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Accelerated actin filament polymerization from microtubule plus ends

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Science  20 May 2016:
Vol. 352, Issue 6288, pp. 1004-1009
DOI: 10.1126/science.aaf1709
  • Fig. 1 CLIP-170 accelerates formin-mediated actin filament elongation.

    (A) CLIP-170 domains. F, FEED sequence; MBD, metal-binding domain. (B) Images from TIRF assays containing 50 pM mDia1 and/or 25 nM CLIP-170-1, taken 215 s after initiation of actin assembly. Scale bar, 20 μm. (C) Kinetics of total actin polymer mass (fluorescence intensity) accumulation averaged from multiple (n ≥ 3) fields of view. Linear fits plotted with 95% confidence intervals are shown as shaded areas. AU, arbitrary units; OG-actin, Oregon Green actin. (D) Representative filament length traces (10 per condition) from TIRF movies. (E) Distributions of elongation rates from TIRF reactions, as in (B), for different concentrations of CLIP-170-1. Distributions are shown from one of three independent experiments (n = 50 filaments each). The red bars show mean elongation rates for subpopulations, measured from all filaments in three separate experiments (n = 150 filaments). (F) Fold increase in mean formin-mediated elongation rate stimulated by 25 nM CLIP-170-1. Error bars indicate SE. (G) Mean elongation rates from TIRF reactions containing different mDia1 constructs with or without profilin. (H) CLIP-170-1 constructs that enhance (+) or fail to enhance (–) the rate of mDia1-mediated elongation. FL, full length; F, FEED sequence; teal box, alternatively spliced region; red dots, FEED1 [445VEEE/AAAA448 (V, Val; E, Glu; A, Ala)] and FEED2 [450ITKGDLE/AAAAAAA456 (I, Ile; T, Thr; K, Lys; G, Gly; D, Asp; L, Leu)] mutants. (I) Distributions of elongation rates measured as in (E) for different CLIP-170-1 constructs. Reactions contained 1 μM globular actin (G-actin) (10% OG-labeled; 0.2% biotin-actin), ± 5 μM profilin, ± 50 pM mDia1, 50 pM mDia2, 50 pM Daam1, 100 nM INF1 or 100 nM INF2, ± 25 nM full-length CLIP-170-1 (WT, FEED1 mutant, or FEED2 mutant). Statistical differences in (E), (G), and (I): ns, not significantly different from control; a, compared with control (actin and profilin) (P < 0.05); b, compared with formin control (actin, profilin, and formin) (P < 0.05).

  • Fig. 2 CLIP-170 and mDia1 form a barbed-end tracking complex that accelerates actin filament elongation.

    (A) Representative time points from a single-molecule TIRF experiment. Reactions contained 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin), 50 pM 649-mDia1, and 25 nM 549-CLIP-170-1. A barbed end is highlighted in each frame (boxed), with insets at bottom right showing individual and merged channels. Arrowheads show two additional growing barbed ends with 649-mDia1 and 549-CLIP-170-1 associated. Scale bar, 10 μm. Insets, 5 μm by 5 μm. (B) Effects of 25 nM CLIP-170-1 or 549-CLIP-170-1 on the rate of mDia1-mediated actin filament elongation. Reactions were as described in (A). Statistical differences: ns, not significantly different from control; a, compared with control (actin and profilin) (P < 0.05); b, compared with formin control (actin, profilin, and formin) (P < 0.05). (C) Formation and dissociation of a CLIP-170–mDia1 complex at a barbed end. (D) Fluorescence intensity profiles for each channel, showing formation and dissociation of the CLIP-170–mDia1 complex in (C). (E) Elongation rates correlate with arrival and dissociation of mDia1 and/or CLIP-170 at the barbed end. (F) Single-molecule colocalization of anchored 649-mDia1 and soluble full-length 549-CLIP-170-1 at different concentrations of unlabeled competitor fragment CLIP-170-1348-460. Data were averaged from three fields of view in each of three independent experiments. Error bars indicate SE. The inset shows a representative field of view from a reaction with no competitor. Scale bar, 5 μm. (G) Representative filament traces from TIRF movies, conditions as in (A) except for the addition of 3 nM CP. Capping events (red arrowheads) and regrowth events (black arrowheads) are highlighted. (H) CLIP-170-1 enhances the duration of mDia1-mediated elongation in the presence of CP. Error bars indicate SE. (I) Cartoon of CLIP-170 joining mDia1 at the barbed end and increasing the rate of elongation and duration of growth in the presence of CP.

