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European Molecular Biology Laboratory (EMBL) Europäisches Laboratorium für Molekularbiologie EMBL Laboratoire Européen de Biologie Moléculaire

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A formula for multiplying by two EMBL researchers identify a key mechanism in cell division

Source article: Ran Induces Spindle Assembly by Reversing the Inhibitory Effect of Importin on TPX2 Activity

Authors: Oliver J. Gruss, Rafael E. Carazo-Salas, Christoph A. Schatz, Giulia Guarguaglini, Jürgen Kast, Matthias Wilm, Nathalie Le Bot, Isabelle Vernos, Eric Karsenti, and Iain W. Mattaj

Scientific contact: Iain W. Mattaj, 49 6221 387 393 (phone), 49 6221 387 518 (fax), [email protected]

Note: Copyright for the text and images from this press release belong to the EMBL except where otherwise noted. They may be freely used and further distributed in if proper attribution to authors and photographers are made. Photographs may only be used in connection to the publication of this story. High-resolution copies of the images can be downloaded from the EMBL web site at: www.embl-heidelberg.de/ExternalInfo/oipa EMBL Press Release - Jan. 12, 2001

A formula EMBL researchers identify a for multiplying key mechanism by two in cell division

Christoph Schatz and Oliver Gruss photo: Doug Young

here are roughly 100 trillion (100,000,000,000,000) cells in each of our bodies, and every one was Tproduced by cell division: the initial fertilized egg split into two daughter cells and then the numbers grew, two by two. While each of these divisions has to happen with absolute precision, a glance into a dividing cell reveals what looks like utter chaos. The immense, sprawling DNA molecules are copied and then knotted up into huge clumps called chromosomes. The membrane that surrounds the nucleus is taken apart and scattered throughout the outer cell compartment, called the cytoplasm, disrupting the normal cell chemistry. And a complicated network of microtubules – railway lines which shuttle millions of molecules to their proper locations – is completely dismantled. The building blocks, called t u b u l i n, are quickly recycled into new microtubules shaped like a spindle. This structure's job is to grab the chromosome copies and pull them towards opposite sides of the cell, where the nuclei of the two new cells will form.

Molecular biologists would like to understand all the minute steps which permit cells to undergo this chaos and then put themselves together again correctly, but the process is so complex that it is hard to know where to begin. The group of Iain Mattaj and their collaborators at the European Molecular Biology Laboratory (EMBL) in Heidelberg have now made an important discovery about a key step: how microtubules reform around chromosomes. In the current issue of the journal Cell, they show that a single active protein is required for the proper construction of some types of spindles. EMBL Press Release - Jan. 12, 2001

The work stems from two long-term lines of research at EMBL. Cell biologist Eric Karsenti has been investigating the process of building spindles. When cells are not dividing, microtubules are constructed in the cytoplasm. They are usually built outwards from a clumpy object called a centrosome. Researchers have traditionally assumed that centrosomes play a central role in division as well, because they usually sit at the poles of spindles, the points towards which newly-copied DNAis drawn – looking very much like spiders extending their long legs to grasp a prey. However, spindles can also be found in some types of cells that don't seem to have centrosomes. This means that there are other recipes for making the structure, and Karsenti and his colleagues went looking for the ingredients in extracts taken from dividing cells. To their great surprise, when they dropped in the mix of DNA and other molecules that usually surround it in the nucleus, called chromatin, spindles formed spontaneously – without centrosomes. Instead of originating at some distant point and reaching out for chromosomes, they seemed to be built from the DNA outwards.

In the meantime, Mattaj and his colleagues were working on a completely diff e re n t p roblem: the ways that molecules manage to pass t h rough the membrane barrier that surrounds the nucleus. This membrane is full of pores, but they do not permit the free passage of most proteins and RNAs. Yet these molecules have to be shuttled in and out at an i n c redible rate to activate genetic information and to synthesize new proteins that perform all sorts of cellular chores. "In a growing mam- malian cell," Mattaj says, "se- Iain Mattaj photo: Doug Young veral million macromolecules must be transported between the nucleus and cytoplasm every minute." The Mattaj group identified proteins that functioned as transport carriers, able to collect cargoes outside the nucleus and navigate them through the pores. Once inside, the carriers are taken apart and the cargo is released.

Related molecules are also used to move cargoes in the opposite direction, out of the nucleus, and the researchers discovered that both loading of export complexes and disassembly of import complexes depends on a molecule called Ran which is packed into the transport carriers. In the nucleus, Ran is found almost exclusively in a high-energy form (called RanGTP), but the chemistry of the outer cytoplasm strips some of this energy and converts it into a low-energy form (RanGDP). This creates an import-export cycle: transport molecules and cargo bind together and the ensemble passes inwards through the membrane. RanGDP moves separately but in parallel. The special chemistry of the nucleus energizes Ran, and this strips the cargo from its import carrier. Other RanGTP molecules can be used to help different molecules escape the nucleus.

