Biologists reverse the engineering of the microtubules that make up the cell walls and pins

Imagine that you are being asked to build a house in a wood yard, without plans or instructions of any kind. The materials are all in front of you, but that doesn't mean you have the first idea of how to get from point A to point B.

This was the situation of Princeton biologists who build microtubules, the skeleton of the cell, from scratch.

"We didn't think it was possible," said Sabine Petry, Assistant Professor of Molecular Biology. For years, Petry and his laboratory's researchers have been dazzling the biological world with videos of what they call "the fireworks of life", which show the branching and growth of these microscopic structures. "From the manufacture of fireworks to the recipe for the manufacture of fireworks? We had imagined and thought about this for five years." Meanwhile, her team had carefully determined the components of the fireworks, one protein at a time, and Akanksha Thawani, a graduate student, had developed a model for the sequence, but it seemed impossible to test it.

But then the review's critics told them that they could not publish their model unless they proved it experimentally.

"Certainly, after seeing Akanksha work on this issue for so long, when the arbitrator asked for more work, I was skeptical about the possibility of sorting the order of molecular attachments in a reasonable time," said Howard Stone, Donald R. Dixon of Princeton69 and Professor of Mechanical and Aerospace Engineering and Co-Guide of Thawani, who holds the Elizabeth W. Dixon Award. "But Akanksha was focused and disciplined, and systematically attacked experiments that identified the order of molecular attachments. It was amazing to follow his detective work."

"They asked us for it, and we wanted it to be published, which did the trick," Petry said. "The evaluation process has bad press, but criticism can sometimes push you to the next level." The results of their work are published in the journal eLife.

Building a house without plans

Microtubules are the bricks and mortar of the cell, used to build the cell walls and the pins of mitosis and meiosis - without them, even unicellular organisms could not reproduce - but until now, no one knew exactly how the microtubules branched. For a decade, researchers have known that branching, caused by the growth of microtubules in each other, was essential to assemble the pins and establish links between cellular components.

"The missing piece over the past decade or so is this branch of microtubules - these microtubules not only grow linearly, but they branch, and they can branch again and again, creating these fireworks," says Petry.

Although Petry's team identified the components needed to make microtubules, they had not developed the sequence - the recipe - that explained exactly how to assemble them, at the molecular level, to make the pins grow and transform them into fireworks. And for the most part, it was good. Biology did it for them. If they assemble the right components, fireworks only grow.

But how did it happen, exactly? That's the question that harassed Thawani, a graduate student in chemical and biological engineering who is doing her research in Petry's laboratory.

"For a long time, I stared at them and wondered how it worked from scratch," said Thawani, who recently won the prestigious Charlotte Elizabeth Proctor Graduate Student Scholarship in her final year. "We start from the total absence of microtubules, then, in 15 minutes, we have these beautiful structures. How to generate a structure from these nanometer proteins? What, in their binding kinetics or organization, could have given rise to the structures we see?"

Thawani was particularly well placed to address these issues, having spent years studying chemical engineering and physics as well as molecular biology. It has essentially invented a new subspecialty between the three areas. "At the intersection of disciplines - that's where the next best science is," she said.

The eLife document is at this unusual crossroads: of the four authors, all but Thawani are principal investigators (PIs) in their own research laboratories, in three generally unrelated areas: Petry in Biology, Stone in Engineering and Joshua Shaevitz, Professor of Physics and Professor of the Lewis-Sigler Institute of Integrative Genomics.

"I don't know of many examples where there is a first author and then three principal investigators," says Petry. "I think it's a Princeton force. I don't know of any other place where it's so easy to bring three teachers together to carry out a project."

The key, Thawani realized, was the creation of a computer model based on accurate measurements of microtubule growth models. This required the use of Total Internal Reflection Fluorescence Microscopy (TIRF), a technique developed by Petry's laboratory to optically isolate a 100 nanometer thick region of the sample so that branched microtubules could be observed in a sea of bottom molecules. (For reference, a human hair is about 500 times wider than that.)

But even then, each pixel recorded by the camera contained thousands of molecules. Thawani had to find a way to disaggregate the visual data to make monomolecular observations, which required months of complicated image analysis - and the help of Shaevitz, who spent years analyzing images.

Ultimately, Thawani measured exactly when and where a single protein binds to an existing microtubule to start a new branch, as well as its growth rate, looking at one molecule at a time.

"The traditional approach, which consists in modifying the quantity of different molecules in the branching reaction, does not determine the order in which things should happen," explains Shaevitz, who is also co-director of the NSF-funded Centre for the Physics of Biological Functions. "By looking at the individual molecules, we can literally look at the assembly piece by piece as it happens."

Thawani then created a computer model using these parameters. Other scientists have already tried to model the branching of microtubules, but none had access to such accurate measurements to test the results of their model. She then tested various sequences that the researchers had studied over the years, and the model excluded all but one of them.

So the research team now had the protein ingredients TPX2, augmin and γ-TuRC, as well as the sequence of steps, but the computer couldn't tell them which protein to add when. And as anyone who has assembled kit furniture or baked bread from scratch knows, making the steps messy simply doesn't work.

The last twist

The experiences requested by the examiners revealed that Thawani and Petry's expectations were exactly retrograde. "We thought it should be augmin first, then TPX2, but it turned out to be the other way around," Thawani said. "It was the twist."

Thanks to this discovery, researchers had the complete recipe for creating microtubule fireworks: If TPX2 is deposited on existing microtubules, followed by binding augmin with γ-TuRC, then the new microtubules will nucleate and branch.

As a final step, they confirmed that the proteins would bind precisely at the speed predicted by the Thawani computer model. "This was the third breakthrough," Petry said, "that these numbers matched, that what had been predicted by his model in the computer was true for biology."

"This work by Petry is really an important addition that will help advance the field," said Daniel Needleman, Gordon McKay Professor of Applied Physics and Professor of Molecular and Cellular Biology at Harvard University. I think that this work, combined with the results of my group and Jan Brugués (at the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden), has really clarified the "rules" of nucleating microtubules into pins. The next step will be to determine the molecular processes that govern these rules. Petry and the universities have put in place a system that should really help to do that."

In retrospect, Petry said, the work has been "full of surprises, both experimentally and in terms of what can be accomplished and how it can be done. Re-examine this long-standing issue, integrating professors from three areas, the review process - the whole system worked."