A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation
- University of Warwick, United Kingdom
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 3 supplements
Microtubules are arranged in stable paraxial arrays during muscle cell differentiation.
(A) Structured illumination microscopy of anti-tubulin-stained C2C12 cells pre/post induction of muscle differentiation as indicated. Microtubule filaments have been manually traced to highlight …
Figure 1—figure supplement 1
Microtubule growth orientation.
Angular distribution of microtubule growth data obtained from EB3-GFP tracks for the cells shown in Figure 1B.
Figure 1—figure supplement 2
Dissipation of photoconverted microtubule labelling.
Dissipation of photoconverted regions of mEos2-Tubulin in C2C12 cells before (undiff) and 48 hr post induction of differentiation. Cells were treated with Taxol to stop dissipation by …
Figure 1—figure supplement 3
Verification of dynein and kinesin depletion.
Immunoblotting of C2C12 cell extracts from GFP-positive FACS-sorted cells treated with individual short hairpin RNAs (shRNAs) for 72 hr probed with antibodies as indicated.
Figure 2 with 6 supplements
oMAP4 is required for myoblast elongation and fusion.
(A) Domain organization of MAP4 isoforms. Green and red boxes represent isoform-specific regions in the projection domains of mMAP4 and oMAP4, respectively. Note that all three isoforms are …
Figure 2—figure supplement 1
Expression of major MAP4 isoforms.
Relative transcript abundances of MAP4 isoforms during myoblast differentiation as determined by analysis of RNA sequencing data from undifferentiated and 60 hr differentiated C2C12 cells (Trapnell …
Figure 2—figure supplement 2
C2C12 cells express three major MAP4 isoforms with variable numbers of MT binding repeats.
RT-PCR from total RNA isolated from C2C12 cells before (0 hr) and 48 hr post induction of differentiation with upstream primers specific for projection domains of uMAP4, mMAP4, and oMAP4 and …
Figure 2—figure supplement 3
Microtubular localisation of eGFP-tagged MAP4 isoforms in C2C12 cells.
Tubulin is used as a marker for microtubules. Scale bars are 20 μm, insets 2 μm.
Figure 2—figure supplement 4
Verification of mMAP4 depletion by immunofluorescence.
Protein depletion was quantified by counting cells that expressed mMAP4 in GFP-Tubulin and shRNA co-expressing cells. Bars are 20 μm. 40–50 cells each were analysed for shControl and sh-mMAP4 …
Figure 2—figure supplement 5
Verification of MAP4 depletion by immunoblotting.
Immunoblotting of C2C12 cell extracts treated with individual shRNAs as indicated for 70–74 hr. Tubulin serves as loading control.
Figure 2—figure supplement 6
Verification of oMAP4 depletion and RNAi-protected rescue construct by immunoblotting.
Immunoblot of whole cell extracts of HeLa cells co-transfected with shRNAs and control or rescue plasmids. Tubulin serves as loading control.
Figure 3 with 2 supplements
oMAP4 is required for the parallel arrangement of microtubules in differentiating muscle cells.
(A and B) C2C12 myoblasts 48 hr after induction of differentiation treated with shRNA as indicated and stained for Myogenin (a marker for differentiating myoblasts, yellow), PCM-1 (red) and DAPI …
Figure 3—figure supplement 1
Microtubule orientation in depleted cells.
Manual segmentation of microtubules (white/black), their angular distribution relative to main cell axis (red), and cumulative frequency of microtubule orientation following depletion of individual …
Figure 3—figure supplement 2
Microtubule growth orientation in depleted cells.
Angular distribution of microtubule growth data from Figure 3J. To aid visualisation of data tails, bin size increases progressively between 0 and ±60° and is then constantly 30°.
Figure 4 with 1 supplement
oMAP4 prevents microtubule sliding in cells.
(A) Microtubule motility (arrows) observed after photoconversion of mEOS2-Tubulin in 48 hr differentiated cells treated with shRNA as indicated. Scale bars 5 μm. See supplementary Videos 10, 11. (B) …
Figure 4—figure supplement 1
Microtubule orientation in depleted cells.
Examples of microtubule traces (yellow) in dynein depleted and dynein + oMAP4-depleted 48 hr differentiated elongated myoblasts, see Figure 3G for examples for control and oMAP4 depletion. Angular …
Figure 5
oMAP4 bundles microtubules in vitro.
(A) SDS-PAGE analysis of oMAP4 protein purification. N-terminally 6xHis tagged full-length oMAP4 was purified by Ni2+-NTA affinity chromatography followed by ion exchange chromatography. (B) …
Figure 6 with 1 supplement
oMAP4 zippers dynamic microtubules with a bias for antiparallel arrangements.
