Combinatorial deployment of F-actin regulators to build complex 3D actin structures in vivo
- Department of Biological Sciences, University of Denver, United States
Figures
Figure 1 with 2 supplements
Rapid formation and dissolution of cortical actin cap structures.
(A) Still images from live-imaging of apical F-actin dynamics (UAS:moeABD:mCherry, cycle 11) at t = 0, 20, 90, 180, 270, 360, 540 s. Scale bar = 5 μm. (B) WT actin cap area dynamics from cycle 11 …
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Figure 1—source data 1
Cap dynamics data.
- https://cdn.elifesciences.org/articles/63046/elife-63046-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Similar actin labeling with different actin markers.
(A) Representative images from live-imaged mCh:MoesinABD (actin-binding domain of Moesin labeled with mCherry), live-imaged GFP:Act88F (Actin directly labeled with GFP) live-imaging, and fixed …
Figure 1—figure supplement 2
WT area dynamics of cortical actin caps in cycle 10–13.
(A) Cycle 11 actin cap area dynamics (n = 15, N = 4) as the standard for analysis. n = 15, N = 4. Cap areas are normalized to the size at t = 0 s. Four different phases are labeled (Exp.: Expansion; …
Figure 2 with 1 supplement
Quantitative dynamics of Formin and Arp2/3-driven actin networks.
(A) Still images from live-imaging of apical F-actin dynamics (UAS:moeABD:mCherry, cycle 11) from control, Dia shRNA and ArpC4 shRNA lines at t = 20, 90, 180, 360, 540 s. Four different phases are …
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Figure 2—source data 1
Arp and Dia disrupted data.
- https://cdn.elifesciences.org/articles/63046/elife-63046-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Apical and furrow-associated actin populations in Dia or Arp2/3 compromised embryos.
(A) Cycle 11 apical cap and furrow actin distributions in Control, Dia shRNA and ArpC4 shRNA embryos at t = 120 s. Scale bar = 5 μm. Cap (0.9 μm below apical most layer) and furrow level (3.9 μm …
Figure 3 with 1 supplement
ANRPs have distinct roles in building cortical actin structures.
(A) Schematics of different ANRPs domain organization. Domains are collected from Flybase (Pfam/SMART) and UniProt. WD40: WD40 repeats; CC: coiled-coil domain; Hs1/Cortactin: Hs1/Cortactin repeats; …
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Figure 3—source data 1
ANRP measurement data.
- https://cdn.elifesciences.org/articles/63046/elife-63046-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
ANRPs have distinct roles in building apical actin structures.
(A, B, C, D, D’, F, F’, H) Still images from live-imaging of apical F-actin dynamics in cycle 11 at t = 180, 270, 360 s, from control (A), DPod1 shRNA (B), Coronin shRNA (C), DPod1 shRNA 2 (D–D’), Co…
Figure 4 with 1 supplement
ANRP localization at apical actin caps.
(A–E) Coronin (UAS:Coronin:GFP), Cortactin (UAS:GFP:Cortactin), DPod1 (UAS:DPod1:GFP), Scar (UAS:Scar:GFP), and Carmil (UAS:Carmil:GFP) localization on apical cap structures at t = 20, 180, 270, and …
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Figure 4—source data 1
RT PCR source data.
- https://cdn.elifesciences.org/articles/63046/elife-63046-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Expression levels and endogenous localization data of ANRPs.
(A, B) Still images from live-imaging of apical CRISPR Arp3:GFP (A) and CRISPR GFP:Cortactin (B) in cycle 11 at t = 20, 180, 270, 360 s. Scale bar = 3 μm. (C) Still images from live-imaging of …
Figure 5 with 1 supplement
Arp2/3 and Formin network analysis suggests competition for free G-actin.
(A) Still images from FRAP recovery of F-actin live-imaging (UAS:moeABD:mCh) in cycle 11 embryos at t = −1s (1 s before photobleaching), 0 s (photobleaching), 4 s, 8 s, 12 s, 24 s, and 32 s. Scale …
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Figure 5—source data 1
FRAP actin network source data.
- https://cdn.elifesciences.org/articles/63046/elife-63046-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
FRAP on Arp2/3 and ANRPs disrupted embryos.
(A, B) FRAP T50 and immobile fraction of F-actin in control and indicated actin regulator compromised embryos in cycle 11. ns: not significant, *: p<0.05, **: p<0.005, ***: p<0.0005. Control: n = 13;…
Figure 6 with 1 supplement
In vivo nucleator recruitment strengths of actin regulatory proteins.
(A, C, E, G, I, K, M) Images of CRISPR Arp3:GFP with mito-tagged mCherry control (A) or mCherry:ANRPs (C, E, G, I, K, M) in cells at stage 12. Scale bar = 3 μm. (B, D, F, H, J, L, N) Intensity line …
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Figure 6—source data 1
Mito-localization source data.
- https://cdn.elifesciences.org/articles/63046/elife-63046-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Mito-tagged ANRPs can direct F-actin polymerization.
