Inscuteable: No longer inscrutable

The structure and function of a controller of stem cell division

An important complex forming the core of the cell division apparatus in stem cells has been imaged using the Macromolecular Crystallography beamlines, I04 and I04-1 at Diamond Light Source. As recently reported in Nature Communications, the spindle orientation protein known as LGN bound to an adapter protein known as Inscuteable in a tetrameric arrangement, which drove asymmetric stem cell division.

Stem cells are undifferentiated cells that have the capacity to differentiate into specialised cells. In a developing embryo, stem cells are the foundation of all other cells, whereas in adults, they can aid repair by replenishing lost tissue. To ensure a physiological balance between differentiated and undifferentiated cells, stem cells undergo asymmetric division to give rise to an identical daughter stem cell and a differentiated cell.

Asymmetric division occurs when there is an unequal segregation of cellular contents. For this to occur, the line of division of the cell (known as the axis) must be carefully positioned. The stem cells use polarity proteins, such as Par3, to determine the location of this axis, and then proteins such as LGN and Inscuteable (Insc) help to align the mitotic spindle to the axis of polarity.

Despite the importance of such a process, little is known about the interactions between the proteins. Dr Marina Mapelli, Group Leader at the European Institute of Oncology in Milan and Dr Simone Culurgioni, Post-Doctoral Research Associate here at Diamond, along with scientists from the Italian Institute of Technology, plus the European Molecular Biology Laboratory in Grenoble set out to solve the crystal structure of LGN bound to Insc. They saw that the proteins were intertwined in a fascinating tetrameric arrangement and found that Insc alone had impressive anti-proliferative properties.


Figure 1: On the left, a stem cell orienting (movement highlighted by the red arrow) its mitotic spindle (in green) in order to partition its cellular components (in pink and yellow) unequally in the two daughter cells; one is retaining the stem state (in pink) and the other one is committed to differentiate (in yellow). On the right, the structure of Insc:LGN complex governing this asymmetric cell division process. Insc:LGN complex assembles in highly stable intertwinned tetrameric structure (Insc in blue and purple, LGN in yellow and orange respectively)

Segregation of cellular material

Stem cells are important because they are vital for tissue regeneration and they can be used for numerous medical approaches. However, they are also implicated in some recurrent cancers, which can originate from pools of stem cells and proliferate at a slow rate that makes them untouchable by traditional chemotherapy drugs.

Stem cells divide in an asymmetric way to yield a cell that can differentiate, plus another stem cell. To have asymmetric cell division, the apparatus that segregates cellular material, known as the mitotic spindle, must be precisely oriented. Par3 determines the polarity of the cell and other proteins such as LGN and Insc come together to form a complex to align the mitotic spindle.

The teams in Milan had a long history of working with stem cells and had already carried out multiple investigations into asymmetric cell division. Dr Mapelli and Dr Culurgioni  previously published the structure of a portion of LGN and Insc, but they were keen to look at a larger complex to capture the functional regions of these proteins.

Dr Culurgioni explained their motivation: “Before, we had a partial structure that only explained part of the story, but we wanted to completely understand it. We wanted to solve the structure of the minimal portion of them to be functional.”

Years in the making

The team embarked on a long-term study that was years in the making. “The optimisation of the crystallisation process took several years because the crystals took a long time to grow and they were not reproducible. One of the reasons why it was so difficult was because the protein complex was very flexible,” explained Dr Culurgioni.

This flexibility was suspected because the complex is found close to the membrane in a dynamic environment, and it was later proven by Small-angle X-ray Scattering (SAXS) conducted at the European Synchrotron Radiation Facility (ESRF).

The researchers solved the structure of LGN bound to Insc using beamlines I04 and I04-1, and a series of experiments in mammary stem cells from mice was conducted to validate the structural insights. In the mammary stem cells the expression levels of the proteins were measured as well as their effects on asymmetric cellular division. These lead to important implications on the connection between Insc and the tumour suppressor protein p53.

Two pools of LGN

The data from Diamond showed that LGN and Insc formed a tight assembly, comprising two of each protein in a tetrameric arrangement. They also saw how LGN related to spindle orientation protein NuMA: “We found out from both the structure and the biological data that Insc and NuMA compete for the same site in LGN, but it is not possible to pass from one to the other. The idea is that there are two pools of LGN: one constantly engaged with Insc and one constantly engaged with NuMA,” summarised Dr Culurgioni.

Amazingly, the team also found that Insc could decrease the proliferation of stem cells. They demonstrated this by knocking out a regulator of proliferation known as p53 to cause aberrant cell growth and then expressing Insc. They saw that Insc drove asymmetric division, thereby reducing the rate of symmetric division to ultimately slow proliferation.

The team next wish to understand how the other pool of LGN coordinates it activities with NuMA, so they will return to Diamond to continue their structural characterisations. They may also explore the interaction between Insc and Par3, to see exactly how the spindle connects with the polarity protein. 


To find out more about I04, or to discuss potential applications, please contact Principal Beamline Scientist Dr Dave Hall: david.hall@diamond.ac.uk and for I04-1, please contact Principal Beamline Scientist Prof Frank von Delft: frank.von-delft@diamond.ac.uk.
 

Related publication:

Culurgioni S et al. Insc:LGN tetramers promote asymmetric divisions of mammary stem cells. Nature Communications 2018; 9: 1025.