Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Germany
- Department of Pathology and Cell Biology, Columbia University Medical Center, United States
- Faculty of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Germany
- NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Germany
Figures
Figure 1 with 1 supplement
Actin dynamics and actin-nucleating mDia1/3 proteins facilitate synaptic vesicle (SV) endocytosis.
(A) Averaged normalized Synaptophysin-pHluorin (Syph-pH) fluorescence traces from transfected hippocampal neurons stimulated with 200 action potentials (APs) (40 Hz, 5 s) at physiological …
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Figure 1—source data 1
Numerical source data for Figure 1A–L.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig1-data1-v2.zip
Figure 1—figure supplement 1
Role of formins and mDia1/3 in synaptic vesicle (SV) endocytosis.
(A) Maxima of background-corrected Synaptophysin-pHluorin (Syph-pHluorin) fluorescence traces (surface normalized) for neurons treated with 0.1% dimethyl sulfoxide (DMSO) (1.7 ± 0.1) or JLY cocktail …
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Figure 1—figure supplement 1—source data 1
Numerical source data of Figure 1—figure supplement 1A, B, C, D, E, F, G, I, J, K, L, M, O.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Original scan for the anti-mDia1 immunoblot from Figure 1—figure supplement 1H.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig1-figsupp1-data2-v2.zip
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Figure 1—figure supplement 1—source data 3
Original scan for the anti-mDia3 immunoblot from Figure 1—figure supplement 1H.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig1-figsupp1-data3-v2.zip
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Figure 1—figure supplement 1—source data 4
Original scan for the anti-tubulin immunoblot from Figure 1—figure supplement 1H.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig1-figsupp1-data4-v2.zip
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Figure 1—figure supplement 1—source data 5
Original scans for immunoblots from Figure 1—figure supplement 1H with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig1-figsupp1-data5-v2.zip
Figure 2 with 1 supplement
Loss of mDia1/3 causes an activity-dependent reduction of the synaptic vesicle (SV) pool.
(A) Representative synaptic electron micrographs from hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3, targeting Diaph1/2 genes. Postsynapse and postsynaptic cleft are …
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Figure 2—source data 1
Numerical source data for Figure 2C, D and G.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig2-data1-v2.zip
Figure 2—figure supplement 1
Synaptic vesicle (SV) depletion in mDia1/3-depleted neurons is activity-dependent.
(A) Representative synaptic electron micrographs from neurons transduced with shCTR or shmDia1 +3 and treated with 0.1% Vehicle or 1 µM TTX for 36 hr before fixation. Postsynapse and postsynaptic …
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Figure 2—figure supplement 1—source data 1
Numerical source data of Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig2-figsupp1-data1-v2.zip
Figure 3 with 1 supplement
mDia1 associates with endocytic proteins and localizes to presynaptic sites.
(A) Schematic representation of functional domains of mDia1. Rho-binding domain (RBD), Diaphanous inhibitory domain (DID), Dimerization domain (DD), Coiled coil domain (CC), Formin homology domain 1 …
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Figure 3—source data 1
Numerical source data for Figure 3B, D, E, H and K.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-data1-v2.zip
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Figure 3—source data 2
Original scan for the anti-mDia1 and anti-Dynamin1 immunoblots from Figure 3F.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-data2-v2.zip
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Figure 3—source data 3
Original scan for the anti-Actin immunoblot from Figure 3F.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-data3-v2.zip
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Figure 3—source data 4
Original scans for immunoblots from Figure 3F with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-data4-v2.zip
Figure 3—figure supplement 1
mDia1 binds membranes and localizes to presynaptic endocytic sites.
