New Research: The Evolution of Multicellularity

We are all multicellular. That is, our bodies are made up of many cells that function in a synergistic manner to create a unified whole, somewhat like how we have millions of people that function to create the ‘organism’ Canada or France. From one fertilized egg cell, we developed all the amazing coterie of cells like nerve cells, blood cells, muscle cells, etc.

Many years ago, life began as a unicellular state, like your average bacteria that lives in your gut. At some point, these unicellular organisms acquired changes that lead to a multicellular state and this was a very critical step in our evolution. With multicellularity, one can have cells that do different things like fight infection or transmit electrical signals (a necessity for cognition).

How did this transition occur? Multicellularity occurred >200 million years ago, at least 25 separate times. Scientists have found that several steps must have been needed. Firstly, the unicellular organisms must gain the ability to form simple clusters of cells. Next, the multicellular state must somehow become favored (or selected for), in comparison to the unicellular state. Finally, the cells will begin to specialize to different things within that organism.

It has long been thought that these steps will take a very long time, yet in this paper, William Ratcliffe shows that it can actually evolve in a very short time. They began with simple baker’s yeast and decided to apply pressure to evolve cell clusters. Since ten cells clustered together are heavier than one individual cell, it should settle faster in a tube due to gravity. With this premise, these scientists decided to transfer cells at the bottom of a culture to another culture, let those grow and then settle, then transfer the bottom to another culture, let them grow and settle, etc.

After 60 transfers, they found the yeast cultures were now dominated by a snowflake-like organism, with multiple cells. Moreover, these snowflake-like organisms were genetically stable; one could not detect a conversion back to a unicellular state. This argues that it had indeed evolved into a new organism. These clusters seemed to arise from an inability to separate after cell-division, rather than re-aggregation of split cells.

Even crazier, Ratcliffe found that these yeast-descendants gained classic multicellular traits. For instance, there was a juvenile phase which grew unimpeded and an adult phase which stopped growing when it reached a certain size and then released a smaller copy of themselves (see arrow on right). Since these clusters didn’t grow forever, it suggests there was some communication between the cells in this organism to arrest growth at some size, another classic multicellular trait. Even more interestingly, they found that these cells had evolved to do different things: there were cells that underwent cell suicide to release progeny.

This study is somewhat flawed however in two reasons: one they applied artificial selection pressure; it is hard to imagine alien’s spinning down yeast and then re-inoculating another culture just to evolve multicellular life on earth. Moreover, baker’s yeast evolved from a multicellular ancestor billions of years ago, so one could argue these genes for multicellularity are already present. However since yeast are not multicellular, those genes have probably been lost over the last billion years of not being used.

In conclusion, earlier in this article, I mentioned how 3 steps were needed to generate multicellularity. In this paper, Ratcliffe shows that with the appropriate selection, all three steps could occur within 60 days (or 720 generations), a remarkable discovery. This suggests that the evolution of multicellularity does not actually require a very long time. This further suggests that only a few genes may be involved. The exciting next step would be to find out exactly which genes were altered in that time period to provide clues into how multicellularity may have arisen.


Ratcliffe, WC et al. (2012) Experimental Evolution of Multicellularity. PNAS onlinePaper

General News: Nobel Prize 2011

This blog is back up and running. For a while, I put it on hold since I was busy helping teach introductory biology, but now that it is over, I am now going to return to working on articles. Many exciting things have happened since so I thought I’d write about two major events that occurred in the last four months.

First, the Nobel Prizes! The 2011 Nobel Prize in Physiology and Medicine were awarded to Bruce Beutler and Jules Hoffmann "for their discoveries concerning the activation of innate immunity" and the late Ralph Steinman, "for his discovery of the dendritic cell and its role in adaptive immunity". This is the first immunology prize since 1996, and so the immunology community is really excited!

Our species is constantly under attack by other microorganisms, which our body defends against using the complex immune system. Mammals have two arms of the immune system, the ancient innate immune response and the more recently-developed adaptive immune response. The innate immune response is the first line of defense, a non-specific response that leads to immune cell recruitment, inflammation and attack of foreign material. The second line of defense is the adaptive immune response, in which the body mounts a more effective, specific attack against the microorganism after studying it for some time. Since this takes time to develop, the innate immune response is critical for immune function.

