Some frogs like it hot, others like it cold

How clawed frog species inhabit different climate zones thanks to small molecular adaptations

African clawed frog species are closely related to each other in evolutionary terms, yet they inhabit different climatic zones across the continent. This is a challenge for the cold-blooded amphibians because other than warm-blooded animals like birds and mammals they rely on external heat. Small differences in a protein of their cytoskeleton enable the frog species to each colonize different temperature zones. This has been discovered by researchers at the Max Planck Institute for Infection Biology in Berlin. Their results show how minimal molecular changes can lead to far-reaching ecological adaptations.

Actually, they are quite similar. The three clawed frog species, Xenopus borealis, laevis and tropicalis, are about as closely related as a horse and a donkey. But when it comes to their comfort temperature, the frogs are very different. For these cold-blooded animals, this is no small matter: amphibians cannot regulate their body temperature and are therefore dependent on a suitable ambient temperature, unlike poikilothermic animals such as humans.

Xenopus tropicalis prefers warm temperatures above 25 degrees Celsius, while Xenopus borealis is most comfortable at 21 degrees Celsius. Xenopus laevis, on the other hand, likes it comparatively cool at 19 degrees Celsius. To put this in perspective, a change of six degrees Celsius in a human's body temperature would result in either a high fever or hypothermia. Both conditions are fatal if they last for too long.

So how did the frogs adapt to such different temperatures despite being closely related? Biochemist Simone Reber and her team set out to answer this question. They have shown that one of the keys to adaptation lies in cytoskeletal proteins called tubulins. In the frog species studied, the researchers were able to detect small differences in these proteins and prove that they are crucial for the adaptation of the cytoskeleton to the ambient temperature.

The structural scaffold of the cell is temperature sensitive

Reber's research group is based at the Max Planck Institute for Infection Biology in Berlin and the Berlin University of Applied Sciences. The group specializes in tubulins, cytoskeletal proteins found in almost all organisms. They are the basic building blocks of microtubules, tubular structures that give cells shape and strength like a scaffold. “We knew that microtubules are sensitive to temperature changes and thus represent a bottleneck for the temperature adaptation of the entire organism,” explains Simone Reber. ”That’s why we wanted to find out how the tubulins have adapted to the temperature preferences of the three clawed frog species in an evolutionarily short time.”

To do this, the researchers first examined how the microtubules of the three frog species behave at different temperatures. They isolated tubulin from the frog species and used it to produce microtubules, which they examined without the external influence of other cellular components. At 18 degrees Celsius, the microtubules of Xenopus tropicalis were no longer able to grow, while those of the cold-adapted Xenopus laevis had no problems.

A small change with a big impact

With this knowledge, the researchers compared the amino acids of the clawed frog tubulins. Proteins are long amino acid chains whose sequence determines their shape and function. “We were able to show that the tubulins of the clawed frog species differ in only 19 of almost 1000 amino acids,” says Reber: “So the key to the temperature adaptation of the tubulins had to lie in these few amino acids.” However, the influence of the different amino acids on the structure of the tubulins and thus of the microtubules could not be deduced from the sequences alone.

In collaboration with Carolyn Moores at Birkbeck University of London, the team therefore used cryo-electron microscopy to decipher the structure of the tubulins. In this technique, proteins are cooled down to -150 degrees Celsius, enabling them to survive the high-energy electron beam without lengthy preparation, which ultimately produces a high-resolution image of the proteins. The researchers were thus able to study the tubulins in their natural state and create three-dimensional structural models of the microtubules.

More flexibility allows microtubules to grow at cold temperatures

Using these models and the results of the microtubule experiments, Simone Reber’s team was able to show that the amino acids involved influence the strength of the microtubule cross-links. Tubular microtubules consist of long strands of individual tubulins connected in parallel. At low temperatures, the microtubules become stiffer and break more quickly. Weak lateral links make the microtubules more flexible and therefore more stable at low temperatures. This allows the clawed frog Xenopus laevis to adapt to a comparatively cool 19 degrees Celsius.

“We were impressed that a small change in the amino acid sequence has such a far-reaching effect,” says Simone Reber, “A difference in the nanometre range allows the clawed frog species to colonise such different habitats.” This finding is not only important for understanding the cytoskeleton, but also for the consequences of climate change. Ambient temperature affects the clawed frogs at the cellular level and therefore more directly than, for example, a lack of water or a change in food supply.

Animal experiments with African clawed frogs
The investigation of African clawed frog tubulins for the present study was carried out on African clawed frog spawn. The researchers used a hormone injection to make the frogs lay eggs in a controlled manner, which were then used in experiments. No further experiments were carried out on the frogs themselves.
Clawed frogs were also known as pharmacist frogs because they were used as pregnancy tests until the 1960s: the frogs react to the pregnancy hormone hCG in the urine within 18 hours by laying eggs. Clawed frogs such as Xenopus laevis have been used in biomedical research since the 1920s. Among other things, Xenopus was used to show that cells can be reprogrammed by exchanging the cell nucleus – a discovery for which the Nobel Prize was awarded in 2012.