(A) Membrane levels of mDia1-WT-mCherry versus mDia1-ΔN-mCherry proteins overexpressed in HEK293T cells. Membrane and cytosolic cellular fractions were isolated by ultracentrifugation and analyzed …
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Figure 3—figure supplement 1—source data 1
Original scan for in-gel mCherry fluorescence from Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 1—source data 2
Original scan for the anti-LAMP1 immunoblot from Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data2-v2.zip
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Figure 3—figure supplement 1—source data 3
Original scans from Figure 3—figure supplement 1A with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data3-v2.zip
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Figure 3—figure supplement 1—source data 4
Numerical source data of Figure 3—figure supplement 1B, C, D, G, I, J.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data4-v2.zip
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Figure 3—figure supplement 1—source data 5
Original scans for mCherry fluorescence in gels used for analysis are shown in Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data5-v2.zip
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Figure 3—figure supplement 1—source data 6
Original scans for mCherry fluorescence in gels used for analysis are shown in Figure 3—figure supplement 1B with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data6-v2.zip
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Figure 3—figure supplement 1—source data 7
Original scan for the anti-mDia1 immunoblot from Figure 3—figure supplement 1E.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data7-v2.zip
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Figure 3—figure supplement 1—source data 8
Original scan for the anti-Myosin IIB immunoblot from Figure 3—figure supplement 1E.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data8-v2.zip
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Figure 3—figure supplement 1—source data 9
Original scan for the anti-Actin immunoblot from Figure 3—figure supplement 1E.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data9-v2.zip
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Figure 3—figure supplement 1—source data 10
Original scans for immunoblots in Figure 3—figure supplement 1E with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig3-figsupp1-data10-v2.zip
Figure 4 with 1 supplement
mDia facilitates synaptic vesicle (SV) endocytosis by regulating presynaptic F-actin.
(A) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures transduced with shCTR or shmDia1 +3, targeting Diaph1/2 genes, fixed and …
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Figure 4—source data 1
Numerical source data for Figure 4B, C, D, E, G and H.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig4-data1-v2.zip
Figure 4—figure supplement 1
mDia1 regulates presynaptic actin and synaptic vesicle (SV) endocytosis.
(A) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures treated with 0.1% dimethyl sulfoxide (DMSO) or 80 µM Dynasore for 10 …
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Figure 4—figure supplement 1—source data 1
Numerical source data of Figure 4—figure supplement 1C, D, E, F, H.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
RhoA/B facilitates presynaptic endocytosis and are regulated by mDia1/3.
(A) Schematic representation of activation of mDia1 by RhoA-GTP and positive feedback loop of mDia1 on RhoA-GTP levels through GEF stimulation. (B) Representative three-channel time-gated STED image …
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Figure 5—source data 1
Numerical source data for Figure 5C, D and F.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data1-v2.zip
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Figure 5—source data 2
Original scans for the anti-mDia1, anti-Tubulin, anti-RhoA, and anti-mDia3 immunoblots from Figure 5E.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data2-v2.zip
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Figure 5—source data 3
Original scans for immunoblots from Figure 5E with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data3-v2.zip
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Figure 5—source data 4
Original scans for the anti-RhoA immunoblots used for analysis are shown in Figure 5F.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data4-v2.zip
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Figure 5—source data 5
Original scans for immunoblots used for analysis are shown in Figure 5F with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-data5-v2.zip
Figure 5—figure supplement 1
RhoA/B regulates synaptic vesicle (SV) endocytosis.
(A) Averaged normalized Synaptophysin-pHluorin fluorescence traces from stimulated (200 APs; 40 Hz, 5 s) hippocampal neurons transfected with plasmids encoding the indicated combinations of …
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Figure 5—figure supplement 1—source data 1
Numerical source data of Figure 5—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig5-figsupp1-data1-v2.zip
Figure 6 with 1 supplement
mDia1/3-Rho and Rac1 signaling facilitates presynaptic endocytosis.
(A) Schematic of the interplay between RhoA and Rac1 signaling via GTPase regulatory proteins (e.g. GTPase activating proteins (GAPs) among others) common for RhoA and Rac1. (B) Analysis of Rac1 …
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Figure 6—source data 1
Original scans for the anti-mDia3, anti-Tubulin, and anti-Rac1 immunoblots from Figure 6B.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data1-v2.zip
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Figure 6—source data 2
Original scan for the anti-mDia1 immunoblot from Figure 6B.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data2-v2.zip
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Figure 6—source data 3
Original scans for immunoblots from Figure 6B with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data3-v2.zip
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Figure 6—source data 4
Numerical source data for Figure 6C, E, F, G and H.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data4-v2.zip
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Figure 6—source data 5
Original scans for the anti-Rac1 immunoblots used for analysis are shown in Figure 6C.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data5-v2.zip
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Figure 6—source data 6
Original scans for immunoblots used for analysis are shown in Figure 6C with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-data6-v2.zip
Figure 6—figure supplement 1
Cooperative action of mDia1/3 and Rac1 pathways in presynaptic endocytosis.
