Journal: bioRxiv / 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 identity of these objects and what causes their accumulation has not been ...Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of 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 (SERCA) 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, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi 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_16685.map.gz / Format: CCP4 / Size: 2.9 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Deconvolved tomogram (binned by four) of an induced protrusion from Drosophila S2 cells. From Dataset1.
Voxel size
X=Y=Z: 11.808 Å
Density
Minimum - Maximum
-0.5459205 - 0.45870563
Average (Standard dev.)
3.5724032e-12 (±0.03461193)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
1124
1124
624
Spacing
1124
1124
624
Cell
A: 13272.191 Å / B: 13272.191 Å / C: 7368.192 Å α=β=γ: 90.0 °
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Supplemental data
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Sample components
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Entire : Tomogram of an induced protrusion of a Drosophila S2 cell.
Entire
Name: Tomogram of an induced protrusion of a Drosophila S2 cell.
Components
Cell: Tomogram of an induced protrusion of a Drosophila S2 cell.
-
Supramolecule #1: Tomogram of an induced protrusion of a Drosophila S2 cell.
Supramolecule
Name: Tomogram of an induced protrusion of a Drosophila S2 cell. type: cell / ID: 1 / Parent: 0 Details: Protrusion formation was induced by treatment with 2.5 uM Cytochalasin D for 4h. Example tomogram from Dataset 1.
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298.15 K / Instrument: FEI VITROBOT MARK III
Details
Cell were treated with 2.5uM Cytochalasin D for 4 h before vitrification.
Sectioning
Other: NO SECTIONING
Fiducial marker
Manufacturer: BBI Solutions / Diameter: 10 nm
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2 Details: Data was collected on Gatan K2 summit (2.952 A/pixel) with a total dose of 123.4 e/A2.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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