(A) Still images of mitochondrial morphology from YFP:mito in stage 12 embryo. Scale bar = 1 μm. (B–E) Staining images of CRISPR Arp3:GFP (anti-GFP) with mito-tagged mCherry:ANRPs (anti-dsRed) and …
Figure 7
Requirement for filamentous actin cap ANRP function in anchoring embryonic nuclei.
(A) Nuclei (marked by Histone:RFP) lose apical anchorage and fall into the embryonic interior in ArpC4 shRNA embryos during cycle 12 at t = 0 s, 90 s, 180 s, 270 s, and 540 s. Medial z-layer (−5 μm …
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Figure 7—source data 1
Nuclear fallout source data.
- https://cdn.elifesciences.org/articles/63046/elife-63046-fig7-data1-v2.xlsx
Author response image 1
Tables
Table 1
Arp2/3 and ANRPs toolkit.
| Construct | Vector | Chromosome |
|---|---|---|
| CRISPR Arp3:GFP | Endogeous | III |
| CRISPR GFP:Cortactin | Endogeous | III |
| CRISPR GFP:DPod1 | Endogeous | X |
| UAS:GFP:Cortactin | pUAST | X, II, III |
| UAS:Cortactin:GFP | pUAST,pUASp | X, II, III |
| UAS:mCherry:Cortactin | pUASp | II, III |
| UAS:DPod1:GFP | pUAST | X, II, III |
| UAS:Coronin:GFP | pUAST | II, III |
| UAS:GFP:Carmil FL | pUAST | X, II, III |
| UAS:Carmil FL:GFP | pUAST | X, II, III |
| UAS:Scar:GFP | pUASp | II, III |
| UAS:mito:mCherry:Cortactin | pUASp | II, III |
| UAS:mito:mCherry:Coronin | pUASp | X, II, III |
| UAS:mito:mCherry:DPod1 | pUASp | II, III |
| UAS:mito:mCherry:Scar | pUASp | II, III |
| UAS:mito:mCherry | pUASp | II, III |
Table 2
Stocks and genetics.
| Stocks | Source | Identifier |
|---|---|---|
| General stocks | ||
| P[mat-tub-Gal4] mat67 | D. St Johnston | |
| P[mat-tub-Gal4] mat15 | D. St Johnston | |
| UAS:mCherry:MoesinABD | T. Millard | |
| Histone:RFP | BDSC | BDSC 23650 III; BDSC 23651 II |
| UAS:GFP:Act88F | BDSC | BDSC #9253 |
| Wasp:sGFP | VDRC | VDRC #318474 |
| Wash:GFP | BDSC | BDSC #81644 |
| YFP:mito | BDSC | BDSC #7194 |
| UAS:mCh:mitoOMM | BDSC | BDSC #66532, 66533 |
| nos-Cas9 | Bestgene | NIG-FLY #CAS-0001, CAS-0003 |
| w1118 | Bestgene | |
| shRNA (Valium) lines | ||
| ArpC4 shRNA | DRSC/TRiP | BDSC #41888 |
| Dia shRNA | DRSC/TRiP | BDSC #35479 |
| DPod1 shRNA | DRSC/TRiP | BDSC #41705 |
| Coronin shRNA | DRSC/TRiP | BDSC #40841 |
| Cortactin shRNA | DRSC/TRiP | BDSC #44425 |
| Carmil shRNA | DRSC/TRiP | BDSC #41686 |
| Scar shRNA | DRSC/TRiP | BDSC #51803 |
| Wasp shRNA | DRSC/TRiP | BDSC #51802 |
| Wash shRNA | DRSC/TRiP | BDSC #62866 |
| Cofilin shRNA | DRSC/TRiP | BDSC #33670 |
| DPod1 shRNA 2 | VDRC/KK | VDRC #108886 |
| Coronin shRNA 2 | VDRC/KK | VDRC #109644 |
| Cortactin shRNA 2 | VDRC/KK | VDRC #105289 |
Table 3
Reagents.
