(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 …
Figure 1 SDS-PAGE gel of the full-length calmodulin-regulated spectrin-associated protein 2.
(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 …
SDS-PAGE gel from a spin-down spontaneous nucleation assay of tubulin showing the total tubulin after 60 min of polymerization at 37°C.
SDS-PAGE gel of depolymerized tubulin after spin-down pelleting assay.
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.
SDS-PAGE gel of depolymerized tubulin with 1 µM calmodulin-regulated spectrin-associated protein 2-FL after spin-down pelleting assay.
Quantification of the depolymerized tubulin concentrations determined by pelleting assay.
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.
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.
Quantification of the depolymerized tubulin concentrations determined by 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).
SDS-PAGE gel of tubulin spin-down pelleting assay without depolymerization (no depoly).
Quantification of the depolymerized tubulin concentrations determined by pelleting assay.
(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 …
Quantification of the fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy.
Quantification of the fluorescence recovery after photobleaching of GFP-calmodulin-regulated spectrin-associated protein 2-FL condensates, acquired via confocal microscopy.
(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 …
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 …
Original image of immunofluorescence visualized by a confocal laser-scanning microscope.
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 …
(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 …
SDS-PAGE gels of full-length GFP-CAMSAP3.
(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 …
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 …
(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 …
Quantifications of fluorescent intensity of calmodulin-regulated spectrin-associated protein 2 condensates at 0 s.
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 …
SDS-PAGE gel of calmodulin-regulated spectrin-associated protein 2 deletion constructs.
SDS-PAGE gel of calmodulin-regulated spectrin-associated protein 2 deletion constructs.
(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 …
SDS-PAGE gel of calmodulin-regulated spectrin-associated protein 2 deletion constructs.
(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 …
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 …
Quantification of the numbers of rings and microtubules at different time points.
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 …
(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 …
(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 …
Tubulin rings, red; intermediates between ring and sheet, orange; tubulin sheets, green; microtubules, blue.
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 …
The video was recorded immediately after photobleaching.
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 …
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.
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 …
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 |
These tags carry modifications. See the Methods for details.