CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation
- Division of Structural Medicine and Anatomy, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Japan
- JST, PRESTO, Japan
- RIKEN Center for Biosystems Dynamics Research, Japan
- Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Japan
- RIKEN SPring-8 Center, Japan
- Japan Synchrotron Radiation Research Institute (JASRI), Japan
- Materials and Structural Analysis, Thermo Fisher Scientific, Japan
- Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Japan
Figures
Figure 1 with 1 supplement
Functional study of recombinant calmodulin-regulated spectrin-associated protein 2 (CAMSAP2).
(A) Schematic representation of the full-length CAMSAP2 constructs used in this study. CH, calponin-homology domain; MBD, microtubule-binding domain; CC, coiled-coil domain; CKK, C-terminal domain …
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Figure 1—source data 1
Figure 1 SDS-PAGE gel of the full-length calmodulin-regulated spectrin-associated protein 2.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig1-data1-v1.jpg
Figure 1—figure supplement 1
Size exclusion chromatography with multi-angle light scattering of the calmodulin-regulated spectrin-associated protein 2-FL.
Figure 2 with 1 supplement
Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) stimulates microtubule nucleation.
(A) SDS-PAGE gels from a spin-down spontaneous nucleation assay of tubulin showing the total tubulin after 60 min of polymerization at 37°C (left gel). Polymerized tubulin was pelleted by …
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Figure 2—source data 1
SDS-PAGE gel from a spin-down spontaneous nucleation assay of tubulin showing the total tubulin after 60 min of polymerization at 37°C.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data1-v1.jpg
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Figure 2—source data 2
SDS-PAGE gel of depolymerized tubulin after spin-down pelleting assay.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data2-v1.jpg
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Figure 2—source data 3
SDS-PAGE gel from a spin-down spontaneous nucleation assay of tubulin with 1 µM calmodulin-regulated spectrin-associated protein 2-FL showing the total tubulin after 60 min of polymerization at 37°C.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data3-v1.jpg
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Figure 2—source data 4
SDS-PAGE gel of depolymerized tubulin with 1 µM calmodulin-regulated spectrin-associated protein 2-FL after spin-down pelleting assay.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data4-v1.jpg
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Figure 2—source data 5
Quantification of the depolymerized tubulin concentrations determined by pelleting assay.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data5-v1.docx
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Figure 2—source data 6
SDS-PAGE gel from a spin-down spontaneous nucleation assay of 10 µM tubulin with 10–1000 nM calmodulin-regulated spectrin-associated protein 2-FL in the PEM.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data6-v1.jpg
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Figure 2—source data 7
SDS-PAGE gel from a spin-down spontaneous nucleation assay of 10 µM tubulin with 10–1000 nM calmodulin-regulated spectrin-associated protein 2-FL in the PEM + 100 mM KCl.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data7-v1.jpg
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Figure 2—source data 8
Quantification of the depolymerized tubulin concentrations determined by pelleting assay.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-data8-v1.docx
Figure 2—figure supplement 1
Comparison of pelleting assay.
Comparison of conventional pelleting assay without depolymerization (no deploy) and modified pelleting assay with depolymerization (depoly, which is the same data as shown in Figure 2C).
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Figure 2—figure supplement 1—source data 1
SDS-PAGE gel of tubulin spin-down pelleting assay without depolymerization (no depoly).
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-figsupp1-data1-v1.jpg
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Figure 2—figure supplement 1—source data 2
Quantification of the depolymerized tubulin concentrations determined by pelleting assay.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig2-figsupp1-data2-v1.jpg
Figure 3 with 1 supplement
Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) forms co-condensate with tubulin in vitro.
(A) Intrinsic disorder prediction of CAMSAP2 by PONDR. CH, calponin-homology domain; MBD, microtubule-binding domain; CC, coiled-coil domain; CKK, C-terminal domain common to CAMSAP1 and two other …
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Figure 3—source data 1
Quantification of the fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig3-data1-v1.docx
Figure 3—figure supplement 1
Three different measurements of fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy and quantification.
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Figure 3—figure supplement 1—source data 1
Quantification of the fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig3-figsupp1-data1-v1.pdf
Figure 4 with 1 supplement
Tubulin is incorporated into calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) condensates to form aster-like structure.
