Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin
- Department of Biochemistry and Molecular Biology, University of Chicago, United States
- Department of Biochemistry and Molecular Biology, The Institute for Biophysical Dynamics, University of Chicago, United States
Figures
Figure 1 with 3 supplements
Hundreds of thousands of Myo10 monomer molecules found in Myo10-transfected U2OS cells.
(A) Epifluorescence image of exogenously expressed HaloTag-Myo10 in U2OS cells. Actin is labeled with phalloidin-AF633 (magenta). Myo10 is labeled with HaloTag ligand-TMR (green). Scale bar = 10 µm. …
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Figure 1—source data 1
Total Myo10 cell signal from all three live-cell bioreplicates.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig1-data1-v1.zip
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Figure 1—source data 2
Uncropped and labeled gels for Figure 1.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig1-data2-v1.zip
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Figure 1—source data 3
Raw unedited gels for Figure 1.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig1-data3-v1.zip
Figure 1—figure supplement 1
HaloTag ligand-TMR specifically labels HaloTag-Myo10 and reliably reflects the total Myo10 present in U2OS cells.
(A) Epifluorescence image of exogenously expressed HaloTag-Myo10 in U2OS cells. Flag-tag was labeled with monoclonal anti-Flag M2 antibody (Sigma, F1804) and goat anti-mouse AF647 (green). HaloTag …
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Figure 1—figure supplement 1—source data 1
Uncropped and labeled gels for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Raw unedited gels for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
HaloTag ligand-TMR robustly labels HaloTag-Myo10.
(A) Left SDS-PAGE lanes: Coomassie blue shows HaloTag standard protein samples prepared to test HaloTag ligand-TMR labeling efficiency. ‘Pre’ refers to protein prior to exposure with TMR ligand and …
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Figure 1—figure supplement 2—source data 1
Uncropped and labeled gels for Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Raw unedited gels for Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig1-figsupp2-data2-v1.zip
Figure 1—figure supplement 3
Calculations to convert signal from SDS-PAGE and epifluorescence microscopy to Myo10 molecule counts.
Figure 2 with 2 supplements
Only a small portion of intracellular Myo10 is activated and enters filopodia, and Myo10 is unevenly distributed around the cell.
The following values are from fixed-cell images. (A) Distribution of the number of Myo10-positive filopodia per cell (N=8733 Myo10-positive filopodia, 150 cells, min = 12, median = 55, max = 116). (B…
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Figure 2—source data 1
Filopodial Myo10 signal, cell body Myo10 signal, and number of filopodia for all three fixed-cell bioreplicates.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig2-data1-v1.xlsx
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Figure 2—source data 2
Data for spatial correlation of Myo10-rich regions of the cell edge for all three fixed-cell bioreplicates.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig2-data2-v1.zip
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Figure 2—source data 3
Data for spatial correlation of Myo10-poor regions of the cell edge for all three fixed-cell bioreplicates.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig2-data3-v1.zip
Figure 2—figure supplement 1
Myo10 is irregularly distributed across the plasma membrane.
In fixed cells: filopodial Myo10 signal distribution in 150 cells. The center of each Myo10 punctum was plotted after two-dimensional PCA. Each Myo10 punctum is colored by signal intensity …
Figure 2—figure supplement 2
Example cell displaying Myo10 density pattern.
(A) Cell 14 from Figure 2—figure supplement 1, though raw signal is plotted. Eight values >100,000 raw signal counts not shown. (B) The fixed-cell image of (A). Myo10 often concentrates in zones at …
Figure 3 with 3 supplements
Hundreds of Myo10 molecules are found in a filopodium, potentially in excess over available actin at the filopodial tip.
The following values are from fixed-cell images. (A) Distribution of the number of Myo10 molecules per filopodium (N=150 cells, 8733 filopodia; min = 6, 95% CI: 5–7; median = 730, 95% CI: 610–850; …
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Figure 3—source data 1
Signal in segmented filopodial Myo10 puncta for all three fixed-cell bioreplicates.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig3-data1-v1.xlsx
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Figure 3—source data 2
Data for local concentration of Myo10 at filopodial tips for all three fixed-cell bioreplicates.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig3-data2-v1.zip
Figure 3—figure supplement 1
HaloTag ligand-TMR labeling to determine Myo10 distributions along filopodia.
(A) In fixed cells: dim Myo10 puncta in filopodia, identified as those containing <35 Myo10 molecules, were examined. 135 total dim puncta across 26 cells were identified. Of the 135 puncta, 7 …
Figure 3—figure supplement 2
96 µM of actin monomers are accessible to Myo10 in a filopodium.
Figure 3—figure supplement 3
Plasma membrane at the filopodial tip can accommodate a portion of Myo10 unbound to actin.
Figure 4 with 5 supplements
Myo10 dynamics from live-cell movies.