(A) Dynamic Rhodamine-labelled microtubules (greyscale) assembled from immobilised Hilyte640-labelled seeds (red) in vitro are zippered in the presence of oMAP4. Arrows highlight bundled …
Figure 6—figure supplement 1
Microtubule length distribution in zippering experiments.
For microtubule encounters between 10 and 30° incident angle in (near) parallel or (near) antiparallel orientation, the length of each microtubule was measured to the attached seed or the closest …
Figure 7
oMAP4 bundles can withstand motor forces.
(A) Microtubule gliding assay on a Drosophila kinesin-1-coated surface. Time colour-coded projections over 30 s are shown for buffer control and 80 nM oMAP4. (B) Instantaneous speeds of microtubule …
Figure 8
oMAP4 and dynein co-operate in the organisation of the paraxial microtubule network in differentiating muscle cells.
(A) Kuiper statistics of traced microtubule filaments as in Figures 3G, 4, Figure 4—figure supplement 1 as a measure for microtubule orderliness is plotted against the mean cell length of 48 hr …
Videos
Video 1
Photoconversion of mEos2-Tubulin in an undifferentiated C2C12 myoblast showing converted (left panel and magenta in right panel) and non-converted channels (middle panel and green in right panel).
Scale bar: 10 μm.
Video 2
Photoactivation of bar-shaped patterns of paGFP-Tubulin in an undifferentiated C2C12 myoblast.
Scale bar: 10 μm.
Video 3
Photoactivation of bar-shaped patterns of paGFP-Tubulin in a 94-hr differentiated C2C12 myoblast.
Scale bar: 10 μm.
Video 4
Photoactivation of bar-shaped patterns of paGFP-Tubulin in an undifferentiated C2C12 myoblast treated with shRNA against dynein heavy chain.
Scale bar: 10 μm.
Video 5
Photoactivation of bar-shaped patterns of paGFP-Tubulin in an undifferentiated C2C12 myoblast treated with shRNA against kinesin heavy chain (Kif5b).
Scale bar: 10 μm.
Video 6
Microtubule orientation in a 48 hr differentiated C2C12 myoblast treated with shControl co-expressing GFP-Tubulin.
Manual tracing of microtubule cytoskeleton (yellow lines) and main cell axis (red line) shown as used for analysis. Scale bar: 10 μm.
Video 7
Microtubule orientation in a 48-hr differentiated C2C12 myoblast treated with sh-oMAP4 co-expressing GFP-Tubulin.
Manual tracing of microtubule cytoskeleton (yellow lines) and main cell axis (red line) shown as used for analysis. Scale bar: 10 μm.
Video 8
Growing microtubules in a 48-hr differentiated C2C12 myoblast treated with sh-Control co-expressing EB3-tdTomato.
Scale bar: 10 μm.
Video 9
Growing microtubules in a 48-hr differentiated C2C12 myoblast treated with sh-oMAP4 co-expressing EB3-tdTomato.
Scale bar: 10 μm.
Video 10
Photoconversion of bar-shaped patterns of mEOS2-Tubulin in a 48-hr differentiated C2C12 myoblast treated with shControl.
Scale bar: 10 μm.
Video 11
Photoconversion of bar-shaped patterns of mEOS2-Tubulin in a 48-hr differentiated C2C12 myoblast treated with sh-oMAP4.
Scale bar: 10 μm.
Video 12
TIRF-based assay showing dynamic Rhodamine-labelled microtubules assembled from immobilised seeds.
Scale bar: 10 μm.
Video 13
TIRF-based assay showing dynamic microtubules in the presence of 80 nM GFP-oMAP4.
Note that antiparallel microtubule encounters result in zippering into an antiparallel bundle in most cases. Scale bar: 10 μm.
Video 14
Microtubule gliding assay on a kinesin-1-coated surface in the presence of 80 nM GFP-oMAP4.
Note that single microtubules move persistently, while bundles don't until they are driven apart. Scale bar: 10 μm.
Additional files
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Supplementary file 1
RNAseq_Myoblast_Myocyte. RNA sequencing data for C2C12 myoblasts and 60 hr differentiated myocytes (Trapnell et al., 2010) extracted for four regions in MAP4 as shown in Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.05697.038
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Supplementary file 2
Affymetrix Exon Arrays. Affymetrix exon array data (Pohl et al., 2009) for four regions in MAP4 as shown in Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.05697.039
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Source code 1
MATLAB code MTdirectionality. Custom MATLAB function to compute directionality of MT growth relative to the main cell axis, generate figure with tracks colour-coded for direction, plot angular histograms and calculate Kuiper statistics relative to a random distribution.
- https://doi.org/10.7554/eLife.05697.040