(A) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal cultures upon inhibition of Rho activity utilizing immobilized PAK as bait. Cells were …
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Figure 6—figure supplement 1—source data 1
Original scan for the anti-Rac1 immunoblots from Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-figsupp1-data1-v2.zip
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Figure 6—figure supplement 1—source data 2
Original scan for the anti-Tubulin immunoblot from Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-figsupp1-data2-v2.zip
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Figure 6—figure supplement 1—source data 3
Original scans for immunoblots in Figure 6—figure supplement 1A with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-figsupp1-data3-v2.zip
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Figure 6—figure supplement 1—source data 4
Numerical source data of Figure 6—figure supplement 1C, D, E, F, G, I, J, K.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-figsupp1-data4-v2.zip
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Figure 6—figure supplement 1—source data 5
Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-figsupp1-data5-v2.zip
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Figure 6—figure supplement 1—source data 6
Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig6-figsupp1-data6-v2.zip
Figure 7 with 1 supplement
Defects in presynaptic ultrastructure induced by interference with mDia1/3-Rho and Rac1 signaling.
(A) Representative synaptic electron micrographs from hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3, targeting Diaph1/2 genes, and treated with 0.1% dimethyl …
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Figure 7—source data 1
Numerical source data from Figure 7B–E.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig7-data1-v2.zip
Figure 7—figure supplement 1
mDia1/3 and Rac1 cooperatively regulate the synaptic vesicle (SV) cycle and presynaptic ultrastructure.
(A) Average number of invaginations per μm2 in WT (0.1±0.1) and Diaph1 KO (mDia1KO; 0.4±0.1; p<0.01, Mann-Whitney test) boutons. Data shown represent the mean ± SEM from nWT = 103 synapses, nmDia1KO …
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Figure 7—figure supplement 1—source data 1
Numerical source data of Figure 7—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/92755/elife-92755-fig7-figsupp1-data1-v2.zip
Author response image 1
Based on this analysis postsynaptic mDia1 levels were also elevated upon Dynasore treatment (new Figure 3-Supplement 1I).
In spite of this and consistent with the fact that the majority of mDia1 is localized at the presynapse, we found that postsynaptic F-actin levels were unchanged in mDia1/3depleted neurons (p = …
Author response image 2
Moreover, we also conducted further analysis with respect to possible effects of Dynasore on synaptic architecture in general.
Neither presynaptic Bassoon nor postsynaptic Homer1 levels were significantly altered by Dynasore treatment (new Figure 3–Supplement 1J).
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | C57BL/6 N wild-type | Leibniz Research Institute for Molecular Pharmacology | RRID:IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | mDia1 KO | Bartolini Lab, Columbia University | Peng et al., 2003; https://doi.org/10.1016/s0960-9822(03)00170–2 | Genetic knockout of Diaph1 |
| Cell line (Homo sapiens) | HEK293T | American Type Culture Collection | Cat# CRL-3216; RRID:CVCL_0063 | |
