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Files in this Data Supplement:
Fig. S1. Endogenous Myo10 is present in neurites, growth cones, and spine-like structures in rat cortical neurons. (A-C) Endogenous Myo10 has a punctate localization in growth cone filopodia but also localizes throughout the growth cone and neurite in primary cultures stained with Myo10 antibody #3568. (D-F) High magnification image from a 2 week culture showing localization of endogenous Myo10 in a neurite as well as at the tip of a dendritic spine or lateral filopodium. Myo10 immunostaining was also observed in axons, dendrites, and cell bodies. Primary rat cortical neurons were prepared by dissociating neural tissue from E17 fetuses and seeding cells onto poly-D-lysine-coated coverslips at a density of 40,000 cells/cm2 in complete medium (MEM with 10% bovine calf serum and 20 mg/ml gentamicin). On day 4 of culture, 1 mM cytosine arabinoside was added to kill non-neuronal cells. After 24 hours, the cytosine arabinoside was removed from the cultures with two washes of Hank's balanced saline solution and the cells were returned to complete medium. The concentration of cytosine arabinoside and time of exposure were chosen to limit the number of non-neuronal cells in the culture while minimizing potential toxic effects on the neurons. Neurons were cultured for at least one additional day in complete medium before use.
The supplemental movies showing Myo10 dynamics in CAD cells are in QuickTime format. If the QuickTime player is not installed on your computer it can be downloaded free of charge (http://www.apple.com/quicktime/) and used on either PC or Macintosh platforms. Each image is time-stamped to indicate the time (minutes:seconds) at which it was acquired. Movements are most clearly seen by setting the QuickTime viewer to loop the movies and it may be helpful to double the screen size. Movies can also be played frame-by-frame using the computer keyboard’s arrow keys. Notice also that movies were scaled using the Metamorph autoscale function and that the ~20-fold compression of the original Metamorph files into QuickTime format leads to some loss of image quality.
Movie 1. HMM-Myo10 can undergo rearward flow along filopodial rootlets and move deep into the lamellipodium. This movie shows undifferentiated CAD cells transfected with the GFP-HMM-Myo10 construct (consisting of head, neck, and coiled coil; green) and CFP-actin (red). Note that the GFP-Myo10 at the tips of filopodia along the leading edge can undergo lateral movements that lead to fusion of the puncta. Fusion events are sometimes followed by rearward movement. Puncta of GFP-Myo10 can also be observed at the tips of filopodia in the region where the two cells contact one another.
Movie 2. Headless Myo10 does not localize to filopodial tips, move laterally along the leading edge, or undergo intrafilopodial motility. This movie shows an undifferentiated CAD cell transfected with headless GFP-Myo10 (green) and CFP-actin (red) and shows the relatively diffuse localization observed with headless Myo10.
Movie 3. Full-length Myo10 can undergo lateral movements along the leading edge. This movie of an undifferentiated CAD cell transfected with GFP-Myo10 (green) and CFP-actin (red) shows that small puncta of GFP-Myo10 along the leading edge can to move laterally and fuse with adjacent tip puncta. The leading edge of this cell was selected for kymographic analysis illustrated in Fig. 7 because (1) the cell did not change shape very much, and (2) most of the tip puncta stayed at the leading edge rather than undergoing rearward movements into the cell.
Movie 4. Dynamics of full-length Myo10 in a CAD cell undergoing neuronal differentiation. This movie shows a CAD cell transfected with GFP-Myo10 (green) and CFP-actin (red). Note that one of the large Myo10 puncta initially at the edge of the lamellipodium forms an intrapodium-like structure and undergoes “rocketing”. Note also that several puncta of Myo10 within the long filopodium/retraction fiber on the right exhibit intrafilopodial motility.
Movie 5. Full-length Myo10 is present at the tips of neuronal filopodia as they move and interact with other cells. This movie shows filopodia from the leading edge of a growth cone like structure from a CAD cell transfected with full-length GFP-Myo10 (green) and CFP-actin (red) as it contacts the neurite of an adjacent but lightly transfected cell. The bottom panel shows the phase images of the same region.
Movie 6. HMM-Myo10 localizes to the tips of filopodia as they extend and retract during neurite extension. This movie shows the growth cone of a differentiating CAD cell transfected with GFP-HMM-Myo10 (green) and CFP-actin (red). Note that the localization and dynamics of HMM-Myo10 are qualitatively similar to those of full-length Myo10 even though the HMM construct lacks most of the tail.
Movie 7. Full-length Myo10 dynamics in a differentiating CAD cell. Numerous filopodia labeled by Myo10 are present on the cell body (lower left), neurite (extending to right), and growth cones emanating from the neurite (right). Puncta of GFP-Myo10 are visible at the tips of filopodia as they probe the substrate and the surrounding non-transfected cells. Note that some puncta of GFP-Myo10 also exhibit intrafilopodial motility.
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