Ser/Thr kinase Trc controls neurite outgrowth in Drosophila by modulating microtubule-microtubule sliding
- Feinberg School of Medicine, Northwestern University, United States
Figures
Figure 1 with 1 supplement
The kinase Trc regulates neurite outgrowth and microtubule sliding.
(A) Example images of timelapse imaging to measure microtubule sliding in Drosophila S2 cells. Microtubules are shown at time zero in green. Magenta indicates photoconverted region. Photoconverted …
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Figure 1—source data 1
Sliding rates for control, Pav RNAi and Trc RNAi-treated S2 cells.
Related to Figure 1A and B.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig1-data1-v2.xlsx
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Figure 1—source data 2
Neurite length for neurons cultured from control larvae or Trc RNAi larvae.
Related to Figure 1C and D.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig1-data2-v2.xlsx
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Figure 1—source data 3
Sliding rates for control, and Trc RNAi neurons.
Related to Figure 1E and F.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
Domain structure of Pavarotti and demonstration of Trc knockdown.
(A) Schematic showing domain structure and location of proposed Trc phosphorylation site inDrosophila Pavarotti and human MKLP1. (B) Lysates from S2 cells demonstrate efficient knockdown of Trc with …
Figure 2 with 1 supplement
Trc regulates microtubule sliding in kinase-dependent manner.
(A) Sliding experiments in S2 cells show increased sliding upon depletion of Trc with dsRNA targeting the non-coding region. The effect can be rescued with overexpression of WT Trc, but not kinase …
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Figure 2—source data 1
Microtubule sliding rates in S2 cells for Trc knockdown and rescue experiments.
Related to Figure 2A and B.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig2-data1-v2.xlsx
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Figure 2—source data 2
Microtubule sliding rates in S2 cells for control and Fry knockdown S2 cells.
Related to Figure 2C and D.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig2-data2-v2.xlsx
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Figure 2—source data 3
Sliding rates for control and Fry RNAi neurons.
Related to Figure 2E and F.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Confirming Fry knockdown.
(A) Western Blot from S2 lysate confirms Trc is knocked down upon treatment with dsRNA targeting a non-coding region. Trc levels remain unchanged upon treatment with Fry dsRNA. (B) Example images …
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Figure 2—figure supplement 1—source data 1
Fry immunostaining intensities.
Related to Figure 2—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 2 supplements
Trc regulates microtubule sliding via phosphorylation of Pavarotti.
(A) Sliding experiments in S2 cells show a decrease in microtubule sliding with Trc overexpression. Trc overexpression in conjunction with depletion of Pavarotti increases sliding beyond control …
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Figure 3—source data 1
Microtubule sliding rates for control, Pav knockdown and Trc overexpressing cells.
Related to Figure 3A and B.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig3-data1-v2.xlsx
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Figure 3—source data 2
Microtubule sliding rates for Pavarotti knockdown and rescue experiments.
Related to Figure 3C and D.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig3-data2-v2.xlsx
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Figure 3—source data 3
Pavarotti localization in control and Trc RNAi-treated cells.
Related to Figure 3E and F.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig3-data3-v2.xlsx
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Figure 3—source data 4
Pavarotti WT and S745A localization.
Related to Figure 3G and H.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig3-data4-v2.xlsx
Figure 3—figure supplement 1
Pavarotti knockdown and rescue.
(A) Western blot of S2 cell lysate confirming Pavarotti knockdown with dsRNA targeting a non-coding region. (B) Immunofluorescence showing expression of WT or S745A GFP Pavarotti. (C) Quantification …
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Figure 3—figure supplement 1—source data 1
Pavarotti WT and S745A expression levels.
Related to B and C.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Method for determining Pav-positive microtubule area.
Figure 4 with 1 supplement
Phospho-Pavarotti brakes microtubule sliding via interaction with 14-3-3 proteins.
(A) Western blot from HEK cell lysate showing co immunoprecipitation of Pavarotti and 14-3-3. The interaction is lost upon mutation of S745 to Alanine and increased upon co-expression of the kinase …
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Figure 4—source data 1
Sliding rates for 14-3-3 RNAi and Pavarotti overexpression experiments.
Related to Figure 4B and C.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig4-data1-v2.xlsx
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Figure 4—source data 2
Pavarotti localization with 14-3-3 RNAi.
Related to Figure 4D and E.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Confirming GFP Pavarotti expression.
Expression of GFP Pavarotti can be detected after photoconversion of EOS tubulin. GFP Pavarotti does not convert upon exposure to UV light so is still visible in the 488 channel. See within …
Figure 5 with 1 supplement
Pavarotti and Trc act in the same pathway to control dendrite outgrowth in vivo.
(A) Example images from timelapse imaging of photoconverted microtubules in neurons under control conditions or upon Pavarotti, Trc or Pavarotti and Trc depletion. Scale bar = 10 µm (B) …
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Figure 5—source data 1
Sliding rates in neurons upon Trc, Pavarotti, or Trc and Pavarotti knockdown.
