The Discovery of Lysosomes and Autophagy

Normally, when you are hungry you look for something to eat, but have you ever wondered what happens inside your cells when no food is available? As incredible as it sounds, eukaryotic cells have evolved a way to resist eating for long periods of time by digesting their own components. When starving conditions are prolonged, cells digest part of their own cytoplasmic components to recycle metabolites needed to synthesize essential molecules. For example, cells can digest long-lived proteins to release amino acids. How did this process of self-eating evolve? How is it controlled by the cell? Today, research on autophagy is a growing field with increasing prominence because understanding the basic mechanisms of autophagy is key to understanding how cells sustain themselves.

Metabolism is the set of chemical reactions that occur in cells (and consequently, in living organisms) that are involved in cell growth, reproduction, and maintenance. Metabolism is a balance of two antagonistic processes: anabolism and catabolism. Anabolism synthesizes molecules and builds structures. On the other side of the spectrum, catabolism breaks down molecules and structures. Autophagy (a Greek word that means "self-eating") is a catabolic process in eukaryotic cells that delivers cytoplasmic components and organelles to the lysosomes for digestion. Lysosomes are specialized organelles that break up macromolecules, allowing the cell to reuse the materials.

In 1949, Christian de Duve, then chairman of the Laboratory of Physiological Chemistry at the University of Louvain in Belgium, was studying how insulin acted on liver cells. He wanted to determine the location of an enzyme (a type of protein involved in chemical reactions) called glucose-6-phosphatase inside the cells. He and his group knew that this enzyme played a key role in regulating blood sugar levels. They obtained cellular extracts by blending rat liver fragments in distilled water and centrifuging the mixture at high speeds. They observed high phosphatase activity in the extracts. However, when they tried to purify the enzyme from cellular extracts, they had an unexpected problem-they could precipitate the enzyme, but they could not redissolve it.

Instead of using cellular extracts, they decided to use a more gentle technique that fractionated the cells with differential centrifugation. This technique separates different components of cells based on their sizes and densities. The researchers ruptured the rat liver cells and then fractionated the samples in a sucrose medium using centrifugation. They succeeded in detecting the enzyme's activity in what was known as the microsomal fraction of the cell. Then serendipity entered the picture.

The scientists were using an enzyme called acid phosphatase as a control for their experiments. To their surprise, the acid phosphatase activity after differential centrifugation was only 10% of the expected enzymatic activity (i.e., the activity they obtained in their previous experiments using cellular extracts). One day, by chance, a scientist purified some cell fractions and then left them in the fridge. Five days later, after returning to measure the enzymatic activity of the fractions, they observed the enzymatic activity levels they were looking for! To ensure there was no mistake, they repeated the experiment a number of times. Each time, the results were the same: if they measured the enzymatic activity using fresh samples, then the activity was only 10% of the activity obtained when they let the samples rest for five days in the fridge. How could they explain these results?

They hypothesized that a membrane-like barrier limited the accessibility of the enzyme to its substrate. Letting the samples rest for a few days gave the enzymes time to diffuse. They described the membrane-like barrier as a "saclike structure surrounded by a membrane and containing acid phosphatase." By 1955, additional hydrolases (enzymes that break chemical bonds) were discovered in these saclike structures, suggesting that they were a new type of organelle with a lytic function (Bainton 1981). De Duve named these new organelles "lysosomes" to reflect their lytic nature.

That same year, Alex Novikoff from the University of Vermont visited de Duve´s laboratory. An experienced microscopist, Novikoff was able to obtain the first electron micrographs of the new organelle from samples of partially purified lysosomes. Using a staining method for acid phosphatase, de Duve and Novikoff confirmed its location in the lysosome using light and electron microscopic studies (Essner & Novikoff 1961).

Nowadays, we know that lysosomes contain hydrolases that are capable of digesting all kinds of macromolecules. Christian de Duve was recognized for his role in the discovery of lysosomes when he was awarded the Nobel Prize in Physiology or Medicine in 1974. The discovery of lysosomes led to many new questions. The most critical question was: what was the physiological function of this "bag" of enzymes?

One of the definitive clues about the function of lysosomes came from the work of Werner Strauss and his group. Strauss wanted to understand how extracellular molecules enter the cell, a process known as endocytosis. He labeled proteins and followed them in their journey through the cell. He observed that the lysosomes described by de Duve contained fragments of the labeled proteins, and concluded that proteins were degraded in the lysosome (Straus 1954). In another series of experiments, Zanvil Cohn fed macrophages (a type of cell in the immune system) radiolabeled bacteria. He observed that all types of radiolabeled bacterial molecules (lipids, amino acids, and carbohydrates) accumulated in the lysosomes (Cohn 1963). Cohn concluded that lysosomes functioned as the digestive system of cells by "eating" compounds that enter the cell from the outside, as well as compounds inside the cell. Therefore, lysosomes are comparable to recycling plants, which are in charge of disposing of waste products and reusing components.