THE ROAD TO INNATE IMMUNITY
Insects have a relatively short lifespan, so it doesn’t seem apparent that they require a powerful immune system to fight pathogens, yet insects are highly resistant to microbial infection. Yet early inquiries into understanding how this happened lead nowhere, because it turns out insects are unlike humans in that they do not have an adaptive immune response. The first glimpse into this mystery occurred in 1981, when Hans Boman discovered that moths induced the antimicrobial peptides Cecropin and Attacin. By the 90s, several genes encoding these antimicrobial compounds were identified in the Drosophila genome. Yet no one understood what was turning on expression of these antimicrobials, a process that Beutler and Hoffmann coined “the black box.”

They explored the promoters of these antimicrobial genes and found they contained binding sites for something similar to the mammalian NF-kB (discovered by Nobel laureate David Baltimore - from MIT!), which has been linked to immune system activation. Previous work by the Nobel laureate Nusslein-Volhard had found a critical development pathway that set up the axes for dorsoventral establishment, the Toll pathway that ended in Dorsal, a NF-kB-like molecule. Through a long line meandering experiments and wrong leads, Dr Hoffmann eventually discovered a mutant, Toll which was highly susceptible to fungal and bacterial infections, for the first time proving that Toll was somewhat essential in immune responses.

In a parallel line of research, septic shock is a debilitating syndrome that causes multiple-organ failure at the end-stage of a bacterial infection. It was recognized that this was induced by a molecule common to most bacterial cells, LPS. Dr Beutler wanted to try to find the receptor that could bind LPS, so they soon discovered that mice resistant to LPS had a mutation in a gene similar to the Toll gene of the fruit fly. They demonstrated that LPS-binding to the Toll receptor activated inflammation, and when the doses were very high, the organ systems in the body would go into inflammation overload and cause septic shock.

Thus, in the grand picture of things, Bruce Beutler and Jules Hoffmann discovered a set of receptors that we now call the pattern-recognition receptors, which recognize a set of molecules that are broadly found in microorganisms but not humans. They demonstrated that it was a very ancient pathway, conserved from flies to humans. Moreover, in later work, they picked apart the pathway, allowing the development of drugs that can target the innate immune response. Indeed the fundamentalness of this pathway is indeed one reason why these two have been awarded Science's Highest Award.


THE ROAD TO DENDRITIC CELLS AND THE ADAPTIVE IMMUNE RESPONSE
One characteristic of the immune system is its incredible diversity of cells; in fact when Dr Steinmann first entered the field in 1970, it was known as God (for generation of diversity). At the time, clonal selection theory suggested that there was an infinite variety of cells in the immune system, each which recognized one antigen. Binding to a specific antigen caused one specific clone to multiply thousand-fold and promote an immune response against that antigen. This hypothesis was further enriched when Nobelist Susumu Tonegawa (also MIT ) demonstrated that the great diversity of clones was generated through genetic recombination. Yet one problem with the theory was that injection of antigens into animals did not always generate an immune response, an idea which would have critical impacts on vaccine generation.

In 1973, Steinman found that purifications of lymphocytes (mixtures of B and T cells) could not produce antibodies unless they were supplemented with “accessory cells.” Looking under the microscope, he explored the accessory cells and found the usual culprits (macrophages) but also, a weird stellate elongated cell, which he named the dendritic cells. Further studies revealed he only needed to mix 1 dendritic cell per 100 lymphocytes to induce strong clonal selection.

We now know that dendritic cells are the critical link between the innate and adaptive immune response. The innate immune response (which half this year’s Nobel is awarded for ) turns on these dendritic cells, which then go on to activate T cells for priming the adaptive immune response. Indeed, these cells have been so influential that pharmaceutical companies are now trying to develop vaccines using dendritic cells as well as treatments for allergies.

However, it is with great sadness that Dr Ralph Steinman passed away on Friday Sept 30, 3 days shy of him finding out about his Nobel Prize. Dr Steinman was a consummate scientist of the highest caliber and many colleagues value his contributions greatly. In one of his essays, he recalls Palade’s quote “For a scientist, it is a unique experience to live through a period in which his field of endeavor comes to bloom—to be witness to those rare moments when the dawn of understanding finally descends upon what appeared to be confusion only a while ago—to listen to the sound of darkness crumbling.” And indeed Dr Steinman helped clear the darkness.


REFERENCES:
Beutler's Discovery of TLRs
Hoffmann's Discovery of TLRs
Steinman's Discovery of Dendritic Cells

New Research: No More Pain from Sunburns?

