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. 2019 Mar 22;38(11):e99630. doi: 10.15252/embj.201899630

Figure 5. Reconstitution of the interplay between F‐actin and microtubules on micropatterns.

Figure 5

  1. Schematic illustration of the micropatterning method used to graft microtubule seeds (green) via neutravidin (yellow) and F‐actin‐nucleation‐promoting complexes (streptavidin‐WA) (orange) on 8‐micron‐wide discoidal micropatterns. A glass coverslip (deep blue) coated with polyethyleneglycol (PEG) (light blue) was placed in contact with a transparency photomask and exposed to deep UV light. The exposed coverslip was then immersed with neutravidin to fix biotinylated microtubule seeds (green) on exposed regions. Streptavidin‐WA was immobilized on microtubule seeds via their interaction with biotin. Tubulin dimers and actin monomers were then added to allow filaments elongation.
  2. Representative images of microtubules (top) and F‐actin (bottom) growth from micropatterns. Scale bars: 20 μm.
  3. Schematic illustration of the assay on micropatterned substrate. Tubulin was first added alone to measure the nucleation capacity of each micropattern, and then washed out. Later on, actin was added followed by actin and tubulin. Finally, actin was rinsed out and gelsolin was added to fully disassemble F‐actin. Representative images showing microtubules (top line) and the merged images of actin filaments and microtubules (bottom line) during the four steps of the assay; in the presence of tubulin only, in the absence of tubulin and presence of actin, in the presence of tubulin and actin, and finally in the presence of tubulin and gelsolin but in the absence of actin (from left to right). Scale bars: 10 μm.
  4. Quantification of the number of microtubules per micropattern in the presence of tubulin only (left), actin and tubulin (middle) and tubulin only after actin filament disassembly (right). Data were pooled from 2 independent experiments; n = 133. ****P < 0.001 Student's t‐test.
  5. The graph shows the same measurements as in panel (E) in an XY representation of individual measurements. It illustrates the differences in the number of microtubules per micropattern between the first to the second step (tubulin only versus actin and tubulin together) with respect to the density of F‐actin per micropattern.