What happens, then, when the membrane around the nucleus dissolves during cell division? This has a radical effect on the cell because it shatters the protective environment of the nucleus and its special chemistry. "The sudden influx of molecules from the cytoplasm means that most or even all of the Ran gets converted to the low-energy form," says postdoctoral fellow Oliver Gruss, who EMBL Press Release - Jan. 12, 2001

works in Mattaj's lab. Last year members of the groups of Mattaj and Karsenti showed that adding RanGTP to their cell extracts, which don't have nuclei, created spindles. Then Rafael Carazo-Salas and Giulia Guarguaglini found that a molecule called RCC1, sitting on the chromatin, is able to reload Ran. And adding only RCC1 to cell extracts, which are full of the GDP form of Ran, creates spindles by converting the molecule to its GTPform. This turned what had been previously thought about the formation of the microtubule structures on its head. Distant centrosomes were not reaching out, spider-like, to grab DNA and pull it away. Instead, RCC1 was creating local concentrations of RanGTP around the DNA. This told the cell where to find the chromosomes and to start building microtubules "backwards" – outwards from the DNA.

"Somehow RanGTP was taking the s c a t t e red tubulin building blocks and stringing them back together into microtubules," Gruss says. "Iain came and said, 'Find out how it works.' We didn't know that much about microtubules – that was the expertise of the Karsenti lab – but we knew a lot about transport mechanisms between the nucleus and the cytoplasm. So we started by testing the components we were familiar with. And as it turns out, the mechanism that launches nucleus cytoplasm m i c rotubule formation is closely related to the machinery that imports molecules into the nucleus." In the cell nucleus, the GDP form of Ran is converted to the high-energy GTP form. RanGTP takes apart import carriers, Since converting Ran to the high- releasing TPX2 and other molecules (above). During cell energy form breaks down transport division, the membrane around the nucleus dissolves. Tubulin carriers, it looked like the process molecules, the building blocks of microtubules, can now was releasing some additional, approach the chromosomes. TPX2 becomes bound to import unknown molecule needed to carriers again. But a molecule called RCC1 on the chromo- m a n u f a c t u re microtubules. Gru s s , somes converts RanGDP to its GTP form, releasing TPX2, Carazo-Salas and colleague which is needed to assemble tubulin into the microtubules that will form the mitotic spindle (below). Christoph Schatz performed a number of experiments to see if they could isolate such a molecule, and their colleagues Jürgen Kast and Matthias Wilm identified the protein they found.

TPX2, as the molecule is called, was already known to play a role in the life of the microtubule railway. Components can be delivere d t h roughout the cell along these routes if they are attached to motor p roteins – engine-like molecules that can walk down the surface of a m i c rotubule, towing cargoes. A number of helper molecules are graphics: Katrin Weigmann EMBL Press Release - Jan. 12, 2001

Rafael Carazo-Salas and Oliver Gruss

photo: Russ Hodge needed to attach all the components that make this happen. By a remarkable coincidence, Torsten Wittmann, Isabel Vernos, and Eric Karsenti had discovered TPX2 just two years before while looking for a molecule that could attach a particular motor protein to the poles of spindles.

"We showed that TPX2 gets packaged into the transport carriers that are brought into the nucleus," Gruss says. "If it stays bound to that group of molecules, its activity is blocked. It doesn't have any effect on microtubules. But if you set it loose, you create spindles. We did a number of tests that proved that the formation of spindles depends on whether TPX2 is caught up in a transport complex or not." The final piece of the puzzle came when Gruss teamed up with Vernos and Natalie Le Bot to prove that Ran has a direct effect on` TPX2 function in microtubule assembly. "When the nucleus breaks down, the Ran gets converted to GDP and this effectively locks up all the transport molecules, including TPX2, into complexes with transport carriers. Then molecules on the DNA start to convert nearby Ran to its GTP form. This breaks down any nearby transport complexes, releases the TPX2, and microtubules start to form." While this is the likely sequence of events in real cells, the researchers went a step further and showed that enough TPX2 alone - even without RCC1 or RanGTP – leads to spindle formation.

In addition to pinning down a mechanism fundamental to the cell cycle, this work may have wider implications. "For spindles to form, cells have to have active TPX2 and the molecules that compose microtubules in the same place," Mattaj says. "Because TPX2 gets attached to import carriers, during the rest of the cell cycle it will be shipped into the nucleus, where there is little or no tubulin. The two components only come together when the nuclear membrane breaks down. Keeping molecules apart in this way may be a general way for cells to inactivate proteins that are specifically needed when cells divide."

TPX2 and very closely-related proteins have drawn attention for another reason: cells seem to produce them mostly near the time that they will divide. Since cancer cells have defects which put them in a nearly permanent state of division, the presence of high levels of such proteins can be used to distinguish cancer cells from healthy cells which divide at a much lower rate.

-- Russ Hodge