Journal: EMBO Rep / Year: 2023 Title: CryoET shows cofilactin filaments inside the microtubule lumen. Authors: Camilla Ventura Santos / Stephen L Rogers / Andrew P Carter / Abstract: Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been ...Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.
Download / File: emd_18475.map.gz / Format: CCP4 / Size: 2.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Cryo-electron tomogram of an induced S2 cell protrusion after treatment with 2.5uM CytD (2h) and 2um thapsigargin (5h).
Voxel size
X=Y=Z: 10.74 Å
Density
Minimum - Maximum
-0.60988736 - 0.43933672
Average (Standard dev.)
0.000000000000655 (±0.0399006)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
1440
1022
376
Spacing
1022
1440
376
Cell
A: 10976.279 Å / B: 15465.6 Å / C: 4038.24 Å α=β=γ: 90.0 °
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Supplemental data
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Sample components
-
Entire : Cytochalasin D-induced protrusion of a Drosophila S2 cell.
Entire
Name: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
Components
Cell: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
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Supramolecule #1: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
Supramolecule
Name: Cytochalasin D-induced protrusion of a Drosophila S2 cell. type: cell / ID: 1 / Parent: 0 Details: Cells were treated with 2uM thapsigargin for 5h and with 2.5uM Cytochalasin D for 2h prior to vitrification.
Model: Quantifoil R3.5/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. Details: Grids were glow discharged for 30s at 20mA and then coated with 0.25ug/mL Concanavalin A. Grids were washed twice with PBS before plating cells.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III
Sectioning
Other: NO SECTIONING
Fiducial marker
Manufacturer: BBI Solutions / Diameter: 10 nm
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.56 sec. / Average electron dose: 2.9 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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