Secret of the body’s self-renewal

DURING starvation a person is able to survive for a considerable stretch of time. Despite the obvious stress, the body is able to cope because of an internal physiological process of “self-cannibalisation” through which the body makes use of its inessential and damaged cellular components by breaking them down and reassembling them into useful proteins and the nutrients needed to sustain its essential functions. This is called autophagy, which literally means “self-devouring” in ancient Greek (auto meaning self, and phagein meaning to eat). It is a process that has been evolutionarily conserved and is intrinsic to all organisms, from unicellular yeast to multicellular mammalian systems like humans.

In fact, without autophagy our cells could not survive. It is an essential part of the body’s self-renewal process. As the scientist Juleen Zierath, a member of the Nobel Committee for Physiology or Medicine, pointed out in a post-announcement interview, every day 200-300 grams of proteins need to be replaced in the human body, but, on average, the intake is only about 70 g of proteins, which is insufficient to take care of the requirement to make new proteins. “Because of this machinery, we are able to rely on some of our own proteins so that we can sustain and we survive,” she said.

Thus, autophagy maintains normal functioning “homeostasis” (the tendency of a biological system to actively maintain the fairly stable internal equilibrium conditions necessary for survival despite changing external conditions) by protein degradation and the recycling of destroyed components from the cytoplasm of cells (the part exterior to the cell nucleus) for new cell formation. This year’s Nobel Prize in Physiology or Medicine has been awarded to the 71-year-old Japanese biologist Yoshinori Ohsumi of the Tokyo Institute of Technology for unravelling in the 1990s the underlying molecular mechanism of autophagy. He was the first to visually observe the process.

The concept of autophagy was known in the 1960s itself. In fact, the phrase was coined in 1963 by the Belgian scientist Christian de Duve, who was awarded the 1974 Nobel Prize in Medicine for his discovery in the 1950s of what is called lysosome . However, the mechanism and physiological relevance remained poorly understood until Ohsumi appeared on the scene. His work dramatically transformed the understanding of this important physiological process.

In the mid 1950s, scientists found specialised cellular compartments called organelles that are bound by lipid membranes (Figure 1). The lysosome is an organelle containing enzymes that can digest proteins, carbohydrates and lipids and it functions as a workstation for breaking down cellular components and plays a crucial part in autophagy. Research in the 1960s revealed a new aspect of these specialised structures within the cell. Sometimes large amounts of cytoplasmic material, including organelles, were found within lysosomes, indicating the existence of a well-organised strategy and machinery to transport large quantities of cellular cargo to the lysosome for degradation. Further biochemical research and microscopic analyses revealed that this transportation agent was yet another kind of double-membraned organelle or vesicle. These were termed autophagosomes . They fused with the lysosome as they delivered the cargo, allowing the process of degradation by the enzymes of the lysosome to take over.

Though in the 1970s and the 1980s, yet another protein-degrading mechanism, which relies on a cellular structure called the proteasome and is mediated by ubiquitin, was discovered (for which discovery the 2004 Nobel Prize was given), it became clear that the proteasome-based process could not degrade damaged or unwanted long-lived large protein complexes and organelles. So, if it was autophagy, the exact mechanism underlying this sophisticated machinery was far from clear, and it remained a biological enigma for three decades. Molecular markers were not available, and the components of the autophagy machinery could not be easily accessed for study as the autophagosome was a transient structure that existed only for 10-20 minutes before fusing with the lysosome, making morphological and biochemical studies on it very difficult.

Ohsumi’s pursuit

There were many unanswered questions about autophagy: How is it initiated? How are autophagosomes formed? How important is autophagy for cellular and organism survival? Does the process have any role in human diseases? Enter Yoshinori Ohsumi. In the late 1980s, Ohsumi, then an assistant professor at the University of Tokyo, embarked on the study of autophagy using baker’s yeast, Saccharomyces cerevisiae , as the model system. In the unicellular yeast, the vesicle corresponding to the lysosome is called the vacuole.