(A) The procedure used to obtain the data in panels (B) and (C). GFP-CAMSAP2-FL, 10 μM tubulin, and 0.5 μM tetramethylrhodamine (TMR)-tubulin were mixed in BRB80 supplemented with 100 mM KCl and …
Figure 4—figure supplement 1
Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) localization in growing microtubule networks in HeLa cells.
Cells were monitored before nocodazole treatment (-nocodazole), just after nocodazole treatment (0 min), and 3 min and 35 min incubation at 37°C after washout. Immunostaining of intrinsic CAMSAP2 …
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Figure 4—figure supplement 1—source data 1
Original image of immunofluorescence visualized by a confocal laser-scanning microscope.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig4-figsupp1-data1-v1.jpg
Figure 5 with 3 supplements
Nucleation and aster formation activity of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2).
Representative electron microscopy (EM) images are shown from at least three independent assays. (A) Negative stain EM micrographs of 1 µM CAMSAP2-FL incubated at 25°C for 30 min. (B) Negative stain …
Figure 5—figure supplement 1
Aster formation activity of recombinant calmodulin-regulated spectrin-associated protein 3 (CAMSAP3).
(A) Schematic representation of the GFP-fused CAMSAP3 constructs used in this study. CH, calponin-homology domain; MBD, microtubule-binding domain; CC, coiled-coil domain; CKK, C-terminal domain …
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Figure 5—figure supplement 1—source data 1
SDS-PAGE gels of full-length GFP-CAMSAP3.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig5-figsupp1-data1-v1.jpg
Figure 5—figure supplement 2
Nucleation and aster formation activity of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2).
(A) Negative stain images of 2 µM tubulin polymerized with 1 µM of CAMSAP2-FL after incubation at 37°C for 30 min in PEM. (B) Negative stain images of 10 µM tubulin polymerized with different …
Figure 5—figure supplement 3
The cryo-electron microscopy micrographs of 30 µM tubulin at different conditions.
No microtubule was observed. (Left) 30 µM tubulin preserved on ice for 30 min. (Middle left) Incubated for 1 min, (Middle right) 3 min, and (Right) 10 min at 37°C after 10 min on ice incubation. The …
Figure 6 with 3 supplements
Functional domain mapping of the microtubule nucleation and aster formation activity of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2).
(A) Microtubule nucleation and aster formation activities of CAMSAP2 deletion constructs evaluated by the results of 10 µM tubulin with 1 µM CAMSAP2. The number of ‘+’ symbols indicates the strength …
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Figure 6—source data 1
Quantifications of fluorescent intensity of calmodulin-regulated spectrin-associated protein 2 condensates at 0 s.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig6-data1-v1.pdf
Figure 6—figure supplement 1
GFP fused calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) constructs used in this study.
Size exclusion chromatography of GFP-CAMSAP2-FL (turquoise green), GFP-CC1-CKK (orange), GFP-CC3-CKK (yellow), GFP-CAMSAP2 ∆CC3 (magenta), GFP-CAMSAP2 ∆CKK (blue), and GFP-CKK (green). The peaks of …
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Figure 6—figure supplement 1—source data 1
SDS-PAGE gel of calmodulin-regulated spectrin-associated protein 2 deletion constructs.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig6-figsupp1-data1-v1.jpg
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Figure 6—figure supplement 1—source data 2
SDS-PAGE gel of calmodulin-regulated spectrin-associated protein 2 deletion constructs.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig6-figsupp1-data2-v1.jpg
Figure 6—figure supplement 2
Functional domain mapping of the microtubule nucleation and aster formation activity of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) deletion mutants.
(A) Schematic representation of the GFP fused CAMSAP2 deletion constructs. CH, calponin-homology domain; MBD, microtubule-binding domain; CC, coiled-coil domain; CKK, C-terminal domain common to …
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Figure 6—figure supplement 2—source data 1
SDS-PAGE gel of calmodulin-regulated spectrin-associated protein 2 deletion constructs.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig6-figsupp2-data1-v1.jpg
Figure 6—figure supplement 3
Functional domain mapping of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) analysed by negative stain electron microscopy (EM).
(A) Ten-micromolar tubulin was incubated alone (left), or with 1 µM GFP-CKK (middle), or with 1 µM GFP-CAMSAP2 ∆CKK (right) at 37°C for 10 min and analysed by negative stain EM. Oligomerization of …
Figure 7
Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) induces tubulin ring formation.
