Figure 3. She1 binds and promotes MT... | Rockefeller University Press

Figure 3.

$Figure 3. She1 binds and promotes MT cross-linking via its C-terminal domain. (A) Schematic diagram and gel images of recombinant She1 constructs. (B) Wide-field images of TMR-labeled She1 constructs incubated with HiLyte 488–labeled, Taxol-stabilized MTs. Red arrows indicate regions of cross-linked MTs with high signals of She1-FL–TMR or She1-C–TMR. (C) Intensity profiles of individual HiLyte 488–labeled MT structures (gray lines) in the presence of She1-FL, She1-C, She1-N, or buffer alone. Mean profiles are shown as a black line. Insets show representative images used for quantification. (D) Turbidity decreases as the ionic strength is increased for a solution containing 10 µM MTs and 8 µM She1-C. Inset shows absorbance of 10 µM MTs with and without added 8 µM She1-C at 50 mM KCl. (E) Low-speed pelleting of 5 µM Taxol-stabilized MTs (at 2,348 g) with and without 1 µM She1-FL, She1-N, or She1-C. S, supernatant; P, pellet. (D and E) Error bars are SEM for three experiments. (F) Low-speed copelleting of 5 µM She1-C–TMR with varying concentrations of Taxol-stabilized MTs (at 15,871 g). The fraction of bound She1-C–TMR was quantified by SDS gel (top) and fitted with a quadratic equation as a function of MT concentration (red dotted curve). Linear regression of the initial slope is indicated by a green dotted line.$

She1 binds and promotes MT cross-linking via its C-terminal domain. (A) Schematic diagram and gel images of recombinant She1 constructs. (B) Wide-field images of TMR-labeled She1 constructs incubated with HiLyte 488–labeled, Taxol-stabilized MTs. Red arrows indicate regions of cross-linked MTs with high signals of She1-FL–TMR or She1-C–TMR. (C) Intensity profiles of individual HiLyte 488–labeled MT structures (gray lines) in the presence of She1-FL, She1-C, She1-N, or buffer alone. Mean profiles are shown as a black line. Insets show representative images used for quantification. (D) Turbidity decreases as the ionic strength is increased for a solution containing 10 µM MTs and 8 µM She1-C. Inset shows absorbance of 10 µM MTs with and without added 8 µM She1-C at 50 mM KCl. (E) Low-speed pelleting of 5 µM Taxol-stabilized MTs (at 2,348 g) with and without 1 µM She1-FL, She1-N, or She1-C. S, supernatant; P, pellet. (D and E) Error bars are SEM for three experiments. (F) Low-speed copelleting of 5 µM She1-C–TMR with varying concentrations of Taxol-stabilized MTs (at 15,871 g). The fraction of bound She1-C–TMR was quantified by SDS gel (top) and fitted with a quadratic equation as a function of MT concentration (red dotted curve). Linear regression of the initial slope is indicated by a green dotted line.