  • Fig. 3 CLIP-170–mDia1 complexes are recruited to MT plus ends by EB1 and stimulate actin polymerization from the MT surface.

    (A) TIRF movies show that 549-CLIP-170-1 binds MT sides and tracks MT plus ends only in the presence of EB1 (17). 488-mDia1 binds MT sides, with or without EB1, and does not track MT plus ends. Yellow arrowheads indicate CLIP-170 molecules; blue arrowheads indicate mDia1 molecules. (B and C) 549-CLIP-170-1 and 488-mDia1 colocalize on MT sides (B) and track MT plus ends together specifically in the presence of EB1 (C). White arrowheads indicate the presence of both CLIP-170 and mDia1 on MT plus ends. Scale bars, 5 μm. Reactions in (A) to (C) contain 15 μM tubulin (30% AlexaFluor649-labeled), biotinylated guanosine monophosphate–CPP MT seeds, and variable components (25 nM 549-CLIP-170-1, 100 pM 488-mDia1, and 500 nM EB1). (D) Percentage of molecules tracking MT plus ends from reactions in (A) to (C). Data were averaged from three experiments (n > 100 molecules). Statistical differences: ns, not different from control; a, compared with CLIP-170-1 (P < 0.05); b, compared with mDia1 (P < 0.05). (E) Co-reconstitution of MTs undergoing dynamic instability and polymerization of actin filaments. Reactions contained all of the same components as in (A) to (C), plus 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin) and 5 μM profilin. Scale bar, 5 μm. (F) Kymographs of MT and actin dynamics. Scale bars, 5 μm. (G) The rate of actin filament elongation does not change significantly in the presence and absence of MTs. Data were averaged from three experiments (n = 50 actin filaments). (H) With the addition of unlabeled EB1 and mDia1, 549-CLIP-170-1 molecules (yellow arrowheads) were recruited to MT plus ends, where they triggered assembly of actin filaments that grew at the accelerated rate. Scale bar, 5 μm. (I) Similar observations as in (H), except using 549-mDia1 and unlabeled CLIP-170-1. Note that, in this panel, the yellow arrowheads indicate 549-mDia1 (rather than CLIP-170). Scale bar, 5 μm. (J) Fraction of formin-generated actin filaments that grew from MT plus ends in reactions as in (H) and (I). Data were from three experiments (n = 50 filaments per condition). (K) Actin elongation rates from reactions as in (H) and (I). Data were from three experiments (n = 50 filaments per condition). Statistical differences: ns, not different from control; a, compared with actin alone or control (P < 0.05); b, compared with EB1 control (P < 0.05). Error bars in (D) and (G) to (K), SE.

  • Fig. 4 CLIP-170 interactions with formins promote dendritic branching in primary neurons.

    (A) Representative images from rat cortical neurons (at 4 or 5 days in vitro) cotransfected with cytomegalovirus promoter–green fluorescent protein (GFP) and pSUPER control or short hairpin RNA (shRNA) against CLIP-170 (shCLIP-170) (16), with or without plasmids expressing shRNA-resistant full-length CLIP-170-1 rescue (WT or FEED1 mutant). Scale bar, 5 μm. (B) Quantification of dendritic branching complexity by Sholl analysis (n = 90 to 120 neurons, from two or more independent experiments). Statistical differences: ns, not significantly different from control; a, compared with scramble shRNA control (P < 0.05). There was never a significant difference between the FEED1 rescue and the shCLIP-170 knockdown alone. Error bars indicate SE. (C) Representative images from rat cortical neurons, as in (A), expressing full-length CLIP-170-1 (WT or FEED1 mutant) without silencing endogenous CLIP-170. Scale bar, 5 μm. (D) Quantification of dendritic branching complexity by Sholl analysis as in (B), except data are from n = 60 neurons in two independent experiments. Statistical differences: ns, not significantly different from control; a, compared with scramble shRNA control (P < 0.05). Error bars indicate SE. (E) Localization of mCherry–CLIP-170-1 (WT or FEED1 mutant) rescue constructs in N2A cells in which endogenous CLIP-170 was silenced. Scale bars, 10 μm. (F) Quantification of mCherry–CLIP-170-1 colocalization with EB1-GFP at MT plus ends from cells as in (E). Error bars indicate SD. (G) Quantitative tracking of mCherry–CLIP-170-1 (WT or FEED1 mutant) comets on growing MT plus ends. Analysis included comet velocity (or MT growth rate), comet lifetime (MT growth duration), and comet “dynamicity” [a general readout of MT dynamics (37)]. Error bars indicate SD.

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.

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