(A–C) Dense Myo10 puncta appear at the start of each phase of the filopodial lifecycle. Distributions of the number of Myo10 molecules in puncta upon: (A) Filopodial initiation (min = 52, 95% CI: …
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Figure 4—source data 1
Trajectories of Myo10 puncta upon retraction from live-cell bioreplicate 1.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data1-v1.zip
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Figure 4—source data 2
Trajectories of Myo10 puncta upon second-phase elongation from live-cell bioreplicate 1.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data2-v1.zip
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Figure 4—source data 3
Trajectories of Myo10 puncta upon filopodial initiation from live-cell bioreplicate 1.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data3-v1.zip
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Figure 4—source data 4
Trajectories of Myo10 puncta upon retraction from live-cell bioreplicate 2.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data4-v1.zip
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Figure 4—source data 5
Trajectories of Myo10 puncta upon second-phase elongation from live-cell bioreplicate 2.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data5-v1.zip
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Figure 4—source data 6
Trajectories of Myo10 puncta upon filopodial initiation from live-cell bioreplicate 2.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data6-v1.zip
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Figure 4—source data 7
Trajectories of Myo10 puncta upon retraction from live-cell bioreplicate 3.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data7-v1.zip
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Figure 4—source data 8
Trajectories of Myo10 puncta upon second-phase elongation from live-cell bioreplicate 3.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data8-v1.zip
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Figure 4—source data 9
Trajectories of Myo10 puncta upon filopodial initiation from live-cell bioreplicate 3.
- https://cdn.elifesciences.org/articles/90603/elife-90603-fig4-data9-v1.zip
Figure 4—figure supplement 1
Myo10 is found along the filopodium shaft and can undergo a second elongation.
(A) Frame stills from Figure 4—video 2. Red arrow points to a Myo10 punctum (red circle) right after emergence from an existing filopodium. Elapsed seconds from the first frame is depicted. Scale …
Figure 4—video 1
Myo10 punctum initiates a nascent filopodium.
Example of a Myo10 punctum (red circle) right as the filopodium shoots from the cell body. 300 ms exposure, 10 fps.
Figure 4—video 2
Myo10 punctum can undergo a second elongation.
Example of a Myo10 punctum (red circle) right after emergence from an existing filopodium. 300 ms exposure, 10 fps.
Figure 4—video 3
Example of Myo10 punctum decreasing in intensity as it returns to cell body.
The trajectory indicated by the blue asterisk in Figure 3E. Example of a Myo10 punctum (purple circle) that decreases in intensity before becoming the site of new filopodium growth. 300 ms exposure, …
Figure 4—video 4
Example of Myo10 punctum in a ‘snapping’ filopodium as it returns to cell body.
The trajectory indicated by the magenta asterisk in Figure 3E. Example of a Myo10 punctum (purple circle) that ‘snaps’ toward the cell body and then diffuses into the cytoplasm. 300 ms exposure, 30 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | U2OS cells | ATCC | HTB-96 | |
| Transfected construct (human) | HaloTag-Myo10-Flag (plasmid) | This paper | See supplemental for DNA sequence | |
| Antibody | Anti-Myosin10 antibody (rabbit polyclonal) | Novus | NBP1-87748 (RRID:AB_11035627) | 1:10,000 for western blot |
| Antibody | Anti-beta-tubulin antibody (mouse monoclonal) | Invitrogen | 22833 (RRID:AB_2533072) | 1:10,000 for western blot |
| Antibody | Anti-Rabbit-HRP antibody (goat polyclonal) | Cell Signaling | 7074 (RRID:AB_2099233) | 1:10,000 for western blot |
| Antibody | Anti-Mouse-HRP antibody (horse polyclonal) | Cell Signaling | 7076 (RRID:AB_330924) | 1:10,000 for western blot |
| Peptide, recombinant protein | Laminin | Sigma-Aldrich | CC095-M | |
| Peptide, recombinant protein | HaloTag standard protein | Promega | G4491 | |
| Commercial assay or kit | SuperSignal West Femto chemiluminescent substrate | Thermo Scientific | 34094 | |
| Other | Gibco 1x DMEM | Thermo Fisher | 11995073 | Cell media |
| Other | Accutase | Corning | MT25058CI | Enzyme cell detachment media |
| Other | FuGENE HD Transfection reagent | Promega | E2311 | Non-liposomal transfection reagent |
| Other | Lipofectamine 2000 | Invitrogen | 11668-027 | Cationic-lipid transfection reagent |
| Other | #1.5 coverglass bottom 35 mm Petri dishes | Cellvis, MatTek | D35-10-1.5-N, P35G-1.5-14-C | Coverglass for live-cell imaging |
| Other | Ibidi eight-well chamber slides | Ibidi | 80807 | Coverglass for fixed-cell imaging |
| Other | 4–20% Mini-PROTEAN TGX Stain-Free protein gel | Bio-Rad | 4568095 | Stain-free precast gels for SDS-PAGE |
| Other | TMR-HaloLigand | Promega | G8251 | Fluorophore-labeled HaloLigand used for visualizing Myo10 |
| Other | Alex Fluor 647 Phalloidin | Invitrogen | A22287 | 1 mM working concentration to label actin |
| Other | Bio-Beads SM-2 | Bio-Rad | 152-8920 | Resin to remove excess dye when testing HaloLigand labeling efficiency |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/90603/elife-90603-mdarchecklist1-v1.pdf
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Source code 1
A descriptive text file explaining each subfolder is included within the zip file.
- https://cdn.elifesciences.org/articles/90603/elife-90603-code1-v1.zip
