Cell Culture For experiment 1 and 3: Human retinal pigment epithelial (RPE1) cells immortalised with hTERT (Clontech) were grown in RPE medium (DM EM/F-12 medium containing 10% FCS, 2.3 g/l sodium bicarbonate, 2mM L-Glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin) at 37 ºC, 5% CO2 in a humidified incubator. The RPE1 GLA6 cell line was generated by transfecting hTERT RPE1 cells (Clontech) with mGFP-LifeAct followed by selection with 500 ug/ml Geneticin (Invitrogen). For experiment 1, small interfering RNA oligonucleotides targeted against Kif1C (5-CCCAUGCCGUCUUUACCAU-[dC]-[dG]-3) or a scrambled control (5-GGACCUGGAGGUCUGCUGU-[dT]-[dT]-3) were transfected using Oligofectamine (Invitrogen) following manufacturer’s instructions. Cells were analysed 48 hours after transfection. Live Cell Imaging A 35mm glass-bottom dishes (Fluorodish) or 2-well chambered coverglass chambers were coated with 10 ug/ml fibronectin. The fibronectin solution was allowed to incubate for 2-12 hours, and was washed twice with ddH2O before equilibrating the chamber with RPE medium. 6000 RPE1 GLA6 cells were seeded into each dish/well and allowed to spread for 4-6 hours. Cell migration experiments were carried out in RPE growth medium in a microscope stage top incubator (Tokai Hit) heated to 37 ºC and providing 5% CO2. In each experiment, numerous fields of migrating cells were imaged every 5 min for 12 hr using a 20x objective on an Olympus Deltavision microscope (Applied Precision, LLC) using a GFP filter set (Chroma) and a Coolsnap HQ camera, controlled by Softworx (Applied Precision, LLC). Frame rate was set at imaging every 5 minutes, The resulting images acquired at every time point were 1024x1024 pixels with 322nm/pixel resolution. For experiment 1, stacks 1-45 contain times series of siKif1C cells and stacks 46-88 contain time series of siControl cells.