Direct observation of microtubule pushing by cortical dynein in living cells

Supplemental Materials

This article contains the following supporting material:

  • Supplemental Materials
  • Movie01 - Movie 1: Directional sliding of microtubules near the cell cortex observed via wide-field (left) and TIRF (right) imaging.
  • Movie02 - Movie 2: Polarity orientation of motile microtubules observed via wide-field and TIRF imaging. This example shows multiple motile microtubules, which were labeled at their plus-tips with mCherry-EB3 and with EGFP-MAP2c along their entire length. Top left: combined mCherry-EB3 TIRF (green) and EGFP-MAP2c TIRF (red); Top right: mCherry-EB3 TIRF; Bottom left: EGFPMAP2c TIRF; Bottom right: EGFP-MAP2c wide-field.
  • Movie03 - Movie 3: Directional sliding of microtubules in COS7 cells transfected with various microtubule associated proteins. COS7 cells were transfected with EGFP-MAP2c, EGFP-tau, or EGFPEMAP115-MTBD. Microtubule motility was observed either via wide-field or TIRF imaging shortly after nocodazole washout.
  • Movie04 - Movie 4: Microtubules labeled directly with mCherry-tubulin in COS7 cells. As microtubules were very dense, dynamic, long and flexible, potential movements were difficult to interpret. Cells were co-transfected with EGFP-Dync1h1 to simultaneously visualize cortical dynein complex dynamics.
  • Movie05 - Movie 5: Dynamic colocalization of microtubules and dynein heavy chain speckles. This example shows a motile dynein speckle colocalized with a microtubule that is first stationary (case 2) and later becomes mobile.
  • Movie06 - Movie 6: Dynamic colocalization of microtubules and dynein heavy chain speckles. This example shows stationary dynein speckles colocalized with motile microtubules (case 3).
  • Movie07 - Movie 7: Dynamic colocalization of microtubules and dynein heavy chain speckles. This is another example of a stationary dynein speckle colocalized with a motile microtubule (case 3).
  • Movie08 - Movie 8: Dynamic colocalization of microtubules and dynein heavy chain speckles. This example shows a motile dynein speckle located at the trailing microtubule end, which moves together with the motile microtubule (case 5).
  • Movie09 - Movie 9: Correlation of microtubule and dynein speckle dynamics. A microtubule slightly rotates while it slides over a speckle of the dynein intermediate chain in the middle of this region. The pivot point of the microtubule rotation is located at the dynein intermediate chain speckle (see also Figure S8 in supplementary material).
  • Movie10 - Movie 10: Correlation of microtubule and dynein speckle dynamics. The disappearance of a dynein heavy chain speckle at timepoint 51 s coincides with the loss of a kink in a microtubule.
  • Movie11 - Movie 11: Stochastic simulation of dynein mediated microtubule motility in cells. Left: Stochastic simulation of microtubule (yellow) movements powered by cortical dynein speckles (blue). Right: Movie of microtubule movements following nocodazole washout.