“As the fundamental unit of life, the question of how dynamic the cell is has always captivated my intellectual curiosity,” said Ohsumi in his address at the post-announcement press conference at his institute. “This strong motivation led me to use yeast, the humble single-cell organism, to address some of the important basic questions of life. My work with yeast stemmed from a personal desire to undertake work that is unique and doesn’t follow the intellectual trends of the day. Yeast is an excellent tool for genetic analysis, so we therefore immediately began the process of identifying genes essential for autophagy to occur.”

According to an interview he gave in December 2012 after winning the Kyoto Prize, Ohsumi stated that back in his postdoctoral days at The Rockefeller University in New York, when he was attempting to isolate the nuclei in yeast cells, he had seen a layer of clearly concentrated organelles at the top layer in the centrifuge tube that was to be discarded and noticed that these were actually vacuoles. He had wondered then whether these vacuoles played an important role in the cells. Upon returning to Japan in 1988, the 43-year-old Ohsumi started his own laboratory at the College of Arts and Sciences at the University of Tokyo with himself as its sole member. And this is where his 28 years of autophagy research began. “I started out with a love of the microscope. Vacuoles are the only organelles visible under the light microscope, and I often observed them. My observations under the microscope were the main reason I was able to discover these hitherto unknown functions of vacuoles,” he said in that interview. “Doing something no one else is doing” was also the motivation for Ohsumi to try and figure out the mechanism of degradation in yeast cells.

“I had a strong interest in the practical degradative function of the vacuole in the cell,” Ohsumi said in his post-announcement speech. “I devised a method to capture this process using a light microscope, which is a ubiquitous piece of equipment in biological laboratories. The work of Misuzu Baba, who joined my lab and conducted electron microscopy of the process in yeast, confirmed that what we could see under the light microscope was actually exactly the same process as a phenomenon that had been observed decades earlier in mammalian cells but was never understood,” Ohsumi said.

When Ohsumi started out, he faced a major challenge: yeasts are small and their inner structures are not easily distinguished under the microscope. He reasoned thus. In the absence of nutrients, yeasts fall into a state of starvation stress. Next they begin to form spores inside the cells, which allows the cells to ward off the starvation. He argued that if vacuoles had a degradative function, they were likely to become most active during starvation when the cells were forming spores. And if he could somehow halt the degradative function in the vacuoles, the autophagosomes would accumulate and he should be able to observe what was being degraded.

Breakthrough experiment

To carry out his ingenious method, he cultured mutant yeasts lacking in vacuolar hydrolytic enzymes (enzymes that use water to break down proteins, carbohydrates and lipids) and simultaneously induced autophagy by starving the cells. Using a light microscope, Ohsumi could, within hours, observe the process in the vacuoles during the state of starvation. The vesicles in motion were autophagosomes, and the experiment demonstrated the existence of autophagy in yeast cells (Figure 2).

“When I observed yeasts that had been starved for several hours,” Ohsumi said in his 2012 interview, “I found that large numbers of small granules accumulate inside the vacuoles and that they were highly mobile. The granules were single-membrane bound structures which contain a portion of cytoplasmic materials. The granules were drawn into the vacuoles and presented the appearance of Brownian movement. The Brownian movement occurred because yeasts contain so many small amount[s] of proteins and are low in viscosity. This made a great impression on me and I spent hours watching them.”

This was the first time anyone had actually seen the autophagous function in action. When the degradative enzymes are present in vacuoles as in a normal working cell, any cytoplasmic material inside is immediately broken down and cannot, therefore, be visually observed. The magnification that present-day microscopes provide are 2,250 times or more, whereas in those days the maximum magnification one could get was 600×. “I am lucky,” Ohsumi said. “I might not have noticed them if they hadn’t been moving.” This was a major breakthrough, and the results were published in 1992.