In the following years, researchers studied different types of cells using electron microscopes and discovered a wide variety of vesicles. Some of the vesicles contained engulfed cytoplasmic material. What did these vesicles do? Marilyn Farquhar and her associates at the University of California, San Francisco, were the first to suggest that these particular vesicles were pre-lysosomes (Smith & Farquhar 1966).

Pre-lysosomes form de novo in the cytoplasm from a cup-shaped membrane called a phagophore. The edges of the phagophore expand while becoming spherical until they seal, enclosing the engulfed pieces of cytoplasm with whatever might lie inside, and giving rise to a double-membrane vesicle. Farquhar observed these closed vesicles, which are known as autophagosomes. Autophagosomes take up damaged molecules or organelles and carry this cargo to the lysosomes. When de Duve observed autophagosomes, he realized that cells could degrade their own components and named the process "autophagy" (Figure 1).

For many years, scientists could only study autophagy by examining cells with electron microscopes. Using this tool, they established that after autophagosomes form, they fuse to the lysosomal membrane to form a structure known as the autolysosome (Figure 1). Then, depending on the stimuli that initiated the autophagy process, the cargo is either degraded or recycled.

In 1992, Yoshinori Ohsumi and his colleagues at the University of Tokyo discovered that autophagy also occurs in yeast. Using a light microscope, they noticed that a few hours after starving yeasts of nutrients, the vacuole (which functions like our lysosomes) was filled with vesicles containing chunks of cytoplasm. These vesicles originate in the cytoplasm and then fuse with the lysosome, exactly as in animal and plant cells. Being able to use yeasts as an experimental model opened the door for studying the molecular biology of the autophagic machinery (and for identifying the key proteins that participate in the process) (Takeshige et al. 1992).

In 2004, fifty years after Novikoff and de Duve observed lysosomes under the electron microscope, Noboru Mizushima and colleagues at the Tokyo Medical and Dental University tracked the formation of autophagosomes by taking pictures every five minutes. For their experiments, they used a fluorescently-labeled protein that localizes specifically in phagophores and observed the real-time formation of autophagosomes (Klionsky 2007) (see video, Figure 2).

One mystery that remained unanswered for many years was: which membrane in the cell gives rise to phagophores? In 2010, Jennifer Lippincott-Schwartz and her colleagues used fluorescently-labeled proteins to study the origin of phagophores. They observed that the outer membrane of the mitochondria was the main membrane source, with some contribution from the endoplasmic reticulum (a network of membranes found in the cytoplasm of the cell) (McEwan & Dikic 2010).

How much and what parts of the cell can be eaten without causing cell death? Scientists hypothesized that the level of autophagy and the cargo specificity must be tightly controlled to ensure the cell's health. For example, when there are plenty of nutrients, the autophagy level should be low, but autophagy must increase upon starvation.

For unicellular organisms, it is essential to maintain a pool of metabolites, such as amino acids. Therefore, starvation-induced autophagy was likely selected first in unicellular organisms, and later retained in multicellular organisms. In mammals, autophagy is not only induced by starvation, but also by physiological stimuli, such as growth factors and hormones, as well as by pathogen invasion. In general, autophagy is used to engulf non-specific components, but it can also selectively degrade damaged organelles, pathogenic inclusions, or invasive bacteria (Nakatogawa et al. 2009). Therefore, autophagy likely evolved as a response to cell starvation, and later it probably served as a primitive immune defense.

The process of autophagy occurs all the time, whether a cell is starving or not, but at a basal level. Under normal conditions autophagy removes damaged proteins and organelles to prevent cell damage. However, under stress (e.g., starvation, the absence of growth factors, or the lack of oxygen), the assembly of phagosomes increases. Under these conditions, intracellular molecules are digested to provide the nutrients the cell needs.

Some years ago, scientists established a link between autophagy and disease. Beth Levine and her colleagues at Columbia University College of Physicians and Surgeons showed that tumors develop after deleting one of the two copies of the cell's Beclin1 gene. Beclin1 is a mammalian homolog of the yeast Atg6 gene, which is necessary for autophagy. In fact, 40-75% of sporadic human breast and ovarian cancers are missing one copy of Beclin1. When Levine and her colleagues increased the expression of Beclin1 in human carcinoma cells, they observed more autophagy, and when these cells were injected into a mouse model they were less capable of developing tumors (Liang et al. 1999). In another set of studies, Eileen White and her colleagues at the University of Medicine and Dentistry in New Jersey found that autophagy protects against DNA damage. When they inhibited autophagy, they observed more chromosomal abnormalities, which are typically associated with tumorigenesis (Mathew et al. 2007).

These discoveries have led many scientists around the world to study the diverse physiological functions of autophagy. Currently, there are over 2,500 publications about autophagy, and several recent findings have linked this cellular process with immune and metabolic diseases. There are still many fundamental questions about the mechanisms governing autophagy that must be addressed. Many scientists believe that studying autophagy regulation is critical to understanding its biological role and to developing alternative therapies for diseases associated with autophagy dysregulation.