Endocytic and exocytic pathways are critical for cellular homeostasis, tissue function, and overall cell survival. Simply put, endocytosis is the process that a cell uses to take in molecules from the extracellular space by folding its membrane around it and forming a vesicle. Exocytosis is the reverse process, which uses vesicles to release substances to the extracellular space. These processes have been suggested to play a critical role in hormone secretion, membrane receptor internalization, pathogen engulfment, and neuronal communications.

Today, we’ll retrace some of the landmark discoveries in the field of endocytosis and exocytosis, highlight some of the unanswered questions, feature notable methods that are used today, and lastly, explore a few specific experiments performed to better understand these processes.

Let’s revisit some of the momentous discoveries that led to the current understanding of endocytosis and exocytosis.

The first documentation related to endocytosis can be mapped back to 1882, when Ilya Metchnikov, using a light microscope, observed that specific cells engulfed invading pathogens. He called this process “phagocytosis,” where cells engulf pathogen via vesicle formation. Almost half a century later, in 1931, Warren Lewis observed a similar process of vesicle formation when cells took in fluid. He named this behavior “pinocytosis.”

Later in 1953, George Palade, while examining the structural and functional organization of the cell, discovered “cave-like” invaginations of the membranes, and called them caveolae. He inferred that these must be required for cell-intake. Soon after, in 1955, Nobel laureate Christian de Duve coined the term endocytosis, which encompassed “phagocytosis” and “pinocytosis.” However, the story of endocytosis wasn’t over yet.

In 1975, Michael Brown and Joseph Goldstein, with electron microscopy expert Richard Anderson, observed that when low density lipoprotein, or LDL, binds to its cell surface receptor, it leads to formation of “coated pits.” These pits are then internalized, and endocytose LDL receptors. This was the discovery of the third type, called “receptor-mediated endocytosis.” In the same year, Barbara Pearse isolated the major coat protein, a triskelion molecule, and named it as clathrin. Therefore, this process is also called “clathrin-mediated endocytosis.”

That was all regarding endocytosis. Now let’s discuss how we learned about exocytosis. In 1980, Randy Schekman’s group generated yeast mutants that were secretion deficient, and revealed the presence of critical genes that coded for proteins necessary for exocytosis.

In 1993, James Rothman identified some of these proteins, and based on their chemical nature they were called SNAREs. In his seminal, “SNARE hypothesis,” he proposed that these helical structures hook on to each other, causing membranes to come closer with sufficient force to fuse and generate exocytosis.

Around the same time, Thomas Südhof established that this process was also tightly controlled in neurons by calcium-sensing proteins called synaptotagmins that fostered and accurately timed vesicle fusion for neurotransmitter release. Together, these scientists were awarded the Nobel Prize in 2013.

Despite the breadth of these discoveries, many intriguing puzzles remain. Let’s take a look at some of the questions being explored today.

Scientists are starting to ask how the functions of endocytosis and exocytosis go beyond acquiring and secreting substances. For example, how are neurotransmitters continuously released by vesicles fusing to the cell membrane without a catastrophic expansion of cell size? They are trying to identify signals that cause cells to internalize membrane, in order to offset the expansion and recycle resources.

Another interesting topic is: what components make up the sophisticated molecular machinery that drives these processes? For example, phagocytosis necessitates large membrane deformations to surround invading pathogens. Scientists are investigating how cytoskeletal proteins like actin contribute to dramatic membrane remodeling.

Lastly, since aberrant endocytosis and exocytosis can result in serious diseases, scientists are interested in understanding what causes this dysregulation. One of the proteins being scrutinized is α-synuclein, whose secretion from neurons has been implicated in the progressive death of nearby neurons. Understanding its exocytosis could provide valuable insights on the treatment of neurodegenerative diseases like Parkinson’s.

Now that we’ve considered some of the key questions being investigated, let’s see what tools are available to answer them.

Researchers use a cell biotinylation assay to track endocytosis of cell surface proteins. This process involves labeling a surface protein with fluorescently tagged biotin, and then allowing the cell to undergo endocytosis. This can be followed by immune blot analysis, revealing protein internalization.

In order to quantify neuronal vesicle recycling, many scientists label cells with membrane-specific fluorescent molecules, like FM dyes. These dyes bind stably to the outer leaflet, and are only internalized by endocytosis. Following sequential stimulation, they are exocytosed. Analyzing release with a fluorescence microscope allows deeper insight into the whole recycling process.

Often, to manipulate and understand the contributions of components that allow exocytosis, scientists set up fusion assays. Two sets of vesicles with distinct fluorescent dye contents are prepared and allowed to come together. Fusion between them results in formation of a new product, which can be monitored using a microplate reader.

Lastly, sophisticated imaging methods, including fluorescence imaging and fluorescence live cell imaging, present researchers with the unique opportunity to image morphological structures and molecular events of endocytosis and exocytosis.

Finally, let’s look at some specific ways in which scientists are implementing these tools in labs today.

Cell biologists are interested in studying how exocytosis helps heal injured membranes. Here, researchers first injured cells in solution of FM dyes by rolling glass beads over them. Subsequent fluorescence imaging shows that if the rate of exocytosis is fast, it will quickly seal up the membrane and stop FM leaking into the cell. When exocytosis is slow, it results in extensive intracellular FM staining.

Researchers can use fusion assays to model the contribution of specific fusion proteins. Here, researchers expressed different vesicle-associated membrane proteins or “VAMPs,” which are SNARE proteins, on the surface of two pools of cells. The fusion was then allowed to take place, and the result was quantified using a spectrometer. Using this setup, scientists were able to compare the fusion efficiencies of multiple VAMPs.

Lastly, researchers are aiming to understand cell surface receptor endocytosis in response to a drug. Here, scientists treated fluorescently tagged cells with a drug, and visualized receptor medicated endocytosis happening in real time using time-lapse microscopy.

You’ve just watched JoVE’s introduction to endocytosis and exocytosis. In this video, we reviewed the historical highlights starting from the discovery of phagocytosis to defining the mechanisms of neurotransmitter release. Next, we introduced a few key questions being asked. We also explored prominent research strategies, and discussed some of their current applications. As always, thanks for watching!