The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle
- Warwick Medical School, United Kingdom
- University of Liverpool, United Kingdom
Figures
Figure 1 with 2 supplements
Intermicrotubule connectors in K-fibers are ‘bridges’ in 2D and a ‘mesh’ in 3D.
(A) 2D views of inter-MT bridges in sections taken orthogonally or longitudinally to the spindle axis. In the annotated version, MTs (green) and inter-MT bridges (yellow) are shown on a blue …
Figure 1—figure supplement 1
The mesh is visible, but not well preserved, in chemically-fixed K-fibers.
Orthoslice of a tomogram generated from a tilt series of a single section through a K-fiber fixed with glutaraldehyde (i). Overlaid is a hand-rendered 3D representation of MTs (green) (ii) and mesh …
Figure 1—figure supplement 2
The mesh is associated with K-fiber MTs.
(A) 3D rendering of ‘non-mesh’ (brown) in a tomogram of a single K-fiber. Single orthoslice (i), with added rendering (MTs, green; mesh, yellow) (ii) and model alone (iii), is shown. Rendering the …
Figure 2
Inter-MT linkages are defined connectors with heterogeneous MT-mesh contacts.
(A) Examples of bipolar, tripolar, and quadrupolar connectors within the mesh. Single orthoslices from tomograms showing examples of different connectors. MTs are hand-rendered (green), mesh is …
Figure 3 with 1 supplement
Analysis of MT packing within a K-fiber.
(A) Box plots showing the number of MTs per K-fiber, the cross-sectional K-fiber area, and the density of MTs in the K-fiber. Nfiber = 26 (control), 37 (TACC3 OE). Box plots show the median, 75th …
Figure 3—figure supplement 1
Inducible expression of GFP-TACC3 in HeLa cells to alter the composition of mesh.
(A) Western blot to show the extent of overexpression of TACC3 caused by inducing the expression of GFP-TACC3 in a stable TetOn HeLa cell line using 0.5 μg/ml doxycycline for 24 hr. The blot was …
Figure 4 with 1 supplement
Analysis of MTs captured by mesh, their connectivity, and proximity.
(A) Histogram to show frequency of MT chain sizes detected in single section tomograms. MT chains are collections of MTs that are connected by mesh within a section. Note that single MTs do not …
Figure 4—figure supplement 1
Examples of MTs chains and associated mesh.
Two example tomograms, which were annotated by 3D rendering and used to make the 2D maps illustrating MT position and chain membership in Figure 4C. MTs are colored as in Figure 4C, mesh associated …
Figure 5 with 2 supplements
Bundling of MTs in vitro by mesh components.
(A) Cartoon to illustrate two potential models for mesh function. In the passive model (left), MT distances are set by some other factor, and the mesh fills in the gaps to connect MTs. In the …
Figure 5—figure supplement 1
MT bundling using purified components.
Representative fluorescence micrographs of rhodamine-labeled MTs incubated with the indicated proteins. In all experiments, control was 210 nM MBP-His6 and 100 nM GST, and the total amount of …
Figure 5—figure supplement 2
MT bundling using mesh complex immunoisolated from mitotic HeLa cells.
(A) Schematic diagram of the procedure to release mitotic spindle proteins from mitotic spindles at metaphase (described in Booth et al., 2011). (B) Western blot to show specific …
Figure 6
Analysis of MT trajectories within a K-fiber.
(A) Representative ‘side views’ of rendered MTs in single tomograms of individual K-fibers from control (left, red, Video 2) or TACC3 OE (right, blue, Video 3) cells. Tomogram thickness, 66.4 nm …
Figure 7 with 1 supplement
Mitotic consequences of TACC3 overexpression.
(A) Cartoon representation of the stages of mitosis captured in live-cell imaging experiments. Cells expressing H2B-mCherry were staged as indicated with the transition to prometaphase marked by …
Figure 7—figure supplement 1
Mitotic consequences of TACC3 depletion.
Box plots to summarize mitotic progression experiments. Time taken to go from prometaphase-to-metaphase (PM-M), metaphase-to-anaphase (M-A), or anaphase-to-telophase (A-T) is shown for control …
Author response image 1
TACC3 mesh complex does not persist in a “spindle matrix” after depolymerization of MTs.
Still images from live-cell imaging experiments to visualize metaphase HeLa cells expressing mCherry-tubulin and the indicated GFP-tagged protein. Cells were exposed to DMSO (control, left) or …
Author response image 2
Fluorescence recovery after photobleaching (FRAP) analysis of TACC3 dynamics in mitotic cells.
(A) Average FRAP traces for metaphase cells expressing GFP-tubulin and either mCherry-clathin light chain a or mCherry-TACC3. Plots show the mean ± s.e.m. fluorescence that is scaled to examine the …
Videos
Video 1
Example of the K-fiber mesh from a normal HeLa cell at metaphase.
Tomogram of a K-fiber. The mesh (yellow) is shown by manual rendering and by automated rendering. MTs (green) were rendered by hand. All segmentation was smoothed in Amira.
Video 2
Example of MT organization in a K-fiber from a control cell.
Tomogram of a K-fiber. MTs (green) were rendered by hand. All segmentation was smoothed in Amira.
Video 3
Example of MT organization in a K-fiber from a GFP-TACC3 expressing cell.
Tomogram of a K-fiber. MTs (green) were rendered by hand. Pale green is used to highlight two highly deviant MTs. All segmentation was smoothed in Amira.
Additional files
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Source code 1
Custom written code in IgorPro 6.36 (Wavemetrics) was used for all analysis and plotting.
- https://doi.org/10.7554/eLife.07635.020
