Extended Data Figure 5. Measurement of cell polarity using orientation of nucleus-centrosome (NC) axis.

a, Schematic of nuclear-centrosome axis in a migrating polarized cell. The NC axis orientation is determined in cells double stained for nuclei (green) and centrosomes (red). This state of polarity is referred here as ‘CN’ when centrosome is placed ahead of nucleus in the direction of migration. When centrosome is placed behind the nucleus, then this state is referred as ‘NC’. Cells with multiple centrosomes and with centrosome either pointing radially inward or outward of the rings were categorized under “No Orientation”. b, NC axis orientation in uncoordinated sub-confluent rings (n=141, 13 rings) compared to confluent coordinated rotating rings (n=131, 11 rings). c-d, Representative immunofluorescence images of nucleus-centrosome positions in conditions of low calcium before the establishment of a coordinated rotation (n=216,12 rings), normal calcium conditions (n=191, 15 rings) and low calcium treatment after the initiation of coordinated rotation (n=385, 23 rings).

* Conditions where rings were not rotating persistently in any direction (sub-confluent and low calcium case before rotation), a fixed reference direction is chosen (clockwise in this case) in order to calculate the alignment of nucleus-centrosome axis. All error bars indicated are standard deviations. All scale bars: 50 μm