Figure 2.
Figure 2. Direct visualization of the key mediators of branching MT nucleation. (A) Representative TIRF micrographs showing expression and localization of γ-tubulin-Tag-RFP-T (red) and mTurquoise2-Dgt5 (blue) in a mid-anaphase Drosophila S2 cell. Inset shows a representative MT branching event where mTurquoise2-Dgt5 and γ-tubulin-Tag-RFP-T colocalized. (B) Still frames from a TIRF time-lapse of MT branching events in a cell coexpressing GFP-α-tubulin (green), mTurquoise2-Dgt5 (blue), and γ-tubulin-TagRFP (red). The events include the following: (a) Localization of two Dgt5 puncta at 10 s on mother MT (one indicated by the red circle and marked 1, and another indicated by the red arrowhead and marked 2 in the mTurquoise2 channel). (b) Dgt5 puncta (1) recruits γ-tubulin (1) (RFP channel, 0:20). The γ-tubulin puncta (1) dissociates from Dgt5 (1) within 30 s without a branch event; although the Dgt5 puncta (1) remains associated with the mother. (c) Dgt5 (2) recruits γ-tubulin (2) (RFP channel, 0:50), and this complex nucleates a daughter within 30 s (1:20). (d) Dgt5 puncta (now denoted 1*) recruits a second γ-tubulin (3) (RFP channel, 1:30), which nucleates a branch within 20 s (1:40). The first daughter MT grows for 20 s (plus end indicated by white arrowheads in GFP channel) while the second daughter MT was born shortly before the mother depolymerizes. The asterisk (GFP channel, 1:20) indicates a rare event in which a daughter with minus end–associated γ-tubulin and Dgt5 dissociates from an intact mother. The unattached daughter depolymerized completely within 20 s. (C) Average lag time between (b/w) mTurquoise2-Dgt5 (augmin complex subunit) binding to the mother MT and recruitment of γ-tubulin-TagRFP-T; n = 21. (D) Histogram of lag time between Dgt5 recruitment to the mother MT and birth of a daughter MT; n = 25. (E) Histogram shows distribution of Dgt5 puncta along the length of mother MTs; n = 110. (F) Dot plot of fluorescence intensities of Dgt5 puncta that supported nucleation of branched MTs (n = 20) versus those that do not support nucleation of branched MTs (n = 48). (G) Dot plot of dwell times of Dgt5 puncta that supported nucleation of branched MTs (n = 23) versus those that do not support nucleation of branched MTs (n = 48). Time: min:s. Scale bars, 5 µm (A), 1 µm (A inset and B). Mean ± SD values are reported. Two-tailed P values of Student’s t tests are reported; n.s., not significant or P > 0.05.

Direct visualization of the key mediators of branching MT nucleation. (A) Representative TIRF micrographs showing expression and localization of γ-tubulin-Tag-RFP-T (red) and mTurquoise2-Dgt5 (blue) in a mid-anaphase Drosophila S2 cell. Inset shows a representative MT branching event where mTurquoise2-Dgt5 and γ-tubulin-Tag-RFP-T colocalized. (B) Still frames from a TIRF time-lapse of MT branching events in a cell coexpressing GFP-α-tubulin (green), mTurquoise2-Dgt5 (blue), and γ-tubulin-TagRFP (red). The events include the following: (a) Localization of two Dgt5 puncta at 10 s on mother MT (one indicated by the red circle and marked 1, and another indicated by the red arrowhead and marked 2 in the mTurquoise2 channel). (b) Dgt5 puncta (1) recruits γ-tubulin (1) (RFP channel, 0:20). The γ-tubulin puncta (1) dissociates from Dgt5 (1) within 30 s without a branch event; although the Dgt5 puncta (1) remains associated with the mother. (c) Dgt5 (2) recruits γ-tubulin (2) (RFP channel, 0:50), and this complex nucleates a daughter within 30 s (1:20). (d) Dgt5 puncta (now denoted 1*) recruits a second γ-tubulin (3) (RFP channel, 1:30), which nucleates a branch within 20 s (1:40). The first daughter MT grows for 20 s (plus end indicated by white arrowheads in GFP channel) while the second daughter MT was born shortly before the mother depolymerizes. The asterisk (GFP channel, 1:20) indicates a rare event in which a daughter with minus end–associated γ-tubulin and Dgt5 dissociates from an intact mother. The unattached daughter depolymerized completely within 20 s. (C) Average lag time between (b/w) mTurquoise2-Dgt5 (augmin complex subunit) binding to the mother MT and recruitment of γ-tubulin-TagRFP-T; n = 21. (D) Histogram of lag time between Dgt5 recruitment to the mother MT and birth of a daughter MT; n = 25. (E) Histogram shows distribution of Dgt5 puncta along the length of mother MTs; n = 110. (F) Dot plot of fluorescence intensities of Dgt5 puncta that supported nucleation of branched MTs (n = 20) versus those that do not support nucleation of branched MTs (n = 48). (G) Dot plot of dwell times of Dgt5 puncta that supported nucleation of branched MTs (n = 23) versus those that do not support nucleation of branched MTs (n = 48). Time: min:s. Scale bars, 5 µm (A), 1 µm (A inset and B). Mean ± SD values are reported. Two-tailed P values of Student’s t tests are reported; n.s., not significant or P > 0.05.

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