Visualizing molecules of functional human profilin
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, United States
- Department of Neuroscience and Physiology, SUNY Upstate Medical University, United States
Figures
Figure 1 with 1 supplement
Strategy for fluorescently tagging and purifying profilin-1 (PFN1).
(A) View of profilin (PFN1; purple) with an actin monomer (gray) and poly-L-proline (PLP; blue), from PDB: 2BTF and 2PAV. (B) Profilin residues that contact actin (teal), microtubules (MT) (M114 and G118; pink), PLP (Y6D; blue) or phosphoinositide (PIP) lipids. (C) Position of genetically encoded tag and linking sequence (SGLRSRAQAS) on PFN1. (D) Coomassie-stained SDS-PAGE gel of PFN1 and mApple-PFN1 (mAp-PFN1). Source file contains uncropped gel in (D).
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Figure 1—source data 1
Full SDS-PAGE gel and gel filtration trace.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig1-data1-v2.zip
Figure 1—figure supplement 1
Gel filtration trace of mApple-profilin-1 (mAp-PFN1).
Trace of mAp-PFN1 (pink) elution from gel filtration column. Source file contains values plotted to obtain the UV trace.
Figure 2 with 1 supplement
Tagged profilin binds phosphoinositide (PIP) lipids.
(A) Schematic of liposome pelleting assay. (B) Blot of supernatant and pellet samples from 1 µM profilin-1 (PFN1) in buffer, PS control, or 0.33 mM PI(3,5)P2 or PI(4,5)P2. Blots were probed with anti-PFN1 primary antibody (1:5000; SantaCruz 137235, clone B-10) paired with goat anti-mouse:IRDye 800CW secondary (1:10,000; LI-COR Biosciences 926–32210). (C) Blot for 1 µM mApple-profilin-1 (mAp-PFN1). Assay as in (B). (D) Band densitometry after background subtraction. (E) Fold change in binding normalized to PFN1. Shaded values are independent data points (n=2–3). Error bars, SE. Not significant by Student’s t-test, ns. Full blots in Figure 2—figure supplement 1. Figure 2—source data 1 contains uncropped blots and quantification values for (D and E).
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Figure 2—source data 1
Full blots associated with PFN1 lipid binding experiments.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig2-data1-v2.zip
Figure 2—figure supplement 1
Full blots associated with tagged profilin-1 (PFN1) binding phosphoinositide (PIP) lipids.
(A) Supernatant and pellet samples from liposome pelleting assays in Figure 2B. (B) Blot from assay in (A) with mApple-PFN1 (mAp-PFN1). Blots were probed with anti-PFN1 antibody (1:5000; SantaCruz 137235, clone B-10) paired with goat anti-mouse:IRDye 800CW (1:10,000; LI-COR Biosciences 926–32210). Source file contains uncropped blot images.
Figure 3 with 4 supplements
Tagged profilin binds actin monomers and stimulates nucleotide exchange.
(A) Fluorescence polarization of 10 nM actin (unlabeled) with concentrations of GFP-thymosin β4 (GFP-Tβ4). (B) Competitive polarization of 10 nM GFP-Tβ4, 10 nM actin, and concentrations of profilin-1 (PFN1; purple) or mApple-PFN1 (mAp-PFN1; pink). (C) Kinetics of 500 nM ATP-ATTO-488–2 µM actin in the presence of MEI buffer (gray), 1 µM PFN1, or mAp-PFN1. Dots represent time resolved means from n=3 replicates. (D) Actin assembly via bulk fluorescence. Reactions contain: 2 µM actin (5% pyrene-labeled), and 3 µM PFN1 or mAp-PFN1. Shaded values are SE from n=3 assays. (E) Time lapse total internal reflection fluorescence (TIRF) of 1 µM actin (20% Oregon Green-labeled, 0.6 nM biotin-actin) in buffer (control), or 3 µM PFN1 or mAp-PFN1. Scale bars, 20 µm. See Figure 3—video 1 and Figure 4—video 2. (F) Actin filament nucleation 100 s following initiation from movies as in (E), n=4 fields of view. (G) Actin filament elongation rates from movies as in (E) (n=51 filaments per condition). Shaded dots are single data points. Error bars, SE. Statistics, one-way ANOVA with Bartlett’s correction: ns, not different; (A) p<0.05 from control. No difference found for mAp-PFN1 to PFN1. Source file contains quantification values for (A–D and F and G).
