Figure 3.

Local Translation Is Needed for Protrusion Stabilization

(A) Widespread translation occurs in protrusions. MDA-MB231 cells seeded on 3-μm transwells for 2 hr were treated with Puromycin (10 μg/ml) for 10 min to label nascent proteins. Equal amounts of isolated protrusion and cell body fractions were then resolved by SDS-PAGE and blotted for puromycinylation, VASP as a protrusion marker, H2AX as a cell body marker, and GAPDH as loading control.

(B) Protrusions initiate but retract in translation-inhibited cells. MDA-MB231 mKate-CAAX cells were seeded on 3-μm transwells in presence or absence of cycloheximide (10 μg/ml), fixed at indicated times, and analyzed by confocal microscopy. Images show protrusions at the bottom of transwell filters. Red, cell membranes; blue, filter. Scale bar, 50 μm. See also Movie S3.

(C) Quantification of protrusions from (B) (n = 4). The significant p values are stated above the bar graph. Error bars are SD.

(D) Translation inhibition switches cell morphology from protrusive to round in 3D. MDA-MB231 cells were seeded on top of 3D collagen-I gels for 4 hr and treated with cycloheximide (10 μg/ml) for indicated times before being fixed and imaged. Scale bar, 50 μm. See also Movie S4.

(E) Quantification of protrusive versus round morphologies from (D) (n = 3). Significant p values are stated above each bar graph. Error bars are SD.

(F) Local inhibition of translation in protrusions. MDA-MB231 cells were seeded on 3-μm transwells for 2 hr. Emetine (1 μg/ml) was then added to the bottom chamber for 5 min as in Figure S2F. Transwells were then washed and treated with Puromycin (10 μg/ml) for 10 min to label nascent proteins before lysis. Equal amounts of isolated protrusion and cell body fractions were then resolved by SDS-PAGE and blotted for puromycinylation, VASP (protrusion marker), H2AX (cell body marker), and GAPDH (loading control). The emetine treatment specifically inhibits translation in protrusions.

(G) Inhibition of translation in protrusions destabilizes protrusions. MDA-MB231 mKate-CAAX cells were seeded on 3-μm transwells for 2 hr before being treated with emetine (1 μg/ml) or vehicle as in (F). The cells were then either fixed immediately (0 min), or left for 1 hr (60 min) before being fixed and analyzed by confocal microscopy. The images show protrusions at the bottom of the filter. Red, cell membranes; blue, filter. Scale bar, 50 μm. See also Movie S5.

(H) Quantification of protrusions from (G) (n = 10). The significant p values are stated above the bar graph. Error bars are SD.

See also Figure S2.