Cellular Organelles I: _{Endosymbiosis and membrane-bound organelles}

The origin of eukaryotic cells

Eukaryotic cells differ from prokaryotic cells in that most of the various organelles in eukaryotic cells are encapsulated in membranes, while prokaryotic cells have only free-floating organelles (Figure 1). The difference is a very obvious one, even when viewed through the simplest microscopes. As early as the late 1800s, scientists were already debating how evolution may have given rise to these two very distinct lineages (Mereschkowski, 1910).

Konstantin Mereschkowski (Figure 2) originally proposed the idea that chloroplasts in plants evolved from a symbiotic relationship like the one he had seen in his research with lichens. Lichens, he had observed, were really composite organisms formed by a fungus and photosynthetic algae living together symbiosis. The fungus part of the organism provides a safe environment for the photosynthetic algae, and the algae photosynthesize ATP for the lichen. Mereschkowski suspected that the chloroplasts in plant cells descended from organisms similar to the algae in his lichens. Unfortunately, he had no real evidence to support his hypothesis, so no one took it very seriously at the time. However, when an American biologist named Lynn Margulis (Figure 2) proposed the idea again in 1967, things were different. Technology was becoming available that would allow scientists to gather data and investigate the claim fully. Mereschkowski's idea finally got some attention, although 100 years later.

The theory of endosymbiosis

The idea proposed by Margulis is called the theory of endosymbiosis. This theory states that modern-day mitochondria and chloroplasts are actually the descendants of ancient bacteria-like organisms that began living inside prokaryotic cells when Earth was very young (Sagan, 1967).

The story begins over two billion years ago when Earth was still hot and mostly barren. There was no oxygen gas (O₂) in the atmosphere, and the only life forms on Earth were single-celled prokaryotic organisms similar to present-day bacteria. Some of these prokaryotes, called cyanobacteria, evolved the ability to capture the energy in sunlight to make organic molecules. Because of their new abilities, these photosynthetic bacteria flourished and began to release free oxygen gas (O₂) into the ocean water.

Oxygen gas was purely a byproduct of photosynthesis and hadn’t previously existed on the planet. Because oxygen is reactive, it was toxic to most of the prokaryotes living at that time and caused many of them to go extinct. However, a small number of prokaryotes evolved the ability to tolerate the oxygen, and some of their descendants later evolved the ability to utilize oxygen to aid their metabolism, much like we do today.

Cells that can use oxygen for metabolism are called aerobic, while those that cannot are called anaerobic. Aerobic cells have a powerful advantage because oxygen allows them to get much more energy out of the food molecules they consume.

The cooperation between the two prokaryotes came about when a large anaerobic cell engulfed (but failed to digest) a smaller, aerobic cell. The aerobic cell, now living inside the anaerobic cell, continued to efficiently metabolize food molecules using oxygen, and shared its excess ATP (chemical storage form of energy) with its host, the large anaerobe. The arrangement would have been similar to the one we see in our own digestive system: Millions of microbes live happily in our intestines, helping us to digest and metabolize the foods we eat. Like the microbes living in our guts today, the aerobic cell lived entirely inside its host.

Over millions of generations, the cells continued to grow, divide, and multiply, and their relationship evolved into a mutually beneficial cooperation – a symbiosis. In time, most (but not all) of the DNA from the smaller aerobic cell made its way into the nucleus of the host cell and the two separate organisms became one – the ancestor of all the eukaryotic cells we see today. The descendants of that small aerobic cell evolved into the organelle we call mitochondria. Mitochondria still pass on some of their own DNA to their daughter cells, just as DNA from the nucleus is passed on to the nucleus of its daughter cells. The fossil record shows us that mitochondria and modern eukaryotic cells look much different than the precursors did because they have continued to evolve and change over two billion years.