| Antibody | β-Actin (mouse monoclonal) | Sigma-Aldrich | Cat# A5441; RRID:AB_476744 | IB (1:5000) |
| Antibody | Bassoon (guinea pig monoclonal) | Synaptic Systems | Cat# 141 318; RRID:AB_2927388 | IC (1:100) |
| Antibody | CDC42 (rabbit polyclonal) | Abcam | Cat# ab64533; RRID:AB_1310067 | IB (1:1000); IC (1:100) |
| Antibody | Dynamin1 (rabbit polyclonal) | Pietro D. Camilli | Shupliakov et al., 2002; DOI: 10.1126/science.276.5310.29 | IB (1:2000) |
| Antibody | GFP (mouse monoclonal) | Thermo Fisher Scientific | Cat# A-11120; RRID:AB_221568 | IC (1:2500) |
| Antibody | Homer1 (mouse monoclonal) | Synaptic Systems | Cat# 160 011; RRID:AB_2120992 | IC (1:200) |
| Antibody | Homer1 (rabbit polyclonal) | Synaptic Systems | Cat# 160 003; RRID:AB_887730 | IC (1:200) |
| Antibody | LAMP1 (rabbit monoclonal) | Cell Signaling Technology | Cat# 9091; RRID:AB_2687579 | IB (1:1000) |
| Antibody | mDia1 (mouse monoclonal) | BD Biosciences | Cat# 610848; RRID:AB_398167 | IB (1:500); IP (2 µg) |
| Antibody | mDia1 (rabbit monoclonal) | Abcam | Cat# ab129167; RRID:AB_11143749 | IC (1:100) |
| Antibody | mDia3 (rabbit polyclonal) | Sigma-Aldrich | Cat# HPA005647; RRID:AB_1078657 | IB (1:1000) |
| Antibody | Myosin IIb (mouse monoclonal) | Abcam | Cat# ab684; RRID:AB_305661 | IC (1:100) |
| Antibody | Myosin IIb (rabbit polyclonal) | Cell Signaling Technology | Cat# 3404; RRID:AB_126421 | IB (1:2000) |
| Antibody | Rac1 (mouse monoclonal) | BD Biosciences | Cat# 610650; RRID:AB_397977 | IB (1:1000); IC (1:50) |
| Antibody | RFP (rabbit polyclonal) | Takara Bio | Cat# 632496; RRID:AB_10013483 | IC (1:500) |
| Antibody | RhoA (rabbit monoclonal) | Cell Signaling Technology | Cat# 2117; RRID:AB_10693922 | IB (1:1000); IC (1:100) |
| Antibody | α-Tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T9026; RRID:AB_47759 | IB (1:5000) |
| Antibody | IgG control (mouse monoclonal) | Thermo Fisher Scientific | Cat# 31903; RRID:AB_10959891 | IP (2 μg) |
| Antibody | vGAT-CypHer5E (rabbit polyclonal) | Synaptic Systems | Cat# 131 103CpH; RRID:AB_2189809 | Live-imaging (1:500) |
| Antibody | Anti-mouse IgG Atto542 (donkey polyclonal) | Martin Lehmann | Gonschior et al., 2022; https://doi.org/10.1038/s41467-022-32533-4 | IC (1:400) |
| Antibody | Anti-rabbit IgG Atto542 (donkey polyclonal) | Martin Lehmann | Gonschior et al., 2022; https://doi.org/10.1038/s41467-022-32533-4 | IC (1:400) |
| Antibody | Anti-mouse Alexa Fluor 594 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11032; RRID:AB_2534091 | IC (1:200) |
| Antibody | Anti-rabbit Alexa Fluor 594 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11037; RRID:AB_2534095 | IC (1:200) |
| Antibody | Anti-guinea pig Atto647N (camelid monoclonal) | Synaptic Systems | Cat# N0602-At647N-S; RRID:AB_2744576 | IC (1:200) |
| Antibody | Anti-mouse Alexa Fluor 647 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-21235; RRID:AB_2535804 | IC (1:200) |
| Antibody | Anti-mouse IRDye 680RD (goat polyclonal) | LI-COR Biosciences | Cat# 925–68070; RRID:AB_2651128 | IB (1:10000) |
| Antibody | Anti-rabbit IRDye 680RD (goat polyclonal) | LI-COR Biosciences | Cat# 926–68071; RRID:AB_10956166 | IB (1:10000) |
| Antibody | Anti-mouse IgG, IRDye 800CW (goat polyclonal) | LI-COR Biosciences | Cat# 926–32210; RRID:AB_621842 | IB (1:10000) |
| Antibody | Anti-rabbit IgG, IRDye 800CW (goat polyclonal) | LI-COR Biosciences | Cat# 926–32211; RRID:AB_621843 | IB (1:10000) |
| Chemical compound, drug | Phalloidin-Alexa Fluor 594 | AAT Bioquest | Cat# ABD-23158 | IC (1:1000) |
