Functional exploration of heterotrimeric kinesin-II in IFT and ciliary length control in Chlamydomonas
- MOE Key Laboratory of Protein Sciences, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, China
- Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, China
- Living Systems Institute, University of Exeter, United Kingdom
Figures
Figure 1 with 3 supplements
Requirement of the heterotrimeric organization of CrKinesin-II for IFT (See also Figure 1—figure supplements 1–3).
(A) Schematic diagram of recombinant CrKinesin-II for expression/purification. (B) Overview of chimeric CrKinesin-II constructs with two identical motor domains. The motor domain of FLA10 was …
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Figure 1—source data 1
Movies and numerical data for Figure 1C.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig1-data1-v2.zip
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Figure 1—source data 2
Movies and numerical data for Figure 1E.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig1-data2-v2.zip
Figure 1—figure supplement 1
Purification of recombinant CrKinesin-II and chimeric Crkinesin-II with identical motor domains.
(A) Strategy for expression and purification CrKinesin-II. (B) Coomassie-stained SDS-PAGE of purified FLA10/FLA8 from an MBP column (left) and KAP from a Ni column (right). (C) Elution profiles of …
Figure 1—figure supplement 2
Cells expressing chimeric CrKinesin-II with identical motor domains of FLA10 form normal cilia.
(A) HA-tagged FLA10’ (motor domain of FLA10 with FLA8 carboxyl tail) was transformed into an aflagellate mutant fla8. The transformants were expected to form a chimeric motor with two identical …
Figure 1—figure supplement 3
Self-dimerization of FLA10 and FLA8.
(A–B) Self-dimerization of FLA10 and FLA8 is not affected by the level of protein expression. Cells were transfected with 7.5-fold differences in the amount of plasmid DNA (high and low as …
Figure 2 with 1 supplement
Chimeric CrKinesin-IIs with motor domains of HsKinesin-II function in vitro and in vivo (see also Figure 2—figure supplement 1).
(A) Overview of chimeric CrKinesin-II constructs. The motor domains of FLA10, FLA8 or both in CrKinesin-II were replaced with their counterparts of HsKinesin-II, respectively. (B) In vitro motility …
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Figure 2—source data 1
Movies and numerical data for Figure 2B.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig2-data1-v2.zip
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Figure 2—source data 2
Representative cell images and numerical data for Figure 2D.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig2-data2-v2.zip
Figure 2—figure supplement 1
Purification of recombinant HsKinesin-II and chimeric CrKinesin-II with motor domains of HsKinesin-II.
(A–C) Expression and purification of recombinant HsKinesin-II. Strategy of expression and purification (A). Purified kinesin-II was subjected to SDS-PAGE analysis followed by coomassie blue staining …
Figure 3
Chimeric KIF3B’/FLA10 motor leads to significant reduction in IFT injection rate but slight decrease in ciliary length.
(A) Velocities of FLA8-YFP and KIF3B’-YFP. The anterograde velocities of FLA8-YFP and KIF3B’-YFP that were expressed respectively in fla8 cells were assayed using TIRF microscopy. ****p<0.0001; …
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Figure 3—source data 1
Movies and numerical data for Figure 3A.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig3-data1-v2.zip
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Figure 3—source data 2
Movies and numerical data related to Figure 3B,C and F.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig3-data2-v2.zip
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Figure 3—source data 3
Numerical data for Figure 3E.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig3-data3-v2.xlsx
Figure 4 with 3 supplements
Mathematical modeling predicts a nonlinear scaling relationship between motor velocity and ciliary length (See also Figure 4—figure supplements 1–3).
(A) A cartoon schematic of kinesin-II mediated IFT in cilia. (B) Simulated kinetics of ciliary assembly during ciliary regeneration. (C) Kinetics of ciliary assembly. Cells were deflagellated by pH …
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Figure 4—source data 1
Representative cells images and numerical data for Figure 4C, and numerical data for Figure 4F.