The endosymbiotic theory goes on to say that chloroplasts evolved in a similar fashion. It proposes that a small cyanobacteria (the photosynthetic bacteria mentioned earlier as the first source of oxygen gas) was engulfed by a larger, aerobic, non-photosynthetic cell. This large non-photosynthetic cell, a descendent of the symbiosis described above, already had mitochondria living inside it. It was either an early eukaryote or an advanced prokaryote that shared some features with eukaryotes. And just as before, when the larger cell engulfed the smaller cell, it failed to digest it. The cyanobacteria, like the aerobic cell before it, lived happily tucked inside the larger cell. The larger cell now had both an aerobic cell and a photosynthetic cell living within it!

The arrangement could have been very similar to what we see in modern day single cell organisms called Paramecium bursaria. P. bursaria commonly live in ponds and eat large quantities of photosynthetic algae that they do not digest. The algae continue to photosynthesize inside the almost completely transparent host, providing the paramecium with an onboard renewable food source. The paramecium host contributes to the symbiosis by transporting the algae to sunny spots in the pond while protecting it from more harmful, less accommodating predators. It also shares food that it is able to find with the algae during times when sunlight is scarce.

Just as with the earlier symbiosis, the cooperative arrangement between the photosynthetic cell and the larger cell was mutually beneficial. The small photosynthesizer was provided with protection and all the nutrients that it needed, including lots of ATP since the large cell was aerobic. The large cell benefited even more. With the small cyanobacteria inside it, the cell no longer had to search around for food to eat – it had a built-in source of high-energy molecules made by its new photosynthetic helper. Over millions of years, the cooperation became closer, and the descendants of the small cyanobacteria are now an entirely dependent organelle called the chloroplast. The large cell, now both aerobic and photosynthetic, gave rise to all plants and algae that we see today.

The theory of endosymbiosis sounds pretty far-fetched, and the scientific community didn't buy it at first. But Lynn Margulis was persistent and worked tirelessly to gather hard evidence to support her theory. She finally got the proof she needed in the late 1970s when scientists developed a new tool for identifying the ancestral lineage of organisms.

Confirming the theory

Margulis originally conceived of the idea of endosymbiosis based on what she observed in the laboratory as she studied Euglena, a single-celled, photosynthetic eukaryotic organism. The chloroplasts inside the Euglena reminded Margulis of bacteria she had studied before. The chloroplasts had DNA that was circular like the DNA in bacteria (Figure 3).

The mitochondria in Euglena also had similarities to free-living bacteria. For example, they pinched themselves in half as a means of reproduction in a process that looks a lot like binary fission (Figure 4) (see our Cell Division I: The Cell Cycle module for more information).

Just because mitochondria look like bacteria wasn’t enough to convince most scientists that the organelles actually descended from bacterial ancestors. Gathering conclusive evidence that present-day mitochondria and chloroplasts are distantly related to bacteria was a very difficult thing to do in the 1960s. However, in the 1970s, scientists developed a method for reading the precise sequence of nucleotides present in an organism’s DNA. The new technique allowed scientists to compare the genome of one species to that of another and look for similarities that indicate relatedness. More similarities between the genomes of two species suggest that they are more related. Fewer similarities suggest that two organisms are less related.

Using the new DNA sequencing techniques, Ford Doolittle and Michael Gray, scientists working at Dalhousie University in Halifax, Nova Scotia, found the evidence needed to convince the scientific community that Margulis was right. They compared the DNA from chloroplasts to DNA from the nucleus of the same cell. Then they compared the chloroplast DNA to an ancient line of free-living photosynthetic bacteria.

They found that the chloroplast DNA appeared to be more closely related to the bacteria than to the nuclear DNA of plants or algae. Not long after that, they showed that mitochondrial DNA, or mtDNA, was more closely related to an ancient line of free-living aerobic bacteria than to nuclear DNA of the eukaryotes that host the mitochondria. The reaction in the scientific community was swift. Margulis's explanation for the origin of mitochondria and chloroplasts in eukaryotic cells quickly became the dominant view. More evidence continued to pour in and by the early 1990s there was solid scientific consensus that the hypothesis of the endosymbiotic origin of mitochondria and chloroplasts was indeed correct (see list below). It was the first documented example of cooperation, rather than competition, driving a major evolutionary innovation.

