immune system response in areas where pathogens and antigens are found. A greater deformation of the membrane is needed in order to lead to the formation of big vesicles. As observed in the video, the membrane extends forming arm-like claws that reconnect resulting in a macropinosome. Once at the cytoplasm, the macropinosome fuses with a lysosome.

Figure 1. Steps of macropinocytosis. Equivalence between schema and 3D Cell Explorer microscope caption. Schema from: Lim, J.P., & Gleeson, P.A. (2011). Macropinocytosis: an endocytic pathway for internalising large gulps. Immunology and cell biology, 89 8, 836-43.

Ellinger, I. & Pietschmann, P. Endocytosis in health and disease—a thematic issue dedicated to Renate Fuchs. Wiener Medizinische Wochenschrift 166, 193–195 (2016).

Kruth, H. S. et al. Macropinocytosis Is the Endocytic Pathway That Mediates Macrophage Foam Cell Formation with Native Low Density Lipoprotein. J. Biol. Chem. 280, 2352–2360 (2005).

Bhattacharya, S., Roxbury, D., Gong, X., Mukhopadhyay, D. & Jagota, A. DNA Conjugated SWCNTs Enter Endothelial Cells via Rac1 Mediated Macropinocytosis. Nano Lett.12, 1826–1830 (2012).

Scientific Publication

Endocytosis-mediated mitochondrial transplantation: Transferring normal human astrocytic mitochondria into glioma cells rescues aerobic respiration and enhances radiosensitivity

This is a publication in the journal Theranostics from users of the Nanolive’s 3D Cell Explorer in the Laboratory of Heavy Ion Radiation Medicine of Chinese Academy of Sciences in Lanzhou, China.

Mitochondrial metabolic abnormalities have been linked to resistance to radiotherapy due to radiation cytotoxicity in cancer cells[1]. In aerobic conditions, an increased glycolysis and lactate production instead of the much more efficient oxidative phosphorylation are observed in cancer cells[2]. This change in metabolism is known as the Warburg effect[2].

Sun and colleagues have studied the radiosensitization effects induced by transplantation of mitochondria from normal human astrocytic into glioma cells. The focus of their study was to identify the mechanism of free mitochondrial transfer into host cells via a NAD+-CD38-cADPR-Ca2+-endocytosis pathway. Their results show that starvation treatment led to a decreased Warburg effect and a recuperation of aerobic respiration. Mitochondrial transplantation into glioma cells could reduce resistance to radiotherapy.

The 3D Cell Explorer was used to obtain live images of the dynamic behaviour of endocytosis during starvation treatment and of the interaction between this process and mitochondria. Nanolive imaging has proven to be a method of choice in organelle dynamics research, due to its non-invasive and phototoxicity free imaging. Their full publication is available here!

Figure: Transplantation of isolated mitochondria into U87 cell through endocytosis

[1]         C. Sun et al., “Endocytosis-mediated mitochondrial transplantation: Transferring normal human astrocytic mitochondria into glioma cells rescues aerobic respiration and enhances radiosensitivity,” Theranostics, vol. 9, p. 12, 2019.

[2]         O. Warburg, F. Wind, and E. Negelein, “THE METABOLISM OF TUMORS IN THE BODY.,” J. Gen. Physiol., vol. 8, no. 6, pp. 519–30, Mar. 1927.