- https://cdn.elifesciences.org/articles/58868/elife-58868-fig4-data1-v2.zip
Figure 4—figure supplement 1
Modeling (case 1): the effect of diffusion constant and the number of motors on ciliary length and the timescale to reach final ciliary length.
(A–B) Contour plots of the steady-state cilium length when N, the total number of motors, is varied with the rate of diffusion (D) at two different motor speeds, when only motors are limiting (case …
Figure 4—figure supplement 2
Modeling (case 2): the effect of diffusion constant and the number of motors on ciliary length and the timescale to reach final ciliary length.
(A–B) Contour plots of the steady-state cilium length when N, the total number of motors, is varied with the rate of diffusion (D) at two different motor speeds, when motors and tubulin are both …
Figure 4—figure supplement 3
Modeling (case 3): the effect of diffusion constant and the number of motors on ciliary length and the timescale to reach final ciliary length.
(A–B) Contour plots of the steady-state cilium length when N, the total number of motors, is varied with the rate of diffusion (D) at two different motor speeds, when only motors are limiting and …
Author response image 1
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (C. reinhardtii, mt+) | 21gr | Chlamdyomonas Resource Center | CC-1690 | |
| Strain, strain background (C. reinhardtii, mt+) | fla8 | Chlamdyomonas Resource Center | CC-829 | |
| Strain, strain background (C. reinhardtii, mt+) | fla10-2 | Chlamdyomonas Resource Center | CC-4180 | |
| Strain, strain background (C. reinhardtii, mt+) | FLA10/FLA10’ | This study | fla8 transformed with a chimeric FLA10’ with FLA10 motor domain and FLA8 tail domain | |
| Strain, strain background (C. reinhardtii, mt+) | FLA10/FLA10’::IFT46-YFP | This study | FLA10/FLA10’ strain expressing IFT46-YFP | |
| Strain, strain background (C. reinhardtii, mt+) | ift46::IFT46-YFP | This study | ift46 rescue strain expressing IFT46-YFP | |
| Strain, strain background (C. reinhardtii, mt+) | wt::IFT46-YFP | This study | Wild-type 21gr strain expressing IFT46-YFP | |
| Strain, strain background (C. reinhardtii, mt+) | fla8::FLA8-YFP | This study | fla8 rescue strain expressing FLA8-YFP | |
| Strain, strain background (C. reinhardtii, mt+) | fla8::FLA8-HA | This study | fla8 rescue strain expressing FLA8-HA | |
| Strain, strain background (C. reinhardtii, mt+) | fla8::KIF3B’-YFP | This study | fla8 transformed with a YFP tagged chimeric KIF3B’ having KIF3B motor domain and FLA8 tail domain | |
| Strain, strain background (C. reinhardtii, mt+) | fla8::KIF3B’-HA | This study | fla8 transformed with an HA-tagged chimeric KIF3B’ having KIF3B motor domain and FLA8 tail domain | |
| Strain, strain background (C. reinhardtii, mt+) | fla8::FLA8-HA/IFT46-YFP | This study | fla8::FLA8-HA strain expressing IFT46-YFP | |
| Strain, strain background (C. reinhardtii, mt+) | fla8::KIF3B’-HA/IFT46-YFP | This study | fla8::KIF3B’-HA strain expressing IFT46-YFP | |
| Cell line (H. sapiens) | HEK293T | ATCC | ATCC CRL-3216 | |
| Cell line (S. frugiperda) | Sf9 | Expression Systems | Sf9 | |
| Transfected construct (S. frugiperda) | pOCC8-KAP-EGFP-His | This study | Junmin Pan’s lab | |
| Transfected construct (S. frugiperda) | pOCC8-KAP3-EGFP-His | This study | Same as above | |
