Golgi positioning during cell migration

Investigation of crucial aspects of cellular function in live cells frequently requires the loss of expression of a specific protein to gain insight into its function. It is also beneficial to combine this with either the enforced re-expression of a tagged version of the protein of interest to validate the phenotype, or the expression of fluorescently tagged marker proteins to investigate a particular phenomenon. Modifications to a short hairpin RNA-mediated knock-down lenti-viral delivery system described here enable the simultaneous expression of short hairpin RNA and a fluorescently-tagged rescue or marker protein to investigate specific cellular processes. The knock-down phenotype of capping protein beta was rescued by the simultaneous expression of a fluorescently tagged knock-down resistant version. Chromophore-assisted laser inactivation of capping protein beta demonstrated the acute loss of function phenotype and highlighted the necessity of loss of endogenous protein expression for the effects to be observed. Another modification in the lenti-viral vector system was used to demonstrate a role for coronin 1B in modulating the rate of actin retrograde flow. A final modification to the lenti-viral system enabled the simultaneous expression of two different fluorescent markers. This was used to investigate Golgi and nucleus positioning during migration. In Rat2 cells at the edge of an artificial wound, the Golgi and centrosome were positioned coincident to one another and were polarized to the front of the nucleus relative to the wound edge. Freely migrating cells expressing fluorescent markers of the Golgi and nucleus did not exhibit such polarity. Instead, Golgi positioning remained fairly constant relative to the nucleus independent of the direction of migration. Nucleus and Golgi positioning relative to the direction of migration was also independent of the speed or directional persistence. Lamellipodial dynamics such as the distance, duration or rate of protrusion were not substantially different along the nuclear-Golgi axis relative to other areas of the cell periphery. Together these data suggest that Golgi positioning in freely migrating cells is independent of the events at the periphery of the cell that are involved in migration.