UNC-45a knockout cells display abnormal cell morphology accompanied by multiple lamellipodial protrusions and deficient tail retraction. (A) WB analysis of endogenous UNC-45a, UNC-45b, and myosin RLC levels in total cell lysates of WT U2OS and UNC-45a knockout (KO) U2OS cells. Human skeletal muscle lysate was used as a positive control for UNC-45b expression, and GAPDH was probed for equal sample loading. (B) Time-lapse images of WT and UNC-45a knockout cells migrating on a laminin-coated surface, and representative examples of endogenous F-actin localization (by fluorescent phalloidin) in WT and UNC45a knockout cells cultured on laminin-coated coverslips. Bars, 50 µm (time-lapse images) and 20 µm (fixed cells stained with fluorescent phalloidin). (C) Lamellipodial-protrusion profiles of randomly migrating WT and UNC-45a knockout cells were tracked for 80 min. The number of lamellipodial protrusions was quantified every 10 min, and the data are presented as the percentage of cells exhibiting one, two, three, or four simultaneous protrusions; means ± SEM, n = 27 WT and 27 UNC-45a knockout cells. (D) Protrusion lengths (vector distance from the edge of nucleus to the furthest cell edge) of randomly migrating WT and UNC-45a knockout cells. The data are presented as protrusion length means ± SEM. n = 20 WT and 20 UNC-45a knockout cells. (E) Random migration velocities of WT and UNC-45a knockout cells. Quantification is based on tracking of the nuclei displacement. The data are presented as velocity obtained from a 10-h cell tracking; means ± SEM. n = 40 for WT and 34 for UNC-45a knockout cells. (C–E) Quantifications were performed from the time-lapse phase-contrast imaging data, exemplified in B, and a Student’s t test was used to derive the p-values. Fig. S5 E provides data for a cell-migration rescue experiment for UNC-45a knockout cells.