| Transfected construct (S. frugiperda) | pOCC25-FLA8-RFP | This study | Same as above | |
| Transfected construct (S. frugiperda) | pOCC25-KIF3B-RFP | This study | Same as above | |
| Transfected construct (S. frugiperda) | pOCC25-KIF3B’-RFP | This study | Same as above | |
| Transfected construct (S. frugiperda) | pOCC52-FLA10-MBP | This study | Same as above | |
| Transfected construct (S. frugiperda) | pOCC52-KIF3A-MBP | This study | Same as above | |
| Transfected construct (S. frugiperda) | pOCC52-KIF3A’-MBP | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-FLA8-EGFP | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-FLA10-EGFP | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-FLA8-MBP | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-FLA10-MBP | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-FLA8 | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-FLA10 | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-KAP | This study | Same as above | |
| Transfected construct (H. sapiens) | pCMV-EGFP (pEGFP-C3) | Addgene | pEGFP-C3 | |
| Antibody | Anti-HA (rat) | Roche | clone 3F10 | 1:1000 |
| Antibody | Anti-MBP (mouse) | CMCTAG | AT0030 | 1:1000 |
| Antibody | Anti-GFP (rabbit) | Abmart | M20004S | 1:2000 |
| Antibody | Anti–α-tubulin (mouse) | Sigma-Aldrich | T6199 | 1:3000 |
| Antibody | Anti-IC69 (mouse) | Sigma-Aldrich | D6168 | 1:20000 |
| Antibody | Anti-IFT140 (rabbit) | Zhu et al., 2017a | 1:2000 | |
| Antibody | Anti-IFT144 (rabbit) | Zhu et al., 2017a | 1:2000 | |
| Antibody | Anti-IFT46 (rabbit) | Lv et al., 2017 | 1:2000 | |
| Antibody | Anti-IFT38 (rabbit) | This study | 1:3000 | |
| Antibody | Anti-IFT57 (rabbit) | This study | 1:2000 | |
| Antibody | Anti-IFTD1bLIC (rabbit) | Meng and Pan, 2016 | 1:1000 | |
| Antibody | Anti-FLA8 (rabbit) | Liang et al., 2014 | 1:3000 | |
| Antibody | Anti-FLA10 (rabbit) | Cole et al., 1998 | 1:3000 | |
| Antibody | Anti-KAP (rabbit) | Liang et al., 2014 | 1:3000 | |
| Antibody | Anti-KIF3A (rabbit) | Abcam | ab11259 | 1:2000 |
| Antibody | Anti-KIF3B (rabbit) | Abcam | ab89278 | 1:500 |
| Antibody | Anti-KIFAP3 (KAP3) (rabbit) | Abcam | ab133537 | 1:5000 |
| Antibody | Anti-Mouse IgG (H and L)-HRP Conjugated (goat) | EasyBio | BE0102 | 1:200 |
| Antibody | Anti-Rabbit IgG (H and L)-HRP Conjugated (goat) | EasyBio | BE0101 | 1:200 |
| Antibody | Anti-Rat IgG (H+L), HRP (goat) | EasyBio | BE0108 | 1:200 |
| Antibody | Alexa Fluor 647–conjugated anti-mouse IgG (goat) | Molecular probes | A21235 | 1:200 |
| Antibody | Alexa Fluor 594–conjugated anti-rabbit IgG (goat) | Molecular probes | A11037 | 1:200 |
| Ahemical compound, drug | Ni-NTA Agarose | Qiagen | 30210 | |
| Chemical compound, drug | Amylose Resin | NEB | E8021 | |
| Chemical compound, drug | Anti-GFP Magarose Beads | Smart-life sciences | SM03801 | |
| Chemical compound, drug | Mini-Complete (EDTA-free) | Roche | 4693132001 | |
| Chemical compound, drug | MG-132 | Selleck | S2619 | |
| Chemical compound, drug | MG-101 (ALLN) | Selleck | S7386 | |
| Chemical compound, drug | Poly-lysine | Sigma-Aldrich | P8920 | |
| Software, algorithm | ImageJ | NIH Image | https://imagej.nih.gov/ | |
| Software, algorithm | GraphPad Prism 7 | GraphPad Software | https://www.graphpad.com/ | |
| Software, algorithm | Adobe Illustrator and Photoshop CS6 | Adobe | https://www.adobe.com/Illustrator |