| Recombinant DNA reagent | Synaptophysin-pHluorin | Leon Lagnado | Gonschior et al., 2022; https://doi.org/10.1038/s41467-022-32533-4 | Expresses rat Synaptophysin-2xpHluorin (inserted between Asn183 – Thr184) under a CMV promotor |
| Recombinant DNA reagent | vGlut1-pHluorin | Volker Haucke | Bolz et al., 2023; https://doi.org/10.1016/j.neuron.2023.08.016 | Expresses rat vGlut1-pHluorin (inserted between Gly99 – Gly100) under a hSyn1 promotor; lentiviral plasmid |
| Recombinant DNA reagent | mCherry | Clontech | Cat# 632523 | Expresses mCherry under a CMV promotor |
| Recombinant DNA reagent | mDia1-WT-mCherry | This study | - | Expresses mouse mDia1-WT-mCherry under a CMV promotor |
| Recombinant DNA reagent | mDia1-ΔN -mCherry | This study | - | Expresses truncation (first 60 AA) variant of mouse mDia1-mCherry under a CMV promotor |
| Recombinant DNA reagent | mDia1-WT-SNAP | This study | - | Expresses mouse mDia1-WT-SNAP under a hSyn1 promotor; lentiviral plasmid |
| Recombinant DNA reagent | mDia1-K994A-SNAP | This study | - | Expresses K994A variant of mDia1-SNAP under a hSyn1 promotor; lentiviral plasmid |
| Recombinant DNA reagent | Dynamin1-WT-SNAP | This study | - | Expresses mouse Dynamin1-WT-SNAP under a hSyn1 promotor; lentiviral plasmid |
| Recombinant DNA reagent | Dynamin1-K44A-SNAP | This study | - | Expresses K44A variant of mouse Dynamin1-SNAP under a hSyn1 promotor; lentiviral plasmid |
| Recombinant DNA reagent | vGlut1-mCherry | Franck Polleux | Kwon et al., 2016; https://doi.org/10.1371/journal.pbio.1002516 | Expresses rat vGlut1-mCherry under a CAG promotor |
| Recombinant DNA reagent | Rac1-CA | Addgene | Cat# 12983; RRID:Addgene_12983 | Expresses Q61L variant of human myc-Rac1 under a CMV promotor |
| Recombinant DNA reagent | Rac1-DN | Addgene | Cat# 12984; RRID:Addgene_12984 | Expresses T17N variant of human myc-Rac1 under a CMV promotor |
| Recombinant DNA reagent | RhoA-WT | Theofilos Papadopoulos | Reddy-Alla et al., 2010; https://doi.org/10.1111/j.1460-9568.2010.07149.x | Expresses human 3xHA-RhoA-WT under a CMV promotor |
| Recombinant DNA reagent | RhoA-DN | Theofilos Papadopoulos | Reddy-Alla et al., 2010; https://doi.org/10.1111/j.1460-9568.2010.07149.x | Expresses T19N variant of human 3xHA-RhoA under a CMV promotor |
| Recombinant DNA reagent | RhoB-WT | Harry Mellor | Mellor, 1998; https://doi.org/10.1074/jbc.273.9.4811 | Expresses human myc-RhoB-WT under a CMV promotor |
| Recombinant DNA reagent | RhoB-DN | Harry Mellor | Mellor, 1998; https://doi.org/10.1074/jbc.273.9.4811 | Expresses T19N variant of human myc-RhoB under a CMV promotor |
| Recombinant DNA reagent | shCTR (transfected) | Sigma-Aldrich | Cat# SHC016 | No murine targets |
| Recombinant DNA reagent | shmDia1 (transfected) | Sigma-Aldrich | Cat# TRCN0000108685 | Targets 3'UTR of NM_007858 |
| Recombinant DNA reagent | shRhoA (transfected) | Sigma-Aldrich | Cat# TRCN0000302388 | Targets CDS of NM_016802 |
| Recombinant DNA reagent | shRhoB (transfected) | Sigma-Aldrich | Cat# TRCN0000294874 | Targets CDS of NM_007483 |
| Recombinant DNA reagent | shCTRmiR | This study | - | Expresses rat Synaptophysin-2xpHluorin under a CMV promotor and shRNA embedded into a microRNA (shRNAmiR) with no murine targets |
| Recombinant DNA reagent | shmDia1miR | This study | - | Expresses rat Synaptophysin-2xpHluorin under a CMV promotor and shRNAmiR against the CDS of mouse Diaph1 (mDia1) |