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Figure 3—source data 1
Polarization readings for binding assays associataed with Figure 3, Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig3-data1-v2.zip
Figure 3—figure supplement 1
mApple-profilin-1 (mAp-PFN1) binds Oregon Green (OG)-actin monomers and is suitable for fluorescence-based binding assays.
(A) Fluorescence polarization of 10 nM OG-actin mixed with concentrations of profilin-1 (PFN1). PFN1 did not elicit a change in polarization (n=1). (B) Polarization of 10 nM actin (unlabeled) with mAp-PFN1 bound actin equivalent to PFN1 (Figure 3B; n=3). Source file contains quantification values.
Figure 3—figure supplement 2
Localization of mApple-profilin-1 (mAp-PFN1) with actin filaments in vitro.
(A) Image and montage from a total internal reflection fluorescence (TIRF) assay containing: 1 µM actin (10% Alexa-647-labeled; 0.6 nM biotin-actin; blue), and 5 µM mAp-PFN1 (pink). (B) Magnified view of (A). (C) Image of 0.5 µM actin (10% Alexa-647-labeled; 0.6 nM biotin-actin; blue) and 5 µM mAp-PFN1. (D) Quantification of mAp-PFN1 with actin. Scale bars, 10 µm. Source file contains quantification values for (D).
Figure 3—video 1
Total internal reflection fluorescence (TIRF) movie of profilin-1 (PFN1) or mApple-PFN1 (mAp-PFN1) on actin assembly.
Reaction contains: 1 µM actin (20% OGoOregon gGreen-labeled; 0.6 nM biotin-actin) with buffer (control) or 3 µM PFN1 or mAp-PFN1. Playback, 10 fps. Scale bars, 10 µm.
Figure 3—video 2
TIRF movie of mAp-PFN1 on actin assembly.Reaction contains: 1 µM actin (20% OG-labeled; 0.6 nM biotin-actin) (cyan) and 3 µM mAp-PFN1 (pink).
Box corresponds to Figure 3—figure supplement 2A Figure S4A inset. Playback, 10 fps. Scale bars, 10 µm.
Figure 4 with 3 supplements
Effects of mApple-profilin-1 (mAp-PFN1) on formin-mediated actin assembly.
(A) Bulk actin assembly: 2 µM actin (5% pyrene-labeled), 25 nM mDia1(FH1-C), and 5 µM profilin-1 (PFN1) or mApple-PFN1 (mAp-PFN1). Shaded values are SE from n=2 assays. (B) total internal reflection fluorescence (TIRF) of 1 µM actin (10% Alexa-647-labeled, 0.6 nM biotin-actin), 25 nM mDia1(FH1-C), and 5 µM PFN1 or mAp-PFN1. Scale bars, 10 µm. See Figure 4—video 1, Figure 4—video 2 . (C) Actin fluorescence from TIRF videos. Shading indicates SE from n = 3 videos. (D) Mean nucleation 100 s after initiation, from n=4 fields of view. (E) Actin filament elongation rates from movies as in (B) (n=51 total filaments per condition). Shaded dots are individual data points. Error bars, SE. Statistics, one-way ANOVA with Bartlett’s correction: ns, not different; (A) p<0.05 from control; (B) p<0.05 from PFN1; (C) p<0.05 from mAp-PFN1; (D) p<0.05 from actin and mDia1 control. No difference was found for mAp-PFN1 to PFN1. Source file contains quantification values for (C–E).
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Figure 4—source data 1
Full views, nucleation, and elognation rate values for experiments exploring the effects of mAp-PFN1 in formin-based actin assembly assays.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig4-data1-v2.zip
Figure 4—figure supplement 1
Full views of mApple-profilin-1 (mAp-PFN1) on formin-mediated actin assembly.