Representative electron microscopy (EM) images are shown from at least three independent assays. (A) Negative stain EM micrographs of 10 µM tubulin polymerization with 1 µM GFP-CC1-CKK at different …
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Figure 7—source data 1
Quantification of the numbers of rings and microtubules at different time points.
- https://cdn.elifesciences.org/articles/77365/elife-77365-fig7-data1-v1.pdf
Figure 8 with 3 supplements
Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) induced microtubule nucleation intermediates visualized by time-lapse cryo-electron microscopy (EM).
Representative EM images are shown from at least three independent assays. (A) Snapshots of growing microtubule intermediates at different time points. The contrast of the micrographs was adjusted …
Figure 8—figure supplement 1
Cryo-electron tomographic reconstruction of the growing Cam2-asters.
(A)-(E) Tomographic reconstruction of a growing Cam2-aster processed by SIRT. See also Video 5. (A) Rendered view of a growing Cam2-aster from representative 120 nm thick tomography slices overlaid …
Figure 8—figure supplement 2
Cryo-electron microscopy (Cryo-EM) visualization of Cam2-aster formation.
(A) Negative stain image of 10 µM tubulin on ice. (B) Cryo-EM image of 30 µM tubulin on ice without calmodulin-regulated spectrin-associated protein 2 (CAMSAP2). (C) Raw micrographs of snapshots of …
Figure 8—figure supplement 3
Segmentation of the structural elements of the micrographs at 60 s.
Tubulin rings, red; intermediates between ring and sheet, orange; tubulin sheets, green; microtubules, blue.
Figure 9
Structural model of microtubule nucleation and Cam2-aster formation induced by calmodulin-regulated spectrin-associated protein 2 (CAMSAP2).
Structural model of tubulin nucleation, polymerization, and aster formation induced by CAMSAP2, as detailed in the main text. CAMSAP2 shifts the equilibrium to the right, as indicated by the arrow …
Author response image 1
Author response image 2
GFP attached on the glass surface represents homogeneous signals, unlike GFP-CAMSAP2-FL.
Videos
Video 1
Fusion of the calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) condensate.
Video 2
Fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy.
The video was recorded immediately after photobleaching.
Video 3
Time-lapse movie of aster formation.
Tubulin (10 μM), TMR-tubulin (0.5 μM), and GFP-calmodulin-regulated spectrin-associated protein 2 (CAMSAP2)-FL (50 nM) were incubated with CAMSAP2 condensates fixed on coverslips. The size of the …
Video 4
Magnified movie of aster formation from 1.
Dynamic microtubules from calmodulin-regulated spectrin-associated protein 2 condensates were observed. The size of the field is 8.2 μm × 8.2 μm. The frame rate is 0.2 frames/s.
Video 5
Cryo-electron tomographic reconstruction of the growing Cam2-aster.
SIRT processed cryo-tomographic reconstruction of the growing Cam2-aster produced by incubation of 10 µM tubulin with 1 µM full-length calmodulin-regulated spectrin-associated protein 2 for 1 min at …
Tables
Table 1
List of protein expression constructs used in this study.
| ID | Construct | Amino acids | Vector | Tag (N-terminal) | Tag (C-terminal) |
|---|---|---|---|---|---|
| RN82 | full length | 1–1472 | pFastBac1 | none | 6× His |
| RN63 | CC1-CKK | 696–1,454 | pGFPS1 | HAT*-GFPS1-TEV* | 7× His |
| RN64 | CC3-CKK | 1157–1454 | pGFPS1 | HAT*-GFPS1-TEV* | 7× His |
| RN136 | CKK | 1332–1472 | pGFPS1 | HAT*-GFPS1-TEV* | 7× His |
| RN189 | full length | 1–1472 | pFastBac1 | His_Strep2-3Csite | none |
| RN282 | CC1-CKK-CT | 696–1472 | pET28 | His_Strep2-3Csite | none |
| RN290 | CC3-CKK-CT | 1157–1472 | pET28 | His_Strep2-3Csite | none |
| RN318 | ∆CKK | 1–1331 | pFastBac1 | His_Strep2-3Csite | none |
| RN328 | ∆CC3 | 1–1472 ∆1154–1250 | pFastBac1 | His_Strep2-3Csite | none |
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*
These tags carry modifications. See the Methods for details.
