tra12017-sup-0001-FigureS1.docWord document66K Figure S1: Sequential images of Qdot-labeled myoVI encountering actin filament intersections and actin bundles. A, B) A single myoVI motor (red dot) travels along an Alexa Fluor 660 phalloidin-labeled actin filament (not visible because of rapid photobleaching) and then switches to (A) or crosses over (B) the intersecting tetramethylrhodamine isothiocyanate (TRITC) phalloidin-labeled actin filament (green). The TRITC actin filaments are draped over the Alexa Fluor 660 actin filaments based on the order of actin filament addition to the flow cell (see Figure 1C). The time sequence is shown in the upper left corner. C, D) A single myoVI motor (red dot) is traveling along a TRITC phalloidin-labeled (green) unipolar bundle for 17 seconds (C) or mixed polarity bundle for 20 seconds, switching directions as it travels (D). Calibration bars in all panels are 1 µm.
tra12017-sup-0002-FigureS2.docWord document71K Figure S2: Fluorescence intensity measurement to estimate the number of actin filaments within an actin bundle. Total intensities were measured for an equal sized area (yellow box) on actin filament bundle (A), single actin filament (B) and background (C). After subtracting the background intensity, the ratio between the bundle and actin filament intensities provided an estimate of the number of actin filaments within the bundle.
tra12017-sup-0003-FigureS3.docWord document134K Figure S3: Relative binding frequency of myoVI and myoVa on single actin filaments and unipolar bundles. Number of Qdot-labeled myoVI and myoVa motors per micron that were bound to actin filament and fascin bundle over 17 seconds for various flowcell motor loading conditions.

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