Ah, it's summer again. You're laying on your patio and fall asleep with a martini on the Ikea mini-table beside you. You doze off, dreaming about that Europe backpacking trip that you had planned with your best friend. Suddenly, you wake up and what's this? Your face hurts! Oh darn, you forgot to put on sunscreen and now your face is burning!

Many of us aren't strangers to sunburns. The characteristic red burning sensation when we've overstayed our time in the sun is a type of inflammation. Inflammation is a component of the many responses to harmful agents, including germs as well as damaged cells. Typically, the process involves the recruitment of immune cells, which destroy damaged tissue as well as germs, in an effort to promote healing. During inflammation, our pain-receptors become sensitized due to chemicals that are released from the white blood cells. Sunlight contains harmful ultraviolet B (UVB) rays, which promotes hypersensitivity to pain 24-48 hrs after exposure. In addition, UVB can cause dangerous mutations in your DNA leading to skin cancer, which is why sunscreen is very important.

John Dawes of Stephen McMahon's lab decided to study this inflammation response towards UVB. After exposing human and rat skin to UVB rays, they found both increased blood flow to the skin as well as the subject's pain thresholds dropping substantially. An analysis of genes that were turned on after UVB irradiation revealed that humans and mice share a similar response towards UVB damage. In addition, the gene that changed the most was a poorly-studied chemokine (chemicals that modulate the immune system) called CXCL5.

CXCL5 has previously been linked with certain chronic pain syndromes, so it looked like an interesting gene. They injected the CXCL5 protein directly into rat feet and saw that they were more susceptible to pain. In addition, they found that CXCL5 could attract white blood cells towards it, similar to a magnet pulling on metal. The cool thing was that blocking the action of CXCL5 raised the pain threshold after UVB treatment, so rats felt less pain in an artificial sunburn. In addition, there was less white blood cells being attracted to the site of injury.

Altogether, this study has found an interesting gene CXCL5 that is associated with the pain in sunburns. Drug companies are now considering developing a sunburn pain remedy that would contain a drug that specifically blocks CXCL5. However, one caveat is that less white blood cells will be attracted to the sunburn site, so one might get delayed healing of the damaged skin.


Dawes, J., et al. (2011) CXCL5 Mediates UVB Irradiation–Induced Pain. Science Translational Medicine 3(90):90ra60. Paper.

New Research: A Link between Cholesterol Metabolism and Lysosome Synthesis

In the immediately previous blog entry, we discussed how proteins are targeted to specific locations in the cell via zip-codes. One location is the lysosome, which functions as a recycling unit, containing enzymes that break apart portions of the cell marked for destruction. The lysosomal enzymes are brought to the lysosome using a specific zip-code, a chemical addition called mannose 6-phosphate (M6P).

One of the two proteins that adds M6P is made of two parts made from the same gene GNPTAB. Initially, a large premature protein is initially made, but it gets chopped into two pieces. Afterwards, the two products combine to form the functional M6P-adding enzyme. Patients with a disease called mucolipidosis II have mutations that prevents proper formation of the enzyme that adds M6P, leading to defective lysosomes and an inability to breakdown damaged portions of a cell. Ultimately, this leads to skeletal abnormalities and mental retardation, similar to Tays-Sachs disease which is also a disease of defective lysosomes.

Katrin Marschner of the Pohl lab wanted to find out what was the protein that caused the chopping of the premature protein. She found that a crucial part of the cleavage site was similar to the sites cut by the protein S1P. Further experiments revealed that S1P could also cleave the precursor to M6P-adding enzyme, creating a mature protein. Both mice with mutations in S1P and mice with mutations in M6P-adding enzyme had similar characteristics, such as defects in cartilage formation. Similar to when a pipe gets clogged, S1P-deficient cells had prominent accumulation of materials, further suggesting that S1P cleaves the M6P-adding enzyme.

S1P also known to be essential for regulating the body's cholesterol levels. At high levels of cholesterol, a molecular switch called SREBP gets anchored to the inside of the cell. However when cholesterol levels are low, S1P cuts off the anchor, freeing SREBP and allowing it to go to the nucleus where it turn on genes that make cholesterol.

As a result, drug companies have been investing money to find chemicals that can block S1P as a way to treat high blood cholesterol. Without S1P, SREBP cannot reach the nucleus and cholesterol is no longer made. However, this study suggests that such drugs would have the unwanted side effect of blocking proper lysosome function. This would lead to accumulation of garbage in the cell, something that is linked also to Alzheimer's and Parkinson's disease.