Related to Figure 5A and B.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig5-data1-v2.xlsx
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Figure 5—source data 2
Dendritic length of DA neurons.
Related to Figure 5C and D.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
Analysis of branch points per cell in DA neurons.
(A) Western blot of Drososphila 3rd instar larvae brain lysate showing dual knockdown of Trc and Pavarotti driven by elav Gal4. (B) Quantification of number of branch points per DA neuron. Ctrl = 221…
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Figure 5—figure supplement 1—source data 1
Number of branch points of DA neurons.
Related to B.
- https://cdn.elifesciences.org/articles/52009/elife-52009-fig5-figsupp1-data1-v2.xlsx
Figure 6
Kinesin-1 slides microtubules along one another to facilitate neurite outgrowth.
The kinase Trc inhibits this process by phosphorylation of Pavarotti – a part of the centralspindlin complex with tumbleweed. Phosphorylated Pavarotti forms a complex with 14-3-3 proteins and …
Videos
Video 1
Pavarotti and Trc RNAi increase microtubule sliding in S2 cells.
Timelapse imaging of photoconverted microtubules in Drosophila S2 cells under control conditions or with depletion of Pavarotti or Trc. Microtubules were labeled with tdEOS alpha Tubulin 84b. 1 …
Video 2
Trc depletion increases microtubule sliding in neurons.
Timelapse imaging of photoconverted microtubules in Drosophila cultured neurons under control conditions or with depletion of Trc. Microtubules were labeled with UAS tdMaple alpha Tubulin 84b under …
Video 3
Microtubule sliding in S2 cells is dependent upon Trc kinase activity.
Timelapse imaging of photoconverted microtubules in Drosophila S2 cells under control conditions or with depletion of Trc using dsRNA against a non-coding region. Expression of WT Trc can rescue the …
Video 4
Fry depletion increases microtubule sliding in S2 cells.
Timelapse imaging of photoconverted microtubules in Drosophila S2 cells under control conditions or with depletion of Furry (Fry). Microtubules were labeled with tdEOS alpha Tubulin 84b. 1 frame per …
Video 5
Fry depletion increases microtubule sliding in neurons.
Timelapse imaging of photoconverted microtubules in Drosophila cultured neurons under control conditions or with depletion Fry. Microtubules were labeled with UAS tdMaple alpha Tubulin 84b under …
Video 6
Trc requires Pavarotti to inhibit microtubule sliding in S2 cells.
Timelapse imaging of photoconverted microtubules in Drosophila S2 cells. Trc overexpression inhibits microtubule sliding only in the presence of Pavarotti. Microtubules were labeled with tdEOS alpha …
Video 7
Pavarotti phosphorylation at Ser745 is required to inhibit sliding.
Timelapse imaging of photoconverted microtubules in Drosophila S2 cells under control conditions or with depletion of Pavarotti using dsRNA against a non-coding region. Expression of WT Pav can …
Video 8
14-3-3 proteins are required for Pavarotti to inhibit microtubule sliding.
Timelapse imaging of photoconverted microtubules in Drosophila S2 cells. Pavarotti can inhibit sliding only in the presence of 14-3-3 proteins. Microtubules were labeled with tdEOS alpha Tubulin …
Video 9
Pavarotti and Trc control microtubule sliding together in neurons.