Major evidence for the endosymbiotic theory

1. Mitochondria and chloroplasts have some of their own DNA, and it is located on a circular chromosome (similar to bacteria).
2. Mitochondria and chloroplasts have their own ribosomes, and they are similar to bacterial ribosomes, not to the eukaryotic ribosomes found in the cytoplasm.
3. Proteins that are made inside mitochondria and chloroplasts begin with N-formyl-methionine, like bacterial proteins, not like eukaryotic proteins, which always begin with regular methionine.
4. Mitochondria and chloroplasts divide and replicate on their own in a manner very similar to how bacteria divide, called binary fission.
5. There are transport proteins found in the membranes of mitochondria and chloroplast, called porins, that are found in bacterial, but not in eukaryotic, plasma membranes.
6. Mitochondrial DNA sequences are more similar to bacteria genes than to any eukaryotic genes.
7. The modern-day bacteria whose DNA is most similar to mitochondrial DNA is the Rickettsia genus. These bacteria live inside large eukaryotic cells as parasites.
8. Chloroplast DNA sequences are more similar to cyanobacteria genes than to any eukaryotic genes. Cyanobacteria are modern-day photosynthetic bacteria.

The advent of DNA sequencing and the ability to compare DNA between different species has also shed light on the likely identity of the large cell that first engulfed the ancestor of the mitochondria. While the mitochondria and the chloroplasts appear to be descended from bacteria, the DNA in the nucleus of eukaryotic cells is more similar to modern-day archaea than to that of bacteria. We tend to think of archaea as arcane organisms relegated to life in extreme environments, far outnumbered by their fellow prokaryotes, the bacteria. However, they were once the dominant life forms on the planet, and scientists today are finding them in more and more surprising places. Due to the similarity of our nuclear DNA to theirs, it is probable that the large cell that engulfed the ancestor of the mitochondria was an archaea, which means that all eukaryotes – including us – are descendents of archaeans through our nucleus, and of bacteria through our mitochondria. It’s almost as if the two prokaryotic domains of life, bacteria and archaea, joined together and gave rise to the eukaryotic branch of the tree of life.

The evolution of other organelles

But what about the nucleus found in eukaryotic cells? How did that evolve? The membrane-bound nucleus, perhaps the single-most defining characteristic of eukaryotic cells, in no way resembles any free-living bacteria or achaean. The nucleus and other organelles evolved in a very different manner than did the mitochondria and chloroplasts (Figure 5).

Biologists cannot say for certain the exact order in which all of the organelles evolved. The fossil record is difficult to read when it comes to tiny, fluid-filled microorganisms from billions of years ago. Further complicating the matter is the fact that some organelles appear to have evolved more than once in different lineages over evolutionary time. In short, there is still some debate about the details of the chronology, but scientists can infer the basic sequence of events based on what we know about how the organelles function today.

Early prokaryotic cells, the first forms of life on Earth, probably had a rigid cell wall, like prokaryotes do today. Inside the cell wall there was a plasma membrane, like all cells have (see our Membranes I: Introduction to Biological Membranes module). Somehow, perhaps as the result of a mutation, the plasma membrane began to fold in on itself, creating a small cavern or – invagination – inside the cell wall (Figure 6). Over many thousands of generations, this invagination grew and eventually surrounded the cell’s DNA, creating a nuclear envelope. This architectural enhancement gave these cells an advantage over other prokaryotic cells because their DNA was now better protected from damaging molecules found in the cytoplasm of the cells.

The nucleus offered another important benefit. Inside the protective environment created by the nucleus, the DNA was able to evolve in ways that it never had before. Free from the interference from the cell's cytoplasm, new chemical reactions that power gene recombination, DNA repair, and gene expression eventually evolved, and the structure of DNA itself began to change. DNA evolved from its ancient form – the simple ring-like structure seen in bacteria – to the long intricate strings of nucleotides that make up our own DNA. With a single evolutionary change – the development of a nucleus – eukaryotic cells were set on a course for greater diversity and specialization than prokaryotic cells could ever achieve.