Marschner, K., et al (2011). A Key Enzyme in the Biogenesis of Lysosomes is a Protease that Regulates Cholesterol Metabolism. Science 33(6038):87-90. Paper

New Research: Nuclear Import of Membrane Proteins

All the cells in the body are made up of different components called organelles. For example, lysosomes recycle components of the cells, the mitochondria generate energy and the nucleus is not only stores the chemical blueprint of life (DNA), it also makes the executive decision of whether to turn genes on and off. Many of these organelles are separated from each other by barriers called membranes. Between all these organelles, proteins are being passed back and forth to different compartments through specific gates.

The nucleus has many gates called the nuclear pore complex (NPC) that regulate what can move across the nuclear membrane. Think of it as a filter; very small molecules can go through whereas large molecules normally cannot. However, the NPC has the ability to recognize a specific instruction on larger molecules called a zip-code. Think of zip-codes as a one-pass key; if a large molecule has a zip-code code for entry, it will be permitted to pass through the gate into the nucleus, but not back out unless it has the zip-code for exit.

We don't quite understand how proteins are moved across the nuclear membrane. Even more puzzling is this. The nuclear membrane actually has two-membranes bridged by the gates. There are proteins embedded in both the inner membrane and the outer membrane, yet it is not well understood how proteins are moved from the outer membrane into the inner membrane.

Anna Meinema from the Veenhoff lab explored this process for a specific inner membrane protein Heh2. She found that the transport of Heh2 into the nucleus depended on how well its zip-code could bind a protein called the karyopherin-α/β1 complex. karyopherin-α/β1 is somewhat like a parent that holds onto a kid as it crosses the street. In the nucleus, it holds onto proteins with nuclear zip-codes and carries it across the nuclear membrane.

She also found something remarkable. Proteins usually have a rigid structure, allowing them to interact with other molecules in specific orientations. However Heh2 transport depended on the presence of an unstructured region in the protein. Not only that, the unstructured region had to be of a sufficient length.

After further studies, she found how Heh2 was being transported across the nuclear membrane (see figure above). Before it crosses the membrane into the nucleus, the nuclear zip-code (NLS) gets bound to the karyopherin-α/β1 complex, which pulls the Heh2 across the nuclear membrane like a parental chaperone. However the NPC is a molecular sieve, with fibers (FG-Nups in the figure) that can block transport. To overcome this, Heh2 has unstructured regions which allows it to change shape for ease of passage through the fibers. After crossing the membrane, karyopherin-α/β1 releases the inner membrane protein Heh2.

Understanding nuclear import is important for several reasons. Firstly, the switches and signals that turn on genes often need to be transported into the nucleus. Secondly, viruses often hitchhike on the nuclear import machinery to be able to hijack the nucleus and produce hundreds of copies of itself. By studying this process, one day we may be able to design drugs to prevent entry of those signals or viruses during diseases.


Meinema, A., et al. (2011). Long Unfolded Linkers Facilitate Membrane Protein Import Through the Nuclear Pore Complex. Science 333(6038): 90-93. Paper.

New Research: Repairing the Heart with Thymosin-β4

The major cause of death in the developing world is cardiovascular disease. This is usually due to the heart's failure to meet the needs of the rest of the body, usually due to a heart attack or due to defects in heart structure. In a heart attack, coronary arteries are blocked, preventing the heart from receiving oxygen, which leads to tissue death and the associated pain. Unlike fish which can regenerate new muscle cells, humans cannot fully regenerate new heart muscle cells to replace the dead ones.

One aim of cardiac regenerative medicine is to repair the damage after a heart attack. Several strategies have been proposed. One includes taking embryonic stem cells and turning them into cardiac muscle cells; these would be grafted back into the damaged tissue to repair the heart. Another strategy is to try and inject stem cells directly into the heart from various organs. However, an ideal strategy would be a less invasive method that involves harnessing the body's own stem cells and turn them into heart cells within the patient.

The heart is surrounded by a layer called the epicardium (epi for around, cardium for heart). The epicardium plays a crucial role in heart development. Firstly, it sends instructions to the heart to program its development. In addition, the epicardium is a source of heart progenitor stem cells, which move into the heart and mature into heart muscle cells during development. One characteristic of these cells is that they have the gene Wt1 turned on. However up to now, none of these cells appeared to be detected in adults.