| Recombinant DNA reagent | shCTR (transduced) | Christian Rosenmund | Watanabe et al., 2014; https://doi.org/10.1038/nature13846 | Expresses NLS-RFP or BFP under a hSyn1 promotor and shRNA against no murine target (msClathrin scrambled) under a U6 promotor |
| Recombinant DNA reagent | shmDia1 (transduced) | This study | - | Expresses NLS-RFP or BFP under a hSyn1 promotor and shRNA against the 3'UTR of mouse Diaph1 (mDia1) under a U6 promotor |
| Recombinant DNA reagent | shmDia3 (transduced) | This study | - | Expresses NLS-RFP or BFP under a hSyn1 promotor and shRNA against the CDS of mouse Diaph2 (mDia3) under a U6 promotor |
| Recombinant DNA reagent | MD2.G | Addgene | Cat# 12259; RRID:Addgene_12259 | Expresses lentiviral VSV-G envelope protein |
| Recombinant DNA reagent | psPAX2 | Addgene | Cat# 12260; RRID:Addgene_12260 | Expresses lentiviral packaging protein |
| Recombinant DNA reagent | pOrange GFP-β-Actin KI | Addgene | Cat#131479; RRID:Addgene_131479 | gRNA and GFP donor for endogenous N-terminal tagging of β-Actin (amino acid position: D2) by targeting Actb |
| Chemical compound, drug | Dynasore | Sigma-Aldrich | Cat# D7693 | 80 μM 10 min |
| Chemical compound, drug | EHT 1864 | MedChemExpress | Cat# HY-16659 | 10 μM acute (CypHer); 2 h (IC/EM) |
| Chemical compound, drug | IMM-01 | Sigma-Aldrich | Cat# SML1064 | 10 μM acute |
| Chemical compound, drug | Jasplakinolide | Sigma-Aldrich | Cat# J4580 | 8 μM acute (JLY); 1 μM 30 min |
| Chemical compound, drug | Latrunculin A | Cayman Chemical | Cat# CAY10684 | 5 µM acute (JLY) |
| Chemical compound, drug | ML141 | MedChemExpress | Cat# HY-12755 | 10 µM acute |
| Chemical compound, drug | Rhosin | MedChemExpress | Cat# HY-12646 | 10 µM 2 h |
| Chemical compound, drug | Tetrodotoxin | Carl Roth | Cat# 6973.1 | 1 µM 36 h |
| Chemical compound, drug | Y-27632 | Tocris | Cat# 1254 | 1 µM acute (JLY) |
| Commercial assay or kit | Rhotekin-Rho binding domain (RBD) beads | Cytoskeleton Inc | Cat# RT02 | 60 μg |
| Commercial assay or kit | PAK-p21 binding domain (PBD) beads | Cytoskeleton Inc | Cat# PAK02 | 20 μg |
| Commercial assay or kit | ProFection Mammalian Transfection System – Calcium Phosphate | Promega | Cat# E1200 | |
| Commercial assay or kit | Gibson Master Mix | New England Biolabs Inc | Cat# E2611L | |
| Commercial assay or kit | Q5 site-directed mutagenesis kit | New England Biolabs Inc | Cat# E0552S | |
| Software, algorithm | Fiji | NIH | RRID:SCR_002285 | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | Version 9.5.1. |
| Software, algorithm | Image Lab Software | Bio-Rad | RRID:SCR_014210 | Version 6.0.1 |
| Software, algorithm | Image Studio Lite | LI-COR Biosciences | RRID:SCR_013715 | Version 5.2.5 |
| Software, algorithm | pHluorin ROI sector | Github | https://github.com/DennisVoll/pHluorin_ROI_selector/; Voll, 2020 | |
| Software, algorithm | SynActJ | Martin Lehmann | Schmied et al., 2021; https://doi.org/10.3389/fcomp.2021.777837 | |
| Software, algorithm | Macro_plot_lineprofile_multicolor | Kees Straatman | Gerth et al., 2017; https://doi.org/10.1016/j.str.2019.03.020 | |
| Software, algorithm | MaxQuant | Jürgen Cox | https://www.maxquant.org/maxquant/ | Version 1.6.1.0 |
| Software, algorithm | Perseus | Jürgen Cox | https://www.maxquant.org/perseus/ | Version 1.6.7.0 |
| Sequence-based reagent | see Supplementary file 1 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/92755/elife-92755-mdarchecklist1-v2.docx
-
Supplementary file 1
List of oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/92755/elife-92755-supp1-v2.xlsx