(A) Full views formin-mediated actin polymerization montage in Figure 4B (white boxes). (B) Multi-color total internal reflection fluorescence (TIRF) montage containing: 1 µM actin (10% Alexa-647-labeled; 0.6 nM biotin-actin), 25 nM mDia1(FH1-C) and 5 µM mAp-PFN1. Scale bars, 20 µm.
Figure 4—video 1
TIRF movie of PFN1 or mAp-PFN1 on formin-mediated actin assembly.
Reaction contains: 1 µM actin monomers (10% Alexa-647-labeled; 0.6 nM biotin-actin) and 5 µM PFN1 or mAp-PFN1. Box corresponds to montage in Figure 4B and Figure 4—figure supplement 1A. Playback, 10 fps. Scale bars, 10 µm.
Figure 4—video 2
Total internal reflection fluorescence (TIRF) video of mApple-profilin-1 (mAp-PFN1) in formin-mediated actin assembly assay.
Reaction contains: 1 µM actin (10% Alexa-647-labeled; 0.6 nM biotin-actin; cyan) and 5 µM mAp-PFN1 (pink). Playback, 10 fps. Scale bars, 10 µm.
Figure 5 with 4 supplements
Profilin binds tubulin dimers and associates with the microtubule lattice.
(A) Total internal reflection fluorescence (TIRF) images of reactions of biotinylated GMP-CPP seeds, 10 µM free tubulin (5% HiLyte-488 labeled), and buffer (control) or 5 µM profilin-1 (PFN1) or mApple-PFN1 (mAp-PFN1). Scale bars, 20 µm. See Figure 5—video 1. (B) Kymographs display dynamics as in (A). Scale bars: length, 10 µm; time, 100 s. (C) Microtubule polymerization (35–58 microtubules from n=3 experiments). (D) Microtubule length (n=35–58 microtubules from n=3 experiments). (E) Stability index: rescue/catastrophe frequency (n=18–46 microtubules from n=3 experiments). (F) Number of microtubules (n=4 experiments). Shaded dots are single data points. Error bars, SE. (G) Polarization of 10 nM tubulin (unlabeled) and mAp-PFN1. (H) TIRF as in (A), but visualizing tubulin (black) and mAp-PFN1 (pink). Scale bars, 20 µm. See Figure 5—video 2. (I) Kymographs display dynamics in (H). Scale bars: length, 15 µm; time, 540 s. (J) Montage of a microtubule (black) and mAp-PFN1 (pink) merged, with the intensity profiles of mAp-PFN1 along the microtubule lattice. See Figure 5—video 3. Scale bar, 10 µm. + and -, microtubule polarity. Statistics, one-way ANOVA with Bartlett’s correction: (A) p<0.05 from control. No difference was found for mAp-PFN1 to PFN1. Source file contains quantification values for (C–G and J).
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Figure 5—source data 1
Full views of TIRF movies and values for measured parameters associated with Figure 5.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig5-data1-v2.zip
Figure 5—figure supplement 1
Additional views of the effects of mApple-profilin-1 (mAp-PFN1) on microtubule dynamics.
(A) Full views of montages present in Figure 5A (black boxes). (B) Full view of multi-color time lapse total internal reflection fluorescence montage present in Figure 5H (black boxes). Scale bars, 20 µm. (C) Montage of a microtubule (black), mAp-PFN1 (pink) and merge from Figure 5I. Scale bar, 10 µm. + and - indicate microtubule polarity.
Figure 5—video 1
TIRF movie of PFN1 or mAp-PFN1 on microtubules.
Reaction contains: 647-biotinylated-GMP-CCP microtubule seeds (not shown), 10 µM tubulin (5% HiLyte 488) in buffer or 5 µM profilin or mAp-PFN1. Box corresponds to Figure 5A and Figure 5—figure supplement 1A. Playback, 10 fps. Scale bars, 20 µm.
Figure 5—video 2
TIRF movie of mAp-PFN1 on microtubules.