A recent paper by Nicola Smart from Paul Riley's group found an important breakthrough in understanding this process. For the first time, they detected cardiac progenitors within the heart. The secret was to add a a special molecule called thymosin-β4 (Tβ4), previously reported to enhance repair of the blood vessels in the heart after heart attacks. Upon addition of Tβ4 to epicardium biopsies, they could detect the presence of progenitors as well as functional heart muscle cells.

Interestingly, she was also able to detect progenitors with the Wt1 gene turned on in adults after a heart attack. They appeared about 7 days after a heart attack, and are linked with the limited cardiac regeneration seen after a heart attack. The stunning thing was an injection of Tβ4 before a heart attack caused the progenitors to appear earlier (2 days later) and at larger numbers. Not only that, these progenitors could turn into functional heart muscle cells that integrated into the damaged heart tissue. Also, the heart with Tβ4 pre-treatment worked better after a heart attack than the heart which had not received any treatment.

What does this mean? Firstly, we now know that there are cells with stem cell capabilities in the heart. Secondly, the addition of Tβ4 can enhance the regeneration abilities of the heart upon suffering a heart attack. While the study only looked at pre-treating hearts with Tβ4, it will be interesting to see if the results translate to patients who receive Tβ4 right after a heart attack. If this ends up holding true, one can see a potential therapeutic use for heart regeneration after heart attacks.


Smart, N. et al. (2011). De novo cardiomyocytes from within the activated adult heart after injury. Nature 474, 640-44. Paper

General News: German E Coli Outbreak

Recent news about the German E coli O104:H4 outbreak has come back into the news, with reports of it in France and other European nations. It began when people in Germany were admitted to hospitals with bloody diarrhea in May 2011, and within a month, over 3700 cases have been reported with 42 deaths. The source of the E coli is likely to be bean sprouts according to the WHO.

E coli is a natural organism of human beings, normally residing in our gut. In fact, a very large portion of human feces is made up of E coli. They are believed to provide some health benefit to humans, perhaps by pre-binding to our intestines they prevent other bacteria from attaching to our intestinal cells. Normally E coli is harmless but sometimes, they can become pathogenic by getting infected with a virus known as a bacteriophage. This specific phage produces Shiga toxin, which is responsible for the severe diarrhea and kidney damage observed in infected patients who develop hemolytic uremic syndrome (HUS). Interestingly, this phage accidentally picked up the Shiga-toxin gene from Shigella, another pathogenic bacteria, while infecting it many years ago.

It is believed that antibiotics may be contributing to the spread of this phage, making it extremely difficult to treat patients who have this disease. Normally under non-stressed conditions, some phages lie lurking in the genome. But when bacteria are stressed by mutagens or even antibiotics, they undergo something known as the SOS response, which is essential a Hail-Mary attempt to survive. Unfortunately, this SOS response also activates the lurking phage, allowing it to exponentially divide and eventually causing the cell to burst, leaking hundreds of phage as well as Shiga toxin into the bloodstream. This is one reason why E coli infections are not usually treated with antibiotics. The other reason is antibiotics also target normal E coli in the gut, removing them and providing other bacteria the opportunity to bind and wreck havoc to the gut.

E coli also finds a home in many of the farm animals we raise, in particular cattle. It is believed that the constant application of antibiotics to cattle is contributing to pathogenic E coli. Firstly, it promotes the acquisition of multiple antibiotic resistant genes. Secondly, it promotes an explosion of phage throughout the cow gut, allowing it to infect many other E coli cells. Since cows don’t get sick from Shiga-toxin producing E coli, it is difficult to tell which cows are affected other than be testing their feces. Indeed, it has been found that over a half of cows in North America possess virulent strains of E coli.

The one thing that has people worried is that the German strain of E coli is new. While it produces Shiga-toxin, it also has multiple antibiotic resistant genes so even if we wanted to try to kill them using antibiotics, we would have a hard time doing is. In addition, bacterial species are very good at picking up genes from other bacteria, so one worry is that these antibiotic genes could transfer to a different bacteria. The other worry is that this bacteria also has some enteroaggregative E coli properties (probably from that gene swapping ability I mentioned earlier), which means it can aggregate cells on a dish. This combination of both Shiga-toxin and enteroaggregative properties has not been observed before, and so has scientists worried.