Timelapse imaging of photoconverted microtubules in Drosophila cultured neurons under control conditions or with depletion Fry. Microtubules were labeled with UAS tdMaple alpha Tubulin 84b under …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | w; elav-Gal4 (III) | C. Doe University of Oregon | ||
| Genetic reagent (D. melanogaster) | yw; wg(Sp)/CyO;Dr(Mio)/TM3, Sb | E. Ferguson, University of Chicago | ||
| Genetic reagent (D. melanogaster) | yw; ppk-CD4-tdtomato (II) | Bloomington Drosophila Stock Center (BDSC) | Stock number 35844; FBst0035844; RRID:BDSC_35844 | Fly base genotype: w1118; P{ppk-CD4-tdTom}4a |
| Genetic reagent (D. melanogaster) | w; D42-Gal4 (III) | BDSC | Stock number 8816; FBst0008816; RRID:BDSC_8816 | Fly base genotype: w*; P{GawB}D42 |
| Genetic reagent (D. melanogaster) | y sc v; UAS-Trc-RNAi | BDSC | Stock number 41591; FBst0041591; RRID:BDSC_41591 | Fly base genotype y1 sc*v1 sev21; P{TRiP.GL01127}attP2 |
| Genetic reagent (D. melanogaster) | y sc v; UAS-Pav-RNAi | BDSC | Stock number 42573; FBst0042573; RRID:BDSC_42573 | Fly base genotype: y1 v1; P{TRiP.HMJ02232}attP40 |
| Genetic reagent (D. melanogaster) | y sc v; UAS-Fry-RNAi | BDSC | Stock number 60103; FBst0060103; RRID:BDSC_60103 | Fly Base Genotype: y1 sc*v1 sev21; P{TRiP.HMC05097}attP40 |
| Genetic reagent (D. melanogaster) | w; UASp-tdMaple3-alpha tubulin 84b | This lab | Described in Lu et al. (2016) Generated by injection of pUASp tdMaple3-alpha tubulin 84b. A second chromosome insertion was used in this study. | |
| Genetic reagent (D. melanogaster) | yw; UASp-tdMaple3-alpha tubulin 84b; UAS-Trc-RNAi | This lab | Generated using an insertion on the second chromosome | |
| Transfected construct (D. melanogaster) | pMT EOS tubulin | This lab | Described in Barlan et al. (2013). For S2 cell expression of EOS tubulin. | |
| Transfected construct (D. melanogaster) | pMT-BFP Trc WT | This lab | S2 cell expression of Trc. Generated from pUASt-Trc WT using EcoRI and NotI, a kind gift from P. Adler. | |
| Transfected construct (D. melanogaster) | pMT-BFP Trc CA (T253E) | This lab | S2 cell expression of Trc. Generated from pUASt-Trc T253E (a kind gift from P. Adler) and pMT vector using EcoRI and NotI. | |
| Transfected construct (D. melanogaster) | pMT-BFP Trc kinase dead (K122A) | This lab | S2 cell expression of Trc. Generated from pUASt-Trc K122A (a kind gift from P. Adler) and pMT vector using EcoRI and NotI. | |
| Transfected construct (D. melanogaster) | pMT-GFP Pavarotti | This lab | S2 cell expression. Generated from pMT-BFP Pavarotti (Del Castillo et al., 2015) | |
| Transfected construct (D. melanogaster) | pMT-GFP Pavarotti S745A | This lab | S2 cell expression. Generated by site directed mutagenesis of WT. | |
| transfected construct (D. melanogaster) | pEGFP Pavarotti WT | This lab | Mammalian expression. Pavarotti constructs were subcloned into pEGFP-C1 using EcoRI and SalI. | |
| Transfected construct (D. melanogaster) | pEGFP Pavarotti S745A | This lab | Mammalian expression. Pavarotti constructs were subcloned into pEGFP-C1 using EcoRI and SalI. | |
| transfected construct (D. melanogaster) | pcDNA Trc T253E | This lab | Mammalian expression. Trc T253E was subcloned into pcDNA 3.1+ using HindIII and NotI. | |
| Transfected construct (D. melanogaster) | pMT mCherry tubulin | This lab | For S2 cell expression. Described in del Castillo et al. (2015) | |
| Cell line (D. melanogaster) | S2 cells | DGRC | FlyBase Report: FBtc0000006 | Cell line maintained in this lab. |
| Cell line (Homo-sapiens) | HEK 293 FT | ATCC | RRID:CVCL_0045; ATCC: CRL-1573 | Cell line maintained in this lab. |
| Antibody | Single chain anti-GFP, GFP-Trap-M | Chromotek | ||
| Antibody | Anti Pavarotti (Rabbit polyclonal) | Scholey lab | Western blot 1:1000 | |
| Antibody | Anti Trc (Rabbit polyclonal) | Emoto lab | Western blot 1:1000 | |
| Antibody | Anti Tubulin (Rabbit polyclonal) | This lab | Affinity purified from immunized rabbit serum. Western blot 1:1000 | |
| Antibody | Anti phospho-Pavarotti S710 (Rabbit polyclonal) | Mishima lab | Western blot 1:1000 | |
| Antibody | Anti GFP (Rabbit polyclonal) | This lab | Affinity purified from immunized rabbit serum. Western blot 1:1000 | |
| Antibody | Anti Furry (Rabbit polyclonal) | Adler lab | Immunofluorescence 1:250 | |
| Antibody | Anti Hsc 70 (Goat polyclonal) | Santa cruz | K-19, RRID:AB_2120291 | Western blot 1:100 |
| Antibody | Anti 14-3-3ζ (Rabbit polyclonal) | Proteintech group | cat no. 14503–1-AP; RRID:AB_2218096 | Western blot 1:250 |
| Antibody | HRP conjugated Anti Rabbit | Jackson | Western blot 1:10,000 | |
| Antibody | HRP conjugated Anti Goat | Jackson | Western blot 1:10,000 |
Additional files
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Source code 1
IJM macro used for sliding analysis.
- https://cdn.elifesciences.org/articles/52009/elife-52009-code1-v2.txt
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Supplementary file 1
Primer sequences used for dsRNA generation.
- https://cdn.elifesciences.org/articles/52009/elife-52009-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52009/elife-52009-transrepform-v2.docx