Reaction contains: 647- biotinylated-GMP-CCP microtubule seeds (not shown), 10 µM free tubulin (5% HiLyte 488) (black) and 5 µM mAp-PFN1 (pink). Box corresponds to montage inset from Figure 5H and Figure 5—figure supplement 1B. Playback 10 fps. Scale bars, 20 µm.
Figure 5—video 3
mAp-PFN1 transiently associates with the microtubule lattice.
Reaction contains: 647-biotinylated-GMP-CCP microtubule seeds (not shown), 10 µM free tubulin (5% HiLyte-488); (black) and 5 µM mAp-PFN1 (pink). +and -, microtubule polarity. Playback, 10 fps. Scale bar, 10 µm.
Figure 6 with 1 supplement
Tagged versions of profilin rescue protein levels and cell-based phenotypes in profilin-1 (PFN1) deficient cells.
(A) Blot confirming knockout and rescue of PFN1 with tag-free PFN1, mApple-PFN1 (mAp-PFN1) or Halo-PFN1 plasmids. Extracts prepared from endogenous PFN1(+/+), PFN1(-/-), and PFN1(-/-) transfected with tag-free (rescue), mAp-PFN1 or Halo-PFN1 plasmids. Blot probed with anti-PFN1 antibody (1:3500; SantaCruz 137235, clone B-10) paired with goat anti-mouse:IRDye 800CW secondary (1:5000; LI-COR Biosciences 926–32210), and anti-α-tubulin (1:10,000; Abcam 18251) paired with donkey anti-rabbit 926–68073 secondary (1:20,000). Coomassie stained membrane used as loading control. Full blot in Figure 6—figure supplement 1A-C. (B) PFN1(+/+) cells proliferate significantly better than PFN1-deficient cell lines. (C) Blot probed as in (A) with known quantities of purified PFN1 and mAp-PFN1. Full blot in Figure 6—figure supplement 1D-F. (D) PFN1 levels in neuroblastoma (N2a) cells. Shaded dots are data points from n=4 experiments. Error bars, SE. Maximum intensity images and quantification of (E–F) cell morphology and (G–H) fluorescence of phalloidin-stained actin filaments or (I–J) microtubules from N2a cells expressing endogenous PFN1 (blue), PFN1(-/-) (pink), PFN1(-/-) transfected with mAp-PFN1 (yellow) or tag-free PFN1 plasmids (Figure 6—figure supplement 1G). Cells were plated on micropatterns and stained with anti-α-tubulin antibody (1:100; Abcam 18251) paired with donkey anti-rabbit conjugated to AlexaFluor-647 (1:100; Life Technologies A31573). Scale bars, 10 µm. Shaded dots are individual cells (n=4–15 cells) from n=3 coverslips. Error bars, SE. Statistics, one-way ANOVA with Bartlett’s correction: ns, not different from PFN1(+/+); (A) p<0.05 from PFN1(+/+). No significant difference was found for mAp-PFN1, Halo-PFN1 or tag-less PFN1 to endogenous PFN1(+/+). Source file contains uncropped blots and quantification values for (B, D, F, H, and J).
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Figure 6—source data 1
Full blots and additional cell views associated with Figure 6.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig6-data1-v2.zip
Figure 6—figure supplement 1
Full blots used to determine profilin-1 (PFN1) levels in neuroblastoma-2a (N2a) cells.
Blots confirming knockout or rescue of PFN1 as in Figure 6A. (A) Blots were probed with anti-PFN1 antibody (1:3500; SantaCruz 137235, clone B-10) paired with goat anti-mouse:IRDye 800CW secondary antibody (1:5000; LI-COR Biosciences 926–32210) and (B) anti-α-tubulin primary (1:10,000; Abcam 18251) paired with donkey anti-rabbit:IRDye 680RD (1:20,000; LI-COR Biosciences 926–68073). (C) Coomassie stained membrane from (A). (D) Example blot used to determine PFN1 concentration N2a cells (n=4 total). Blot probed as in (A–C) for (D) PFN1 and (E) α-tubulin, and (F) Coomassie stained. (G) Morphology measurements related to Figure 6 for tag-free PFN1. Source file contains uncropped blots.
