By Jovana Drinjakovic

A team of Toronto scientists have zoomed in on an elusive part of the cell to uncover thousands of molecular events that underlie some of the most common human diseases.

Dr. Laurence Pelletier

Dr. Laurence Pelletier

It was a tough nut to crack. The centrosome - a tiny structure in the cell with an out sized role in human health – has eluded scientists for decades because they lacked the right tools to study it. But now a team of researchers led by Dr. Laurence Pelletier, Molecular Genetics professor and Senior Scientist at the Lunenfeld-Tanenbaum Research Institute (LTRI), and Dr. Brian Raught, a Senior Scientist at Princess Margaret Cancer Centre in Toronto, have successfully teased apart the centrosome to reveal an extraordinary degree of molecular complexity. The study, out December 3 in Cell, will guide future discovery into the basic processes in the cell and inform how they may go awry when disease strikes.

The centrosome is a small structure, composed of two barrel-shaped assemblies, called centrioles. Its main job is to sprout cable-like microtubules that take on different roles. They move cargo between different places inside the cell by acting as superhighways, and help to cleanly separate chromosomes into the two daughter cells when a cell divides. Microtubules can also make up a whip-like cilium that can act as an antenna, informing the cell about the outside world, or a beating rod to move liquid and floating chemicals around the cell’s exterior.

Given the different roles that microtubules have, it is not surprising that a faulty centrosome has been linked to seemingly unrelated diseases. For example, microcephaly, an abnormally reduced size of the head, and polycystic kidney disease stem from cells’ inability to form a working centriole or a cilium, respectively, while many cancers start when dividing cells fail to correctly segregate their DNA.

Only two micrometers across, the centrosome is a crowded place, with about 200 distinct proteins that make up its core. Sticking together to make larger structures is just one way that proteins work. The majority of contacts between proteins are more fleeting and it is these transient interactions that drive the biochemistry of life. Scientists surmised that if they could unpick all of the centrosome’s proteins and their contacts, they would then have a strong handle on some of the key processes that are controlled by this critical structure in the cell. The trouble is that centrosomal proteins are notoriously sticky and separating them with traditional laboratory methods has proven to be difficult.

This all changed with the arrival of proximity-dependent biotinylation (BioID), a new tool that enabled Pelletier and Raught to record proteins in and around the centrosome in previously unimaginable detail. BioID works by engineering a protein of choice – a bait — in a way that allows it to attach tags onto other proteins it comes into contact with, a bit like dipping your hand in red paint before shaking hands with those around you. Scientists then collect all of the tagged proteins to reconstruct how they interacted with the bait.

Pelletier’s team used almost 60 different bait proteins that were known to be important at the centrosome, to reveal an astonishing 7,000 protein contacts between 1,700 distinct proteins.

“More than 70 per cent of these protein interactions have never been detected before. This greatly expands what we know about protein interaction at the centrosome-cilium interface,” says Pelletier.

But just because a protein is in contact with the centrosome does not mean that it has a biologically relevant role. To test this, the researchers removed from the cells, one by one, 500 newly discovered proteins to see what happened. They discovered that more than half of the proteins they tested play important roles in keeping the centrosome intact, or helping to build the cilium. But the researchers were surprized to find that most of the proteins are actually involved in more than one process, painting a far more dynamic picture of the centrosome than we have previously imagined.

“The degree to which these processes are intertwined is surprising and much more pronounced than we anticipated,” says Pelletier.

In the future, Pelletier plans to study in more detail some of the newly discovered proteins to understand how they affect cilia function, a long-standing research interest of his. “Of course, we are also very interested to determine whether these interactions are altered in disease and what role they might play. We can do the same BioID experiments in cells with mutations in genes that cause cilia/centriole dysfunction and are linked to kidney or brain diseases. If we can understand the molecular basis of the disease then we can think of ways to fix it,” says Pelletier.

References:
http://www.sciencedirect.com/science/article/pii/S009286741501421X