Later on in their evolution, when eukaryotic cells acquired mitochondria and chloroplasts, they gained another advantage. Eukaryotic cells could now find and utilize food sources better than their prokaryotic cousins. Eukaryotic cells began to grow larger. (The average eukaryotic cell today is 100 to 1,000 times larger than a prokaryote.) And as the cells became larger, their outer membrane continued folding in on itself in the same way that it had when the nucleus was formed. More folds created more channels inside the cell, and the same process of invagination that formed the nucleus began to fashion the remaining membrane-bound organelles.

The compartments (or organelles) created spaces where new processes could evolve without interference from the rest of the cell. Membrane-bound organelles gave eukaryotic cells the same benefit that a proper laboratory gives a chemist – an environment where reactions can be controlled. Inside the newly formed organelles, complex processes like protein synthesis were able to evolve without chemical disruption from other cell functions like respiration or photosynthesis. Eventually, pathways and other features developed in cells that enabled them to communicate with each other. And once cells were able to pass signals and cooperate, they began to develop larger symbiotic relationships that ultimately gave rise to the tissues and organs that make up our bodies (Figure 7).

While the explanation above is an educated guess, it is bolstered by evidence from modern-day prokaryotes, the bacteria. Many bacteria have invaginations in their membranes that they use for a variety of purposes. In fact, most bacteria have extensive in-foldings of their plasma membranes that process food molecules the same way that mitochondria metabolize food in eukaryotic cells. What this shows is that the evolution of membrane folds into internal compartments is not a far-fetched possibility. In fact, it still happens today and can provide clear advantages for cells.

The membranes surrounding the organelles of eukaryotic cells do more than just provide a barrier between organelles and cytoplasm, however. They serve as a network that provides a means of communication and transport throughout the cell. The endomembrane system, also thought to have evolved via the process of invagination, illustrates this point nicely.

The endomembrane system

Camillo Golgi, an Italian physician working in the late 1800s, is said to have discovered the Golgi apparatus when he was looking at cells from the body's central nervous system. The internal reticular apparatus, as he called it, appeared to be an individual structure when viewed through his microscope, which was the cutting edge technology of the day (Figure 8). Today, we know that the Golgi apparatus is connected to a larger endomembrane system.

The endomembrane system divides the cell's cytoplasm into separate compartments, or organelles, that each performs specialized tasks within the cell. The separate compartments, however, aren't entirely separate. Some are actually connected by shared membranes, as is the case with the rough endoplasmic reticulum and the nuclear membrane. This particular network forms a pathway for large molecules and signals to pass between the nucleus and the environment outside the cell.

Compartments that don't share a direct physical connection pass signals, proteins, and waste via tiny membrane-bound sacs called vesicles. Vesicles form when part of an organelle's membrane pinches off, forms a lipid-bound sac, and floats through the cytoplasm to deliver its cargo between organelles. The vesicles, being formed of the same plasma membrane that surrounds the cell and all the organelles, easily merges with the membranes surrounding each compartment. Vesicles containing basic proteins synthesized in the rough endoplasmic reticulum travel to the Golgi apparatus for final processing via vesicles. Vesicles containing the finished protein leave the Golgi apparatus and deliver the final product out to another organelle (Figure 9).

Our current understanding of the membranes surrounding organelles has come from new techniques in biochemistry that give researchers greater access to the inner workings of cells than the scientists of Margulis's day had. Researchers today can sift through cell samples using centrifuges and isolate individual organelles for closer scrutiny. They can also track the movement of specific chemicals and proteins through a cell's system and witness first-hand the flow of chemicals and signals from one organelle to another. The result has been a greater understanding of the true spirit of cooperation that was the basis of the evolution of the eukaryotic cell in the first place. As Lynn Margulis and her son wrote in one of their many books, “Life did not take over the globe by combat, but by networking."