Figure 7
The profilin-1(G118V) amyotrophic lateral sclerosis (ALS) variant does not rescue morphology or cytoskeletal phenotypes present in profilin-1 (PFN1) deficient cells.
(A–B) Maximum intensity images and quantification of cell morphology and (C–D) fluorescence of phalloidin-stained actin filaments (E–F) or microtubules from neuroblastoma-2a PFN1(-/-) transfected with Halo-PFN1 (blue), PFN1(-/-) (pink), or PFN1(-/-) cells transfected with Halo-PFN1(G118V; lime). Cells plated and stained as in Figure 6I–J. Scale bars, 10 µm. Shaded dots are individual cells (n=16–25 cells) from at least n=3 coverslips. Error bars, SE. Statistics, one-way ANOVA with Bartlett’s correction: ns, not different from PFN1(-/-) expressing Halo-PFN1 (control); (A) p<0.05 from control. Source file contains quantification values for (B, D, and F).
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Figure 7—source data 1
Source values for cell morphology, and total fluorescence of actin filaments and microtubules.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig7-data1-v2.zip
Figure 8 with 3 supplements
Live-cell visualization of individual molecules of profilin-1 (PFN1).
(A) Maximum intensity projections of PFN1(-/-) expressing markers for actin (green), microtubules (pink) or Halo-PFN1 (blue; top) visualized with JF-646 and imaged as in Figure 6. Titration of JF-646 to illuminate PFN1 molecules. (B) PFN1(-/-) transfected with Halo-PFN1 plasmids (wild-type, R88E, Y6D, and G118V) as in (A). Scale bars, 10 µm. (C) Halo-PFN1-microtubule overlap in cells from (B). Overlap analysis was performed in n=3–4 experiments. Averages from 10 to 25 cells per experiment. Statistics, one-way ANOVA with Bartlett’s correction: ns, not different from PFN1(-/-) expressing Halo-PFN1 (control); (A) p<0.05 from control; (B) p<0.05 from Halo-PFN1(R88E) or Halo-PFN1(Y6D); (C) p<0.05 from Halo-PFN1(G118V). (D) Kymographs display microtubule dynamics (see Figure 8—figure supplement 2 and Figure 8—video 1). (E) Model of PFN1 distribution in cells. Source file contains quantification values for (C).
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Figure 8—source data 1
Halo-profilin microtubule localization counts.
- https://cdn.elifesciences.org/articles/76485/elife-76485-fig8-data1-v2.zip
Figure 8—figure supplement 1
Localization of Halo-profilin-1 (Halo-PFN1) in different cell types.
(A) Maximum intensity images of different PFN1(+/+) cells transiently expressing GFP-actin (green), a marker for microtubules (EMTB-2× mCherry; pink), and either Halo-PFN1 (Halo-PFN1) or Halo-PFN1(R88E) (light blue), visualized with 10 nM JF-646 ligand. Scale bars, 10 µm. (B–D) Quantification of Halo-PFN1-microtubule overlap from different cell types from (A) (n=15–49 cells). (E) No difference was found Halo-PFN1 or Halo-PFN1(R88E)-microtubule colocalization (n=42–46 cells). Source file contains quantification values for (B–E).
Figure 8—figure supplement 2
Live cell localization of constructs of Halo-profilin-1 (Halo-PFN1).
View of kymograph line drawn on cells expressing Halo-PFN1 mutants in Figure 8D. Scale bars, 10 µm.
Figure 8—video 1
Halo-profilin-1 (Halo-PFN1) dynamics in live neuroblastoma-2aN2a cells.
4D-spinning disk confocal movie of cells transiently expressing markers for actin (GFP-actin; cyan), microtubules (EMTB; yellow), and Halo-PFN1 plasmids labeled with 10 nM JF-646 (magenta). Playback, 10 fps. Scale bar, 10 µm.
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Visualizing molecules of functional human profilin
eLife 11:e76485.
https://doi.org/10.7554/eLife.76485
