Supplementary Materials

Accelerated actin filament polymerization from microtubule plus ends

Jessica L. Henty-Ridilla, Aneliya Rankova, Julian A. Eskin, Katelyn Kenny, Bruce L. Goode

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S9
  • Captions for Movies S1 to S11
  • Full Reference List

Images, Video, and Other Other Media

Movie S1
Reaction components: 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin) and 5 μM profilin. Variable components: 25 nM full-length CLIP-170-1 and/or 50 pM mDia1 (FH1-FH2-tail). Video playback is 10 frames per s. Scale bar, 20 μm.
Movie S2
Reaction components: 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin), 5 μM profilin, 50 pM mDia1 (FH1-FH2-tail), and 25 nM full-length CLIP-170-1. Arrows highlight three filaments in a single field of view, each growing at a distinct rate. One filament grows slowly (~10 subunits s-1 μM-1), at a rate consistent with a free barbed-end (white arrow). A second filament grows faster (~55 subunits s-1 μM-1), at a rate consistent with mDia1-mediated stimulation (blue arrow). A third filament grows at an even faster or ‘accelerated’ rate (~180 subunits s-1 μM-1), consistent with the combined effects of mDia1 and CLIP-170-1 (red arrow). Video playback is 10 frames per s. Scale bar, 20 μm.
Movie S3
Reaction components: 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin), 5 μM profilin, and 50 pM 649-mDia1 (FH1-FH2-tail), with or without 25 nM 549-CLIP-170-1. Left panel shows a growing filament with 649-mDia1 on its barbed-end (purple arrows). Right panel shows a growing filament with 649-mDia1 and 549-CLIP-170-1 on its barbed-end (white arrows). Video playback is 10 frames per s. Scale bar, 10 μm.
Movie S4
Reaction components: 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin), 5 μM profilin, 50 pM 649-mDia1 (FH1-FH2-tail), and 25 nM 549-CLIP-170-1. Panels show individual channels and merge, as indicated. Video playback is 10 frames per s. Scale bar, 10 μm.
Movie S5
Reaction components: 15 μM tubulin (30% AlexaFluor649 labeled), biotinylated GMP-CPP MT seeds, 25 nM 549-CLIP-170-1, with or without 500 nM EB1. Video playback is 10 frames per s. Scale bar, 5 μm.
Movie S6
Reaction components: 15 μM tubulin (30% AlexaFluor649 labeled), biotinylated GMP-CPP MT seeds, and 100 pM 488-mDia1. Variable components: 25 nM 549-CLIP-170-1 and 500 nM EB1. Video playback is 10 frames per s. Scale bar, 5 μm.
Movie S7
Reaction components: 15 μM tubulin (30% AlexaFluor649 labeled), biotinylated GMP-CPP MT seeds, and 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin). Video playback is 10 frames per s. Scale bar, 5 μm.
Movie S8
Reaction components: 15 μM tubulin (30% AlexaFluor649 labeled), biotinylated GMP-CPP MT seeds, 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin), 25 nM 549-CLIP-170-1, 100 pM mDia1, 5 μM profilin, and 500 nM EB1. Video playback is 10 frames per s. Scale bar, 5 μm.
Movie S9
Reaction components: 15 μM tubulin (30% AlexaFluor649 labeled), biotinylated GMP-CPP MT seeds, 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin), 25 nM CLIP-170-1, 100 pM 549-mDia1, 5 μM profilin, and 500 nM EB1. Video playback is 10 frames per s. Scale bar, 5 μm.
Movie S10
N2A cells were depleted of endogenous CLIP-170 by expression of an shRNA construct, and rescued either with mChr-marked wild type or FEED1 mutant CLIP-170-1. Cells were co-transfected with a GFP-EB1 plasmid. Living cells were imaged by TIRF microscopy. GFP-EB1 comets shown in cyan; mChr-CLIP-170-1 comets shown in magenta. Images were acquired at 1 s intervals with 25 ms exposure times. Video playback is 25 frames per s. Scale bar, 10 μm.
Movie S11
N2A cells were depleted of endogenous CLIP-170 by expression of an shRNA construct, and rescued either with mChr-marked wild type or FEED1 mutant CLIP-170-1. Cells were co-transfected with a GFP-EB1 plasmid. Living cells were imaged by TIRF microscopy. GFP-EB1 comets are shown in grayscale with MT trajectory particle tracking output overlaid as red lines. Images were acquired with 1 s intervals with 25 ms exposure times. Video playback is 25 frames per s. Scale bar, 10 μm.