But, more importantly, this breakthrough experiment laid the basis for Ohsumi to identify and characterise the genes involved in the process. The identification of the mutant yeast strains, in which autophagosomes accumulated, also meant that the genes responsible for autophagy had been inactivated in them. Ohsumi exposed yeast cells to a chemical that randomly introduced mutations in many genes, and then he induced autophagy through starvation stress. Using this strategy and armed with thousands of mutant strains, within a year Ohsumi was able to identify the genes essential for autophagy. “Fortunately,” Ohsumi said in his post-announcement talk, “we were able to identify many indispensable autophagy genes in a very short period of time thanks to the efforts of a graduate student who joined my lab, Miki Tsukada.” These were called ATG (AuTophaGy-related) genes, and Ohsumi succeeded in identifying as many as 15 of them (Figure 3). These results were published in 1993.

In the series of elegant studies that followed, Ohsumi characterised the proteins encoded by these genes by cloning the gene products. The results revealed that autophagy induced by stress signals is regulated by a cascade of proteins, and protein complexes, each controlling a distinct stage of autophagosome initiation and formation (Figure 4). Following the identification of the machinery for autophagy in yeast, Ohsumi and others set out to find out whether there was a corresponding mechanism in other organisms. It became clear that virtually identical mechanisms operate in all organisms, including humans. Ohsumi and colleagues were, in fact, the first to identify the mammalian homologues of the yeast ATG genes, which formed the basis for studies on autophagy in higher eukaryotes. Studies of knockout mouse models lacking different components of the autophagy machinery confirmed the importance of the process in a variety of mammalian cells.

“Our… experiments revealed that these genes are in fact responsible for the membrane rearrangements that are an essential part of the autophagy process. We also found that these genes are very well conserved and that homologues for most exist in human and plant cells. The identification of genes required for autophagy has really had a lasting impact on the quality of autophagy research that continues to this day. After that point, the mechanisms and function of autophagy genes were studied by us and numerous other researchers and laboratories around the world, with many fascinating insights that continue to emerge today,” Ohsumi said. The path-breaking studies of Ohsumi and his associates have, in fact, unleashed an explosion of research in the field today with an exponential increase in the number of publications since the early 2000s.

In the early pre-Ohsumi days, autophagy was thought of as a “waste dump”, Juleen Zierath said in her interview. Ohsumi’s work has shown that rather than being a waste dump, the physiological process of autophagy is a “recycling plant”, with a sophisticated machinery that recycles damaged long-lived proteins, which could then be reused. Insights gained from research following Ohsumi’s work in the molecular characterisation of autophagy have also been instrumental in advancing the understanding of this process and its involvement in cell physiology and various pathological states.

Initially, autophagy was thought to be a cellular response to stress, but Ohsumi’s work has shown that the system operates continuously at basal levels. As pointed out earlier, unlike the ubiquitin-proteasome protein-degrading process, which preferentially degrades only short-lived proteins, autophagy removes long-lived proteins and is the only process capable of destroying whole organelles, such as mitochondria, preoxisomes and endoplasmic reticulum. Moreover, autophagy participates in a variety of physiological processes, such as cell differentiation (which gives rise to the different types of cells that constitute the human body) and embryogenesis, that require the disposal of large amounts of cytoplasmic material. Autophagy also has a cytoprotective function: by rapidly responding to various kinds of stress, it provides the cellular machinery the capacity to counteract cell injury and many diseases associated with ageing (Figure 5). By capturing invading viruses and bacteria (a phenomenon called xenophagy), autophagy is essential for activating immune responses to infectious diseases.

Conversely, defects in autophagy are associated with many human diseases. Failure of autophagy is associated with ageing and many diseases of old age, neurodegenerative disorders such as Alzheimer’s disease, epilepsy and motor disorders and also type 2 diabetes. While a properly functioning autophagy machinery can prevent cancer, by degrading cancerous cells, too much autophagy may have undesirable effects. For example, a high degree of autophagy can promote the growth of tumour cells and resistance to cancer drugs. Thanks to Ohsumi’s findings, it is now possible to study exactly how the mechanism works in each case, and this constitutes an entirely new research area.

Thus, Ohsumi’s discoveries have been instrumental in revealing the mechanism and significance of a fundamental physiological process, and this gives rise to the hope that this knowledge will lead to the development of new strategies for the treatment of many human diseases. Indeed, already many clinical trials of strategies exploiting autophagy to treat cancer are under way.