| Reagent | Source | Identifier |
|---|---|---|
| Antibodies and dyes | ||
| Rabbit anti-GFP | Invitrogen | A11122 |
| Mouse anti-dsRed | Clontech | 632393 |
| Alexa Fluor Goat anti rabbit 488 | Invitrogen | A11034 |
| Alexa Fluor Goat anti mouse 568 | Invitrogen | A11031 |
| Alexa 568-Phalloidin | Invitrogen | Cat# A12380 |
| Alexa 647-Phalloidin | Invitrogen | Cat# A22287 |
| Rabbit anti-Dia | Wasserman lab | |
| Chemicals and kits | ||
| Halocarbon oil 27 | Cat# H8773 | |
| Halocarbon oil 700 | Cat# H8898 | |
| Paraformaldehyde | Electron Microscopy Sciences | Cat# 15714 |
| ProLong Gold | Invitrogen | Cat# P36931 |
| Jasplakinolide | Santa Cruz Biotech | Cat# sc202191 |
| Latrunculin B | Sigma | Cat# L5288 |
| QIAShredder | QIAGEN | Cat# 79654 |
| Quick-RNA MicroPrep | Zymo Research | Cat# R1050 |
| QuantiTech Reverse Transcription Kit | QIAGEN | Cat# 205310 |
| QuantiTech SYBR Green RT-PCR | QIAGEN | Cat# 204141 |
| Q5 site-directed mutagenesis EZNA insect DNA kit | NEB Omega Biotek | Cat# E0554S Cat# D0926-01 |
| Software | ||
| iQ5 | Bio-Rad | bio-rad.com |
| FIJI/ImageJ | Schindelin et al., 2012 | Fiji.sc |
| Micromanager 1.4 | Edelstein et al., 2014 | micro-manager.org |
| OriginPro | OriginLab | originlab.com |
| Photoshop | Adobe | adobe.com |
| Illustrator | Adobe | adobe.com |
| Peptides and oligonucleotides | ||
| Coronin peptide for antibody | GenScript | CLPAKKAGNILNKPR |
| TOM70-HA | S. Munro lab | |
| qPCR primers | ||
| Sqh(MRLC) | QuantiTect | Cat# QT00499065 |
| Rh3 | QuantiTect | Cat# QT00978481 |
| DPod1 set1 | QuantiTect | Cat# QT00499464 |
| DPod1 set2 | Eurofins | 5′-TCCTCACCAAGAACCACTGC |
| Eurofins | 5′-GTGGGTGGGAACAGATCGTC | |
| Coronin set1 | QuantiTect | Cat# QT00940737 |
| Coronin set2 | Eurofins | 5′-ACAGGCTTCAACCGTAGCTC |
| Eurofins | 5′-GAACATTACGCCGTTGGACG | |
| Cortactin set1 | QuantiTect | Cat# QT00979020 |
| Cortactin set2 | Eurofins | 5′-TTCGGAGTGCAAGAGGATCG |
| Eurofins | 5′-GCACTCCAAATTTGCCTCCG | |
| Arp14D | QuantiTect | Cat# QT00923419 |
| ArpC1(sop2) | QuantiTect | Cat# QT00936222 |
| Dia set1 | QuantiTect | Cat# QT00939477 |
| Dia set2 | Eurofins | 5′-CAAATCGAAGGAGGAGCGACA |
| Eurofins | 5′-CCCATTCTGCAGGTATTCCAC | |
| Wasp set1 | QuantiTect | Cat# QT00984641 |
| Wasp set2 | Eurofins | 5′-ATGGCATGGAGGTGGTCAAG |
| Eurofins | 5′-TTACGCGTCTCTATGGTGGC | |
| Scar set1 | QuantiTect | Cat# QT00934584 |
| Scar set2 | Eurofins | 5′-ACGATCCATAGAACCCGTGC |
| Eurofins | 5′-GGCGAATGATGTTCGTCAGC | |
| Carmil set1 | Eurofins | 5′-CCACTGGTGGGTCGTAAGTC |
| Eurofins | 5′-GGCATAGACGTCTCCTCAGC | |
| Carmil set2 | Eurofins | 5′-GCTGAGGAGACGTCTATGCC |
| Eurofins | 5′-ATAACACTACCCTCGCCTGC | |
| Wash | Eurofins | 5′-GCGTAGGAAGAGTGTGGGAC |
| Eurofins | 5′-GTGATGGAATTGCGCTCGTC | |
| Guide RNAs for CRISPR | ||
| Arp3:GFP | ||
| chiRNA1 | Eurofins | 5′-CTTCGCTATCAGGTGTGTCACACGA |
| Eurofins | 5′-AAACTCGTGTGACACACCTGATAGC | |
| chiRNA2 | Eurofins | 5′-CTTCGCCAGTTCAACCCCCTATCTA |
| Eurofins | 5′-AAACTAGATAGGGGGTTGAACTGGC | |
| GFP:Cortactin | ||
| chiRNA1 | Eurofins | 5′-CTTCGGGGCCGACAAAGCCGGATC |
| Eurofins | 5′-AAACGATCCGGCTTTGTCGGCCCC | |
| chiRNA2 | Eurofins | 5′-CTTCGGTGGCCTGAATCTGGTGAC |
| Eurofins | 5′-AAACGTCACCAGATTCAGGCCACC | |
| GFP:DPod1 | ||
| chiRNA1 | Eurofins | 5′-CTTCGAGCGACTGAGAGGGAGCCAC |
| Eurofins | 5′-AAACGTGGCTCCCTCTCAGTCGCTC | |
| chiRNA2 | Eurofins | 5′-CTTCGCGATGTTGTTACCGTACGTC |
| Eurofins | 5′-AAACGACGTACGGTAACAACATCGC | |
| DPod1 mutated PAM sites in homologous constructs | This study | 5′-CCACCGGACTAGTGACACTCGAC 5′-GCAGCGCACAACTGACACTCGAC |
| This study | 5′-GTGGGCAGCTACCAGACGTACGG 5′-GTGGGCAGTTATCAAACCTATGG | |
