Plasmids encoding different fragments of LGN fused to YFP were nucleofected into the canine kidney cell line MDCK T23, which expresses the tetracycline repressible transactivator, and stably expressing cells were selected.
Other plasmids coding for NuMA or Galphai1 fused to YFP, respectively, were nucleofected into MDCK II cells. In addition, a fragment of NuMA with an HA-tag was overexpressed by nucleofection of the coding plasmid into MDCK II cells.
During asymmetric cell divisions, mitotic spindles align along the axis of polarization. In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit. A mammalian Pins, called LGN, binds Galphai and also interacts through an N-terminal domain with the microtubule binding protein NuMA. During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain. Using a FRET biosensor, we find that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization. Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements. We propose that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode.