Direct visualization of augmin-mediated MT branching. Time-lapse TIRF microscopy showing two MT branching events in a cell coexpressing EGFP-α-tubulin (green), γ-tubulin-Tag-RFP-T (red), and mTurquoise2-Dgt5 (blue) in an anaphase Drosophila S2 cell. Two Dgt5 puncta bind to a mother MT (one indicated by an arrow, ←, and another by a left caret, <, in the Dgt5 channel). One Dgt5 puncta (<) recruits a γ-tubulin (left caret, <, in RFP channel, indicated by 1) at 00:20; however, this γ-tubulin dissociates from Dgt5 at 00:50 without nucleating a daughter. The second Dgt5 puncta (←) recruits γ-tubulin at 00:50 (indicated by an arrow, ←, and designated 2) and nucleates a daughter MT at 01:20. The first Dgt5 puncta (<) remains localized to the mother and recruits another γ-tubulin (left caret, <, in RFP channel, indicated by 3) at 01:30, which nucleates a daughter MT at 01:40 shortly before the mother MT depolymerizes (01:50–02:10). In the montage, γ-tubulin-Tag-RFP-T is in the upper left panel, mTurquoise2-Dgt5 is in the upper right panel, EGFP-α-tubulin is in the lower left panel, and the merge is in the lower right panel. Time: min:s. Scale bar, 1 µm.
