Structural studies of RIC-8A GEF activity in oriented cell divisions

Oriented cell divisions of epithelial cells are essential for tissue development during embryogenesis and for homeostasis of adult organisms. Consistently, disruption of orientation mechanisms in stem cells leads to tissue disorganization and tumor-like proliferation. The placement of the mitotic spindle within the cell is orchestrated by a non-canonical G-protein signalling pathway, which controls the onset of cortical pulling forces acting on astral microtubules. Specifically, in mitosis the Galphai subunit of heterotrimeric G-proteins anchored at the plasma membrane via myrystoil groups recruits the spindle orientation protein LGN to the cortex by direct binding. In turn, LGN associates with the Dynein-adaptor NuMA whose motor activity generates traction forces orienting the spindle. Interestingly LGN binds only to the GDP-bound form of Galphai, suggesting that a GTP-cycle likely catalysed by the Guanine nucleotide Exchange Factor (GEF) RIC-8A is required for the activation of productive MT-pulling forces. Whether Ric-8A is involved in dissociating Galphai from Gbeta/gamma this way enabling the onset pf pulling forces, or in dissociating the LGN:Galphai complexes to stop pulling forces is to date unclear. To understand the working principles of the GEF RIC-8A, we set out to determine the crystallographic structure of RIC-8A in complex with nucleotide-free Galphai started. We first mapped the minimal interacting domains between the two proteins by limited proteolysis coupled to mass spectrometry, and designed constructs of Ric-8A and Galphai that assembled in monodispersed samples. The crystals obtained with these samples diffracted poorly likely due to an intrinsic conformational variability of the empty Galphai subunit. To stabilize the assembly and improve the crystal packing, we generated a pool of camelid nanobodies recognizing the complex, and performed nanobody-assisted crystallization experiments with samples of Ric-8A:Galphai complexes bound to up to three nanobodies. We are currently using these crystals to determine the architecture of the Ric-8A:Galphai interaction. The structural information will allow us to dissect the catalytic mechanism of the GEF Ric-8A, and to address in living cells how the Galphai GTP-